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Frontiers Section Editor (Bioinformatics and Biophysics) for the Current Drug Targets ISSN: 1873-5592

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Section Editor (Bioinformatics in Drug Design and Discovery) for the Current Medicinal Chemistry ISSN: 1875-533X

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Member of the Editorial Board for the PeerJ ISSN: 2167-8359

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Member of the Editorial Board for the Current Bioinformatics ISSN: 2212-392X (Online) ISSN: 1574-8936 (Print)

 

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Protein-Ligand Interacions

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Molecular Docking

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Bioinspired Computing

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Computational Systems Biology

 

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Volkart PA et al. Curr Drug Targets. 2019;20(7):716-726

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Russo S, De Azevedo WF. Curr Med Chem 2019. 26(10):1908-1919

                                  

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Russo S, de Azevedo WF Jr. Computational Analysis of Dipyrone Metabolite 4-Aminoantipyrine as a Cannabinoid Receptor 1 Agonist. Curr Med Chem. doi: 10.2174/0929867326666190906155339

 

BACKGROUND: Cannabinoid receptor 1 has its crystallographic structure available in complex with agonists and inverse agonists, which paved the way to establish an understanding of the structural basis of interactions with ligands. Dipyrone is a prodrug with analgesic capabilities and which is widely used in some countries. Recently it was shown some evidence of a dipyrone metabolite acting over the Cannabinoid Receptor 1. OBJECTIVE: Our goal here is to explore the dipyrone metabolite 4-aminoantipyrine as a Cannabinoid Receptor 1 agonist, reviewing dipyrone characteristics, and investigating the structural basis for its interaction with Cannabinoid Receptor 1. METHOD: We reviewed here recent functional studies related to the dipyrone metabolite focusing on its action as a Cannabinoid Receptor 1 agonist. We also analyzed protein-ligand interactions for this complex obtained through docking simulations against the crystallographic structure of the Cannabinoid Receptor 1. RESULTS: Analysis of the crystallographic structure and docking simulations revealed that most of the interactions present in the docked pose were also present in the crystallographic structure of Cannabinoid Receptor 1 and agonist. CONCLUSION: Analysis of the complex of 4-aminoantipyrine and Cannabinoid Receptor 1 revealed the pivotal role played by residues Phe 170, Phe 174, Phe 177, Phe 189, Leu 193, Val 196, and Phe 379, besides the conserved hydrogen bond at Ser 383. The mechanistic analysis and the present computational study suggest that the dipyrone metabolite 4-aminoantipyrine interacts with Cannabinoid Receptor 1.

Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

KEYWORDS: 4-aminoantipyrine; cannabinoid receptor 1; dipyrone; docking; metamizole; molecular interactions

PMID: 31490743 DOI: 10.2174/0929867326666190906155339

de Ávila MB, Bitencourt-Ferreira G, de Azevedo Jr. WF. Structural Basis for Inhibition of Enoyl-[Acyl Carrier Protein] Reductase (InhA) from Mycobacterium tuberculosis. Curr Med Chem. doi: 10.2174/0929867326666181203125229

 

BACKGROUND: The enzyme trans-enoyl-[acyl carrier protein] reductase (InhA) is a central protein for the development of antitubercular drugs. This enzyme is the target for the pro-drug isoniazid, which is catalyzed by the enzyme catalase-peroxidase (KatG) to become active. OBJECTIVE: Our goal here is to review the studies on InhA, starting with general aspects and focusing on the recent structural studies, with emphasis on the crystallographic structures of complexes involving InhA and inhibitors. METHOD: We start with a literature review, and then we describe recent studies on InhA crystallographic structures. We use this structural information to depict protein-ligand interactions. We also analyze the structural basis for inhibition of InhA. Furthermore, we describe the application of computational methods to predict binding affinity based on the crystallographic position of the ligands. RESULTS: Analysis of the structures in complex with inhibitors revealed the critical residues responsible for the specificity against InhA. Most of the intermolecular interactions involve the hydrophobic residues with two exceptions, the residues Ser 94 and Tyr 158. Examination of the interactions has shown that many of the key residues for inhibitor binding were found in mutations of the InhA gene in the isoniazid-resistant Mycobacterium tuberculosis. Computational prediction of the binding affinity for InhA has indicated a moderate uphill relationship with experimental values. CONCLUSION: Analysis of the structures involving InhA inhibitors shows that small modifications on these molecules could modulate their inhibition, which may be used to design novel antitubercular drugs specific for multidrug-resistant strains.

Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

KEYWORDS: Crystal Structure; Drug Design.; Enoyl-[Acyl Carrier Protein] Reductase; Protein-Ligand Interactions

PMID: 30501592 DOI: 10.2174/0929867326666181203125229

da Silva AD, Bitencourt-Ferreira G, de Azevedo WF Jr. Taba: A Tool to Analyze the Binding Affinity. J Comput Chem. 2019 doi: 10.1002/jcc.26048.

 

Evaluation of ligand-binding affinity using the atomic coordinates of a protein-ligand complex is a challenge from the computational point of view. The availability of crystallographic structures of complexes with binding affinity data opens the possibility to create machine-learning models targeted to a specific protein system. Here, we describe a new methodology that combines a mass-spring system approach with supervised machine-learning techniques to predict the binding affinity of protein-ligand complexes. The combination of these techniques allows exploring the scoring function space, generating a model targeted to a protein system of interest. The new model shows superior predictive performance when compared with classical scoring functions implemented in the programs Molegro Virtual Docker, AutoDock4, and AutoDock Vina. We implemented this methodology in a new program named Taba. Taba is implemented in Python and available to download under the GNU license at https://github.com/azevedolab/taba.

© 2019 Wiley Periodicals, Inc.

KEYWORDS: binding affinity; drug design; machine learning; protein-ligand interactions; scoring function

PMID: 31410856 DOI: 10.1002/jcc.26048

Russo S, De Azevedo WF. Advances in the Understanding of the Cannabinoid Receptor 1 - Focusing on the Inverse Agonists Interactions. Curr Med Chem. 2019; 26(10): 1908–1919.

 

BACKGROUND: Cannabinoid Receptor 1 (CB1) is a membrane protein prevalent in the central nervous system, whose crystallographic structure has recently been solved. Studies will be needed to investigate CB1 complexes with its ligands and its role in the development of new drugs. OBJECTIVE: Our goal here is to review the studies on CB1, starting with general aspects and focusing on the recent structural studies, with emphasis on the inverse agonists bound structures. METHOD: We start with a literature review, and then we describe recent studies on CB 1 crystallographic structure and docking simulations. We use this structural information to depict protein-ligand interactions. We also describe the molecular docking method to obtain complex structures of CB 1 with inverse agonists. RESULTS: Analysis of the crystallographic structure and docking results revealed the residues responsible for the specificity of the inverse agonists for CB 1. Most of the intermolecular interactions involve hydrophobic residues, with the participation of the residues Phe 170 and Leu 359 in all complex structures investigated in the present study. For the complexes with otenabant and taranabant, we observed intermolecular hydrogen bonds involving residues His 178 (otenabant) and Thr 197 and Ser 383 (taranabant). CONCLUSION: Analysis of the structures involving inverse agonists and CB 1 revealed the pivotal role played by residues Phe 170 and Leu 359 in their interactions and the strong intermolecular hydrogen bonds highlighting the importance of the exploration of intermolecular interactions in the development of novel inverse agonists.

Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

KEYWORDS: Cannabinoid receptor; docking; drug design; inverse agonist.

PMID: 29667549 DOI: 10.2174/0929867325666180417165247

Bitencourt-Ferreira G, de Azevedo WF Jr. How Docking Programs Work. Methods Mol Biol. 2019; 2053: 35–50.

 

Protein-ligand docking simulations are of central interest for computer-aided drug design. Docking is also of pivotal importance to understand the structural basis for protein-ligand binding affinity. In the last decades, we have seen an explosion in the number of three-dimensional structures of protein-ligand complexes available at the Protein Data Bank. These structures gave further support for the development and validation of in silico approaches to address the binding of small molecules to proteins. As a result, we have now dozens of open source programs and web servers to carry out molecular docking simulations. The development of the docking programs and the success of such simulations called the attention of a broad spectrum of researchers not necessarily familiar with computer simulations. In this scenario, it is essential for those involved in experimental studies of protein-ligand interactions and biophysical techniques to have a glimpse of the basics of the protein-ligand docking simulations. Applications of protein-ligand docking simulations to drug development and discovery were able to identify hits, inhibitors, and even drugs. In the present chapter, we cover the fundamental ideas behind protein-ligand docking programs for non-specialists, which may benefit from such knowledge when studying molecular recognition mechanism.

KEYWORDS: Docking; Drug design; Ligand; Molecular recognition; Protein

PMID: 31452097 DOI: 10.1007/978-1-4939-9752-7_3

Bitencourt-Ferreira G, de Azevedo WF Jr. SAnDReS: A Computational Tool for Docking. Methods Mol Biol. 2019; 2053: 51–65.

 

Since the early 1980s, we have witnessed considerable progress in the development and application of docking programs to assess protein-ligand interactions. Most of these applications had as a goal the identification of potential new binders to protein targets. Another remarkable progress is taking place in the determination of the structures of protein-ligand complexes, mostly using X-ray diffraction crystallography. Considering these developments, we have a favorable scenario for the creation of a computational tool that integrates into one workflow all steps involved in molecular docking simulations. We had these goals in mind when we developed the program SAnDReS. This program allows the integration of all computational features related to modern docking studies into one workflow. SAnDReS not only carries out docking simulations but also evaluates several docking protocols allowing the selection of the best approach for a given protein system. SAnDReS is a free and open-source (GNU General Public License) computational environment for running docking simulations. Here, we describe the combination of SAnDReS and AutoDock4 for protein-ligand docking simulations. AutoDock4 is a free program that has been applied to over a thousand receptor-ligand docking simulations. The dataset described in this chapter is available for downloading at https://github.com/azevedolab/sandres.

KEYWORDS: AutoDock4; Binding affinity; Docking; Drug design; Molecular recognition; SAnDReS

PMID: 31452098 DOI: 10.1007/978-1-4939-9752-7_4

Bitencourt-Ferreira G, Veit-Acosta M, de Azevedo WF Jr. Electrostatic Energy in Protein-Ligand Complexes. Methods Mol Biol. 2019; 2053: 67–77.

 

Computational analysis of protein-ligand interactions is of pivotal importance for drug design. Assessment of ligand binding energy allows us to have a glimpse of the potential of a small organic molecule as a ligand to the binding site of a protein target. Considering scoring functions available in docking programs such as AutoDock4, AutoDock Vina, and Molegro Virtual Docker, we could say that they all rely on equations that sum each type of protein-ligand interactions to model the binding affinity. Most of the scoring functions consider electrostatic interactions involving the protein and the ligand. In this chapter, we present the main physics concepts necessary to understand electrostatics interactions relevant to molecular recognition of a ligand by the binding pocket of a protein target. Moreover, we analyze the electrostatic potential energy for an ensemble of structures to highlight the main features related to the importance of this interaction for binding affinity.

KEYWORDS: Binding affinity; Drug design; Electrostatic interactions; Molecular recognition; Shikimate pathway

PMID: 31452099 DOI: 10.1007/978-1-4939-9752-7_5

Bitencourt-Ferreira G, Veit-Acosta M, de Azevedo WF Jr. Van der Waals Potential in Protein Complexes. Methods Mol Biol. 2019; 2053: 79–91.

 

Van der Waals forces are determinants of the formation of protein-ligand complexes. Physical models based on the Lennard-Jones potential can estimate van der Waals interactions with considerable accuracy and with a computational complexity that allows its application to molecular docking simulations and virtual screening of large databases of small organic molecules. Several empirical scoring functions used to evaluate protein-ligand interactions approximate van der Waals interactions with the Lennard-Jones potential. In this chapter, we present the main concepts necessary to understand van der Waals interactions relevant to molecular recognition of a ligand by the binding pocket of a protein target. We describe the Lennard-Jones potential and its application to calculate potential energy for an ensemble of structures to highlight the main features related to the importance of this interaction for binding affinity.

KEYWORDS: Binding affinity; Drug design; Lennard-Jones potential; Shikimate pathway; van der Waals interactions

PMID: 31452100 DOI: 10.1007/978-1-4939-9752-7_6

Bitencourt-Ferreira G, Veit-Acosta M, de Azevedo WF Jr. Hydrogen Bonds in Protein-Ligand Complexes. Methods Mol Biol. 2019; 2053: 93–107.

 

Fast and reliable evaluation of the hydrogen bond potential energy has a significant impact in the drug design and development since it allows the assessment of large databases of organic molecules in virtual screening projects focused on a protein of interest. Semi-empirical force fields implemented in molecular docking programs make it possible the evaluation of protein-ligand binding affinity where the hydrogen bond potential is a common term used in the calculation. In this chapter, we describe the concepts behind the programs used to predict hydrogen bond potential energy employing semi-empirical force fields as the ones available in the programs AMBER, AutoDock4, TreeDock, and ReplicOpter. We described here the 12-10 potential and applied it to evaluate the binding affinity for an ensemble of crystallographic structures for which experimental data about binding affinity are available.

KEYWORDS: Binding affinity; Drug design; Hydrogen bond interactions; Molecular recognition; Shikimate pathway

PMID: 31452101 DOI: 10.1007/978-1-4939-9752-7_7

Bitencourt-Ferreira G, de Azevedo WF Jr. Molecular Dynamics Simulations with NAMD2. Methods Mol Biol. 2019; 2053: 109–124.

 

X-ray diffraction crystallography is the primary technique to determine the three-dimensional structures of biomolecules. Although a robust method, X-ray crystallography is not able to access the dynamical behavior of macromolecules. To do so, we have to carry out molecular dynamics simulations taking as an initial system the three-dimensional structure obtained from experimental techniques or generated using homology modeling. In this chapter, we describe in detail a tutorial to carry out molecular dynamics simulations using the program NAMD2. We chose as a molecular system to simulate the structure of human cyclin-dependent kinase 2.

KEYWORDS: Cyclin-dependent kinase 2; Drug design; Force fields; Molecular dynamics; Molecular recognition; NAMD2

PMID: 31452102 DOI: 10.1007/978-1-4939-9752-7_8

Bitencourt-Ferreira G, de Azevedo WF Jr. Docking with AutoDock4. Methods Mol Biol. 2019; 2053: 125–148.

 

AutoDock is one of the most popular receptor-ligand docking simulation programs. It was first released in the early 1990s and is in continuous development and adapted to specific protein targets. AutoDock has been applied to a wide range of biological systems. It has been used not only for protein-ligand docking simulation but also for the prediction of binding affinity with good correlation with experimental binding affinity for several protein systems. The latest version makes use of a semi-empirical force field to evaluate protein-ligand binding affinity and for selecting the lowest energy pose in docking simulation. AutoDock4.2.6 has an arsenal of four search algorithms to carry out docking simulation including simulated annealing, genetic algorithm, and Lamarckian algorithm. In this chapter, we describe a tutorial about how to perform docking with AutoDock4. We focus our simulations on the protein target cyclin-dependent kinase 2.

KEYWORDS: AutoDock; Cyclin-dependent kinase 2; Drug design; Molecular docking; Protein-ligand interactions

PMID: 31452103 DOI: 10.1007/978-1-4939-9752-7_9

Bitencourt-Ferreira G, de Azevedo WF Jr. Molegro Virtual Docker for Docking. Methods Mol Biol. 2019; 2053: 149–167.

 

Molegro Virtual Docker is a protein-ligand docking simulation program that allows us to carry out docking simulations in a fully integrated computational package. MVD has been successfully applied to hundreds of different proteins, with docking performance similar to other docking programs such as AutoDock4 and AutoDock Vina. The program MVD has four search algorithms and four native scoring functions. Considering that we may have water molecules or not in the docking simulations, we have a total of 32 docking protocols. The integration of the programs SAnDReS ( https://github.com/azevedolab/sandres ) and MVD opens the possibility to carry out a detailed statistical analysis of docking results, which adds to the native capabilities of the program MVD. In this chapter, we describe a tutorial to carry out docking simulations with MVD and how to perform a statistical analysis of the docking results with the program SAnDReS. To illustrate the integration of both programs, we describe the redocking simulation focused the cyclin-dependent kinase 2 in complex with a competitive inhibitor.

KEYWORDS: Cyclin-dependent kinase 2; Drug design; MolDock; Molecular docking; Molegro Virtual Docker; Protein-ligand interactions

PMID: 31452104 DOI: 10.1007/978-1-4939-9752-7_10

Bitencourt-Ferreira G, de Azevedo WF Jr. Docking with GemDock. Methods Mol Biol. 2019; 2053: 169–188.

 

GEMDOCK is a protein-ligand docking software that makes use of an elegant biologically inspired computational methodology based on the differential evolution algorithm. As any docking program, GEMDOCK has two major features to predict the binding of a small-molecule ligand to the binding site of a protein target: the search algorithm and the scoring function to evaluate the generated poses. The GEMDOCK scoring function uses a piecewise potential energy function integrated into the differential evolutionary algorithm. GEMDOCK has been applied to a wide range of protein systems with docking accuracy similar to other docking programs such as Molegro Virtual Docker, AutoDock4, and AutoDock Vina. In this chapter, we explain how to carry out protein-ligand docking simulations with GEMDOCK. We focus this tutorial on the protein target cyclin-dependent kinase 2.

KEYWORDS: Cyclin-dependent kinase 2; Drug design; GEMDOCK; Molecular docking; Protein-ligand interactions

PMID: 31452105 DOI: 10.1007/978-1-4939-9752-7_11

Bitencourt-Ferreira G, de Azevedo WF Jr. Docking with SwissDock. Methods Mol Biol. 2019; 2053: 189–202.

 

Protein-ligand docking simulation is central in drug design and development. Therefore, the development of web servers intended to docking simulations is of pivotal importance. SwissDock is a web server dedicated to carrying out protein-ligand docking simulation intuitively and elegantly. SwissDock is based on the protein-ligand docking program EADock DSS and has a simple and integrated interface. The SwissDock allows the user to upload structure files for a protein and a ligand, and returns the results by e-mail. To facilitate the upload of the protein and ligand files, we can prepare these input files using the program UCSF Chimera. In this chapter, we describe how to use UCSF Chimera and SwissDock to perform protein-ligand docking simulations. To illustrate the process, we describe the molecular docking of the competitive inhibitor roscovitine against the structure of human cyclin-dependent kinase 2.

KEYWORDS: Cyclin-dependent kinase 2; Drug design; Molecular docking; Protein-ligand interactions; SwissDock

PMID: 31452106 DOI: 10.1007/978-1-4939-9752-7_12

Bitencourt-Ferreira G, de Azevedo WF Jr. Molecular Docking Simulations with ArgusLab. Methods Mol Biol. 2019; 2053: 203–220.

 

Molecular docking is the major computational technique employed in the early stages of computer-aided drug discovery. The availability of free software to carry out docking simulations of protein-ligand systems has allowed for an increasing number of studies using this technique. Among the available free docking programs, we discuss the use of ArgusLab ( http://www.arguslab.com/arguslab.com/ArgusLab.html ) for protein-ligand docking simulation. This easy-to-use computational tool makes use of a genetic algorithm as a search algorithm and a fast scoring function that allows users with minimal experience in the simulations of protein-ligand simulations to carry out docking simulations. In this chapter, we present a detailed tutorial to perform docking simulations using ArgusLab.

KEYWORDS: ArgusLab; Cyclin-dependent kinase 2; Drug design; Molecular docking; Molecular recognition; Protein-ligand interactions

PMID: 31452107 DOI: 10.1007/978-1-4939-9752-7_13

Bitencourt-Ferreira G, de Azevedo WF Jr. Homology Modeling of Protein Targets with MODELLER. Methods Mol Biol. 2019; 2053: 231–249.

 

Homology modeling is a computational approach to generate three-dimensional structures of protein targets when experimental data about similar proteins are available. Although experimental methods such as X-ray crystallography and nuclear magnetic resonance spectroscopy successfully solved the structures of nearly 150,000 macromolecules, there is still a gap in our structural knowledge. We can fulfill this gap with computational methodologies. Our goal in this chapter is to explain how to perform homology modeling of protein targets for drug development. We choose as a homology modeling tool the program MODELLER. To illustrate its use, we describe how to model the structure of human cyclin-dependent kinase 3 using MODELLER. We explain the modeling procedure of CDK3 apoenzyme and the structure of this enzyme in complex with roscovitine.

KEYWORDS: Cyclin-dependent kinase 3; Drug design; Homology modeling; MODELLER; Molecular recognition

PMID: 31452109 DOI: 10.1007/978-1-4939-9752-7_15

Bitencourt-Ferreira G, de Azevedo WF Jr. Machine Learning to Predict Binding Affinity. Methods Mol Biol. 2019; 2053: 251–273.

 

Recent progress in the development of scientific libraries with machine-learning techniques paved the way for the implementation of integrated computational tools to predict ligand-binding affinity. The prediction of binding affinity uses the atomic coordinates of protein-ligand complexes. These new computational tools made application of a broad spectrum of machine-learning techniques to study protein-ligand interactions possible. The essential aspect of these machine-learning approaches is to train a new computational model by using technologies such as supervised machine-learning techniques, convolutional neural network, and random forest to mention the most commonly applied methods. In this chapter, we focus on supervised machine-learning techniques and their applications in the development of protein-targeted scoring functions for the prediction of binding affinity. We discuss the development of the program SAnDReS and its application to the creation of machine-learning models to predict inhibition of cyclin-dependent kinase and HIV-1 protease. Moreover, we describe the scoring function space, and how to use it to explain the development of targeted scoring functions.

KEYWORDS: Binding affinity; Cyclin-dependent kinase; HIV-1 protease; Machine learning; Regression; SAnDReS; Scoring function space

PMID: 31452110 DOI: 10.1007/978-1-4939-9752-7_16

Bitencourt-Ferreira G, de Azevedo WF Jr. Exploring the Scoring Function Space. Methods Mol Biol. 2019; 2053: 275–281.

 

In the analysis of protein-ligand interactions, two abstractions have been widely employed to build a systematic approach to analyze these complexes: protein and chemical spaces. The pioneering idea of the protein space dates back to 1970, and the chemical space is newer, later 1990s. With the progress of computational methodologies to create machine-learning models to predict the ligand-binding affinity, clearly there is a need for novel approaches to the problem of protein-ligand interactions. New abstractions are required to guide the conceptual analysis of the molecular recognition problem. Using a systems approach, we proposed to address protein-ligand scoring functions using the modern idea of the scoring function space. In this chapter, we describe the fundamental concept behind the scoring function space and how it has been applied to develop the new generation of targeted-scoring functions.

KEYWORDS: Binding affinity; Chemical space; Machine learning; Protein space; SAnDReS; Scoring function; Scoring function space

PMID: 31452111 DOI: 110.1007/978-1-4939-9752-7_17

Volkart PA, Bitencourt-Ferreira G, Souto AA, de Azevedo WF. Cyclin-Dependent Kinase 2 in Cellular Senescence and Cancer. A Structural and Functional Review. Curr Drug Targets. 2019; 20(7): 716–726.

 

BACKGROUND: Cyclin-dependent kinase 2 (CDK2) has been studied due to its role in the cell-cycle progression. The elucidation of the CDK2 structure paved the way to investigate the molecular basis for inhibition of this enzyme, with the coordinated efforts combining crystallography with functional studies. OBJECTIVE: Our goal here is to review recent functional and structural studies directed to understanding the role of CDK2 in cancer and senescence. METHOD: There are over four hundreds of crystallographic structures available for CDK2, many of them with binding affinity information. We use this abundance of data to analyze the essential features responsible for the inhibition of CDK2 and its function in cancer and senescence. RESULTS: The structural and affinity data available CDK2 makes possible to have a clear view of the vital CDK2 residues involved in molecular recognition. A detailed description of the structural basis for ligand binding is of pivotal importance in the design of CDK2 inhibitors. Our analysis shows the relevance of the residues Leu 83 and Asp 86 for binding affinity. The recent findings revealing the participation of CDK2 inhibition on senescence opens the possibility to explore the richness of structural and affinity data to start a new age the development of CDK2 inhibitors, now targeting on cellular senescence. CONCLUSION: Here we analyzed structural information for CDK2 in complex with inhibitors and mapped the molecular aspects behind the strongest CDK2 inhibitors for which structures and ligand-binding affinity data were available. From this analysis, we identified the significant intermolecular interactions responsible for binding affinity. This knowledge may guide the future development of CDK2 inhibitors targeting cancer and cellular senescence.

Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

KEYWORDS: CDK2; Cyclin; cancer. ; cellular senescence; drug design; protein-ligand interactions

Bitencourt-Ferreira G, de Azevedo Jr. WF. Development of a machine-learning model to predict Gibbs free energy of binding for protein-ligand complexes. Biophys Chem. 2018; 240: 63–69.

 

Abstract The possibility of using the atomic coordinates of protein-ligand complexes to assess binding affinity has a beneficial impact in the early stages of drug development and design. From the computational view, the creation of reliable scoring functions is still an open problem in the simulation of biological systems, and the development of a new generation machine-learning model is an active research field. In this work, we propose a novel scoring function to predict Gibbs free energy of binding (ΔG) based on the crystallographic structure of complexes involving a protein and an active ligand. We made use of the energy terms available the AutoDock Vina scoring function and trained a novel function using the machine learning methods available in the program SAnDReS. We used a training set composed exclusively of high-resolution crystallographic structures for which the ΔG data was available. We describe here the methodology to develop a machine-learning model to predict binding affinity using the program SAnDReS. Statistical analysis of our machine-learning model indicated a superior performance when compared to the MolDock, Plants, AutoDock 4, and AutoDock Vina scoring functions. We expect that this new machine-learning model could improve drug design and development through the application of a reliable scoring function in the analysis virtual screening simulations.

Copyright © 2018 Elsevier B.V. All rights reserved.

PMID: 29906639 DOI: 10.1016/j.bpc.2018.05.010

de Ávila MB, de Azevedo WF Jr. Development of machine learning models to predict inhibition of 3-dehydroquinate dehydratase. Chem Biol Drug Des. 2018; 92: 1468–1474.

 

In this study, we describe the development of new machine learning models to predict inhibition of the enzyme 3-dehydroquinate dehydratase (DHQD). This enzyme is the third step of the shikimate pathway and is responsible for the synthesis of chorismate, which is a natural precursor of aromatic amino acids. The enzymes of shikimate pathway are absent in humans, which make them protein targets for the design of antimicrobial drugs. We focus our study on the crystallographic structures of DHQD in complex with competitive inhibitors, for which experimental inhibition constant data is available. Application of supervised machine learning techniques was able to elaborate a robust DHQD-targeted model to predict binding affinity. Combination of high-resolution crystallographic structures and binding information indicates that the prevalence of intermolecular electrostatic interactions between DHQD and competitive inhibitors is of pivotal importance for the binding affinity against this enzyme. The present findings can be used to speed up virtual screening studies focused on the DHQD structure.

© 2018 John Wiley & Sons A/S.

KEYWORDS: 3-dehydroquinate dehydratase; crystallographic structures; drug design; machine learning; protein-ligand interactions; systems biology

PMID: 29676519 DOI: 10.1111/cbdd.13312

Amaral MEA, Nery LR, Leite CE, de Azevedo Junior WF, Campos MM. Pre-clinical effects of metformin and aspirin on the cell lines of different breast cancer subtypes. Invest New Drugs. 2018; 36(5): 782–796.

 

Background Breast cancer is highly prevalent among women worldwide. It is classified into three main subtypes: estrogen receptor positive (ER+), human epidermal growth factor receptor 2 positive (HER2+), and triple negative breast cancer (TNBC). This study has evaluated the effects of aspirin and metformin, isolated or in a combination, in breast cancer cells of the different subtypes. Methods The breast cancer cell lines MCF-7, MDA-MB-231, and SK-BR-3 were treated with aspirin and/or metformin (0.01 mM - 10 mM); functional in vitro assays were performed. The interactions with the estrogen receptors (ER) were evaluated in silico. Results Metformin (2.5, 5 and 10 mM) altered the morphology and reduced the viability and migration of the ER+ cell line MCF-7, whereas aspirin triggered this effect only at 10 mM. A synergistic effect for the combination of metformin and aspirin (2.5, 5 or 10 mM each) was observed in the TNBC cell subtype MDA-MB-231, according to the evaluation of its viability and colony formation. Partial inhibitory effects were observed for either of the drugs in the HER2+ cell subtype SK-BR-3. The effects of metformin and aspirin partly relied on cyclooxygenase-2 (COX-2) upregulation, without the production of lipoxins. In silico, metformin and aspirin bound to the ERα receptor with the same energy. Conclusion We have provided novel evidence on the mechanisms of action of aspirin and metformin in breast cancer cells, showing favorable outcomes for these drugs in the ER+ and TNBC subtypes.

KEYWORDS: Aspirin; Breast cance; Drug repurposing; Metformin

PMID: 29392539 DOI: 10.1007/s10637-018-0568-y

Levin NMB, Pintro VO, Bitencourt-Ferreira G, Mattos BB, Silvério AC, de Azevedo Jr. WF. Development of CDK-targeted scoring functions for prediction of binding affinity. Biophys Chem. 2018; 235: 1–8.

 

Cyclin-dependent kinase (CDK) is an interesting biological macromolecule due to its role in cell cycle progression, transcription control, and neuronal development, to mention the most studied biological activities. Furthermore, the availability of hundreds of structural studies focused on the intermolecular interactions of CDK with competitive inhibitors makes possible to develop computational models to predict binding affinity, where the atomic coordinates of binary complexes involving CDK and ligands can be used to train a machine learning model. The present work is focused on the development of new machine learning models to predict binding affinity for CDK. The CDK-targeted machine learning models were compared with classical scoring functions such as MolDock, AutoDock 4, and Vina Scores. The overall performance of our CDK-targeted scoring function was higher than the previously mentioned scoring functions, which opens the possibility of increasing the reliability of virtual screening studies focused on CDK.

Copyright © 2018 Elsevier B.V. All rights reserved.

KEYWORDS: Bioinformatics; CDK; Docking; Drug design; Machine learning; Protein

PMID: 29407904 DOI: 10.1016/j.bpc.2018.01.004

Freitas PG, Elias TC, Pinto IA, Costa LT, de Carvalho PVSD, Omote DQ, Camps I, Ishikawa T, Arcuri HA, Vinga S, Oliveira AL, Junior WFA, da Silveira NJF. Computational Approach to the Discovery of Phytochemical Molecules with Therapeutic Potential Targets to the PKCZ protein. Lett Drug Des Discov. 2018; 15(5): 488–499.

 

Background: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies in humans and the average 5-year survival rate is one of the lowest among aggressive cancers. Protein kinase C zeta (PKCZ) is highly expressed in head and neck tumors, and the inhibition of PKCZ reduces MAPK activation in five of seven head and neck tumors cell lines. Considering the world-wide HNSCC problems, there is an urgent need to develop new drugs to treat this disease, that present low toxicity, effective results and that are relatively inexpensive. Methods: A unified approach involving homology modeling, docking and molecular dynamics simulations studies on PKCZ are presented. The in silico study on this enzyme was undertaken using 10 compounds from latex of Euphorbia tirucalli L. (aveloz). Results: The binding free energies highlight that the main contribution in energetic terms for the compounds-PKCZ interactions is based on van der Waals. The per-residue decomposition free energy from the PKCZ revealed that the compounds binding were favorably stabilized by residues Glu300, Ileu383 and Asp394. Based on the docking, Xscore and molecular dynamics results, euphol, ß-sitosterol and taraxasterol were confirmed as the promising lead compounds. Conclusion: The present study should therefore play a guiding role in the experimental design and development of euphol, ß-sitosterol and taraxasterol as anticancer agents in head and neck tumors. They are potential lead compounds, better than other ligands based on the best values of docking and MM-PBSA energy.

Keywords: HNSCC, PKCZ, molecular marker, euphorbia tirucalli, homology modeling, molecular docking, molecular dynamics.

Pintro VO, Azevedo WF. Optimized Virtual Screening Workflow. Towards Target-Based Polynomial Scoring Functions for HIV-1 Protease. Comb Chem High Throughput Screen. 2017; 20(9): 820–827.

 

BACKGROUND: One key step in the development of inhibitors for an enzyme is the application of computational methodologies to predict protein-ligand interactions. The abundance of structural and ligand-binding information for HIV-1 protease opens up the possibility to apply computational methods to develop scoring functions targeted to this enzyme. OBJECTIVE: Our goal here is to develop an integrated molecular docking approach to investigate protein-ligand interactions with a focus on the HIV-1 protease. In addition, with this methodology, we intend to build target-based scoring functions to predict inhibition constant (Ki) for ligands against the HIV-1 protease system. METHODS: Here, we described a computational methodology to build datasets with decoys and actives directly taken from crystallographic structures to be applied in evaluation of docking performance using the program SAnDReS. Furthermore, we built a novel function using as terms MolDock and PLANTS scoring functions to predict binding affinity. To build a scoring function targeted to the HIV-1 protease, we have used machine-learning techniques. RESULTS: The integrated approach reported here has been tested against a dataset comprised of 71 crystallographic structures of HIV protease, to our knowledge the largest HIV-1 protease dataset tested so far. Comparison of our docking simulations with benchmarks indicated that the present approach is able to generate results with improved accuracy. CONCLUSION: We developed a scoring function with performance higher than previously published benchmarks for HIV-1 protease. Taken together, we believe that the approach here described has the potential to improve docking accuracy in drug design projects focused on HIV-1 protease.

Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

KEYWORDS: Docking; HIV-1 protease; SAnDReS; drug design; machine learning; virtual screening

PMID: 29165067 DOI: 10.2174/1386207320666171121110019

de Ávila MB, Xavier MM, Pintro VO, de Azevedo WF. Supervised machine learning techniques to predict binding affinity. A study for cyclin-dependent kinase 2. Biochem Biophys Res Commun. 2017; 494: 305–310.

 

Here we report the development of a machine-learning model to predict binding affinity based on the crystallographic structures of protein-ligand complexes. We used an ensemble of crystallographic structures (resolution better than 1.5 Å resolution) for which half-maximal inhibitory concentration (IC50) data is available. Polynomial scoring functions were built using as explanatory variables the energy terms present in the MolDock and PLANTS scoring functions. Prediction performance was tested and the supervised machine learning models showed improvement in the prediction power, when compared with PLANTS and MolDock scoring functions. In addition, the machine-learning model was applied to predict binding affinity of CDK2, which showed a better performance when compared with AutoDock4, AutoDock Vina, MolDock, and PLANTS scores.

Copyright © 2017 Elsevier Inc. All rights reserved.

KEYWORDS: Bioinformatics; CDK2; Docking; Drug design; Kinase; Machine learning

PMID: 29017921 DOI: 10.1016/j.bbrc.2017.10.035

de Azevedo WF. Meet Our Editorial Board Member. Author(s): Walter Filgueira de Azevedo. Curr Bioinform. 2017; 12(5):385–386.
Heck GS, Pintro VO, Pereira RR, de Ávila MB, Levin NMB, de Azevedo WF. Supervised Machine Learning Methods Applied to Predict Ligand-Binding Affinity. Curr Med Chem. 2017; 24(23): 2459–2470.

 

BACKGROUND: Calculation of ligand-binding affinity is an open problem in computational medicinal chemistry. The ability to computationally predict affinities has a beneficial impact in the early stages of drug development, since it allows a mathematical model to assess protein-ligand interactions. Due to the availability of structural and binding information, machine learning methods have been applied to generate scoring functions with good predictive power. OBJECTIVE: Our goal here is to review recent developments in the application of machine learning methods to predict ligand-binding affinity. METHOD: We focus our review on the application of computational methods to predict binding affinity for protein targets. In addition, we also describe the major available databases for experimental binding constants and protein structures. Furthermore, we explain the most successful methods to evaluate the predictive power of scoring functions. RESULTS: Association of structural information with ligand-binding affinity makes it possible to generate scoring functions targeted to a specific biological system. Through regression analysis, this data can be used as a base to generate mathematical models to predict ligandbinding affinities, such as inhibition constant, dissociation constant and binding energy. CONCLUSION: Experimental biophysical techniques were able to determine the structures of over 120,000 macromolecules. Considering also the evolution of binding affinity information, we may say that we have a promising scenario for development of scoring functions, making use of machine learning techniques. Recent developments in this area indicate that building scoring functions targeted to the biological systems of interest shows superior predictive performance, when compared with other approaches.

Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

KEYWORDS: Machine learning; binding affinity; drug; enzyme; ligand-binding affinity; medicinal chemistry; regression

PMID: 28641555 DOI: 10.2174/0929867324666170623092503

Levin NM, Pintro VO, de Ávila MB, de Mattos BB, De Azevedo WF Jr. Understanding the Structural Basis for Inhibition of Cyclin-Dependent Kinases. New Pieces in the Molecular Puzzle. Curr Drug Targets. 2017; 18(9): 1104–1111.

 

BACKGROUND: Cyclin-dependent kinases (CDKs) comprise an important protein family for development of drugs, mostly aimed for use in treatment of cancer but there is also potential for development of drugs for neurodegenerative diseases and diabetes. Since the early 1990s, structural studies have been carried out on CDKs, in order to determine the structural basis for inhibition of this protein target. OBJECTIVE: Our goal here is to review recent structural studies focused on CDKs. We concentrate on latest developments in the understanding of the structural basis for inhibition of CDKs, relating structures and ligand-binding information. METHOD: Protein crystallography has been successfully applied to elucidate over 400 CDK structures. Most of these structures are complexed with inhibitors. We use this richness of structural information to describe the major structural features determining the inhibition of this enzyme. RESULTS: Structures of CDK1, 2, 4-9, 12 13, and 16 have been elucidated. Analysis of these structures in complex with a wide range of different competitive inhibitors, strongly indicate some common features that can be used to guide the development of CDK inhibitors, such as a pattern of hydrogen bonding and the presence of halogen atoms in the ligand structure. CONCLUSION: Nowadays we have structural information for hundreds of CDKs. Combining the structural and functional information we may say that a pattern of intermolecular hydrogen bonds is of pivotal importance for inhibitor specificity. In addition, machine learning techniques have shown improvements in predicting binding affinity for CDKs. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

KEYWORDS: Cyclin-dependent kinase; binding affinity; drug design; inhibitors; machine learning; neurodegenerative disease

PMID: 27848884 DOI: 10.2174/1389450118666161116130155

Xavier MM, Heck GS, de Avila MB, Levin NM, Pintro VO, Carvalho NL, Azevedo WF Jr. SAnDReS a Computational Tool for Statistical Analysis of Docking Results and Development of Scoring Functions. Comb Chem High Throughput Screen. 2016; 19(10): 801–812.

 

BACKGROUND: Docking allows to predict ligand binding to proteins, since the 3D-structure for the target is available. Several docking studies have been carried out to identify potential ligands for drug targets. Many of these studies resulted in the leads that were later developed as drugs. OBJECTIVE: Our goal here is to describe the development of an integrated computational tool to assess docking accuracy and build new scoring functions to predict ligandbinding affinity. METHOD: We carried out docking simulations using MVD program for a data set available on CSAR 2014 database (coagulation factor Xa) for which ligand-binding information and structures are available. These docking results were analyzed using SAnDReS available at www.sandres.net. Machine learning methods were applied to build new scoring functions and our results were compared with previously published benchmarks. RESULTS: Our integrated docking strategy generated poses with docking accuracy higher than previously published benchmarks. In addition, the new scoring function developed using SAnDReS shows better performance than well-established scoring functions such the ones available in Autodock, Autodock- Vina, Gold, Glide, and MVD. CONCLUSION: The big data generated during docking lacked an integrated computational tool for statistical analysis of the influence of structural parameters on docking and scoring function performance. Here we describe methods to evaluate docking results using SAnDReS, a computational environment for statistical analysis of docking results and development of scoring functions. We believe that SAnDReS is a computational tool with potential to improve accuracy in docking projects.

Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

KEYWORDS: Dock; drug; machine learning.; protein; target

PMID: 27686428 DOI: 10.2174/1386207319666160927111347

de Azevedo Jr. WF. Meet Our Editorial Board Member Author(s): Walter Filgueira de Azevedo Jr. Curr Bioinform. 2016; 11(4):393.
de Azevedo Jr. WF. Opinion Paper: Targeting Multiple Cyclin-Dependent Kinases (CDKs): A New Strategy for Molecular Docking Studies. Curr Drug Targets. 2016;17(1):2.
Teles CB, Moreira-Dill LS, Silva Ade A, Facundo VA, de Azevedo WF Jr., da Silva LH, Motta MC, Stábeli RG, Silva-Jardim I. A lupane-triterpene isolated from Combretum leprosum Mart. fruit extracts that interferes with the intracellular development of Leishmania (L.) amazonensis in vitro. BMC Complement Altern Med. 2015; 15(1):165.

 

BACKGROUND: 3beta,6beta,16beta-trihydroxylup-20(29)-ene is a lupane triterpene isolated from Combretum leprosum fruit. The lupane group has been extensively used in studies on anticancer effects; however, its possible activity against protozoa parasites is yet poorly known. The high toxicity of the compounds currently used in leishmaniasis chemotherapy stimulates the investigation of new molecules and drug targets for antileishmanial therapy. METHODS: The activity of 3beta,6beta,16beta-trihydroxylup-20(29)-ene was evaluated against Leishmania (L.) amazonensis by determining the cytotoxicity of the compound on murine peritoneal macrophages, as well as its effects on parasite survival inside host cells. To evaluate the effect of this compound on intracellular amastigotes, cultures of infected macrophages were treated for 24, 48 and 96 h and the percentage of infected macrophages and the number of intracellular parasites was scored using light microscopy. RESULTS: Lupane showed significant activity against the intracellular amastigotes of L. (L.) amazonensis. The treatment with 109 μM for 96 h reduced in 80 % the survival index of parasites in BALB/c peritoneal macrophages. At this concentration, the triterpene caused no cytotoxic effects against mouse peritoneal macrophages. Ultrastructural analyses of L. (L.) amazonensis intracellular amastigotes showed that lupane induced some morphological changes in parasites, such as cytosolic vacuolization, lipid body formation and mitochondrial swelling. Bioinformatic analyses through molecular docking suggest that this lupane has high-affinity binding with DNA topoisomerase. CONCLUSION: Taken together, our results have showed that the lupane triterpene from C. leprosum interferes with L. (L.) amazonensis amastigote replication and survival inside vertebrate host cells and bioinformatics analyses strongly indicate that this molecule may be a potential inhibitor of topoisomerase IB. Moreover, this study opens major prospects for the development of novel chemotherapeutic agents with leishmanicidal activity.

PMID: 26048712 PMCID: PMC4457080 DOI: 10.1186/s12906-015-0681-9

de Avila, MB, de Azevedo, WF. Data Mining of Docking Results. Application to 3-Dehydroquinate Dehydratase. Curr Bioinform. 2014; 9(4): 361–379.

 

Abstract: In this article, we describe a computational methodology that analyzes the results generated in ligand docking and evaluates the correlation between simulation results and intrinsic characteristics present in the crystallographic structures used in the simulation, such as thermal parameter, resolution, and overall quality of the X-ray diffraction data. We focus our analysis on molecular docking data obtained from application of differential evolution implemented in the program Molegro Virtual Docker. As a protein target, we selected the enzyme 3-dehydroquinate dehydratase (DHQD). This enzyme is part of the shikimate pathway, and a protein target for development of anti-tubercular drugs. We used a set with 20 DHQD crystallographic structures with ligands bound to the active site. In order to identify the best approach to molecular docking, we analyzed crystallographic parameters and looked for correlation between the docking results and structural features present in the protein target. Analysis of docking results helps to identify the best approach to use in ligand docking and identify structural features important for the success of this methodology. Analysis of results generated by ligand docking focused on DHQD made possible to assess the best docking protocol for this enzyme and use this optimized approach in the more computational demanding methodology of virtual screening (VS). We used a data set of natural products to identify structural features important for ligand-binding affinity.

Keywords: 3-dehydroquinate dehydratase, bioinformatics, data mining, ligand docking, Mycobacterium tuberculosis, shikimate pathway.

Coracini JD, de Azevedo WF Jr. Shikimate kinase, a protein target for drug design. Curr Med Chem. 2014; 21(5):592–604.

 

ATP: shikimate 3-phosphotransferase catalyzes the fifth chemical reaction of shikimate pathway. This metabolic route is responsible for the production of chorismate, a precursor of aromatic amino acids. This especially interesting enzymatic step is indispensable for the survival of the etiological agent of tuberculosis and not found in animals. Therefore the enzyme ATP: shikimate 3-phosphotransferase has been classified as a target for chemotherapeutic development of antitubercular drugs. The ATP:shikimate 3-phosphotransferase has also the denomination of shikimate kinase. This review highlights the available crystallographic studies of shikimate kinases that have been used to identify structural features for ligand-biding affinity. We also describe molecular docking studies focused on shikimate kinase. These computational studies were performed in order to identify the new generation of antitubercular drugs and several potential inhibitors have been described. In addition, a structural comparison of shikimate kinase ATP-binding pocket with human cyclin-dependent kinase 2 (CDK2) is described. This analysis shows the structural similarities between both enzymes, and the potential beneficial aspects of abundant structural studies of CDK2 and their inhibitors to bring further understanding of the ligand-binding specificity for shikimate kinase.

Lombardi FR, Anazetti MC, Santos GC, Polizelli PP, Olivieri JR, de Azevedo WF, Bonilla-Rodriguez GO. Oxygen Binding Properties and Tetrameric Stability of Hemoglobins From the Snakes Crotalus Durissus Terrificus and Liophis Miliaris (Chapter 1). Frontiers in Protein and Peptide Sciences. 2014; 1, 3–30.

 

Hemoglobins (Hbs) from the South American snake Liophis miliaris undergo a reversible oxygen-linked dissociation phenomenon α2β2 + 4O2 ↔ 2αβ(O2), due to substituted glutamate residues (43β and 101β) at the α1β2- interface, according to Matsuura et al., 1989. This work reports the oxygen-binding properties and analysis of tetrameric stability from the major hemoglobins of two snakes: a terrestrial species (Crotalus durissus terrificus), and a semiaquatic one, L. miliaris. The major hemoglobins from both species were analyzed by classic functional experimental approaches and other analytical techniques: dimer-tetramer association constant by analytical gel filtration chromatography and small angle x-ray scattering (SAXS). In addition, we performed molecular modeling of the hemoglobin from L. miliaris. The functional results display cooperative oxygen binding, alkaline Bohr Effect, and conformational transitions, a behavior which is compatible with tetrameric Hbs. Also, the estimated dimer-tetramer association constant 4K2, radius of gyration, and maximum dimension exhibit similar compatibility. A molecular modeling of the structure of L. miliaris Hb, on the other hand, allows us to conclude that the residues Glu43β(CD2) and Glu101β(G3) are not essential in the maintenance of the tetrameric form, supporting our findings and allowing us to rule out the hypothesis proposed in the literature for the Hb of Liophis miliaris and other species of snakes. Therefore, the results reported in the paper suggest that the major Hbs of Liophis miliaris and Crotalus durissus terrificus are stable tetramers in vivo and in vitro.

Keywords: Crotalus durissus terrificus, Dimer-tetramer association constant, Hemoglobin, Liophis miliaris, Oxygen binding properties and SAXS.

Azevedo LS, Moraes FP, Xavier MM, Pantoja EO, Villavicencio B, Finck JA, Proenca, AM, Rocha, KB, de Azevedo, WF. Recent Progress of Molecular Docking Simulations Applied to Development of Drugs. Curr Bioinform. 2012; 7(4): 352–65.

 

In order to obtain structural information about intermolecular interactions between a protein target and a drug we could either solve the structure by experimental techniques (protein crystallography or nuclear magnetic resonance), or simulate the protein-drug complex computationally. Molecular docking is a computer simulation methodology that can predict the conformation of a protein-drug complex, with relatively high accuracy when compared with experimental structures. Although a plethora of algorithms has been applied to the problem of molecular docking simulation, recent results show that the most successful approaches are those based on evolutionary algorithms. Evolution as a source of inspiration has been shown to have a great positive impact on the progress of new computational methodologies. In this scenario, analyses of the interactions between a protein target and a drug can be simulated by these evolutionary algorithms. These algorithms mimic evolution to create new paradigms for computation. This review provides a description of evolutionary algorithms and applications to molecular docking simulation. Special attention is dedicated to differential evolutionary algorithm and its implementation in the program molegro virtual docker. Recent applications of these methodologies to protein targets such as acetylcholinesterese, cyclin-dependent kinase 2, purine nucleoside phosphorylase, and shikimate kinase are described. Keywords: Differential evolution, evolutionary algorithms, molecular docking, protein-drug, simulation, structure-based virtual screening, chromosomes, Shoemake’s methodology, complex protein-ligand, crossdocking

Rosado LA, Caceres RA, de Azevedo WF Jr, Basso LA, Santos DS. Role of Serine140 in the mode of action of Mycobacterium tuberculosis ß-ketoacyl-ACP Reductase (MabA). BMC Res Notes. 2012; 5:526.

 

BACKGROUND: Tuberculosis (TB) still remains one of the most deadly infectious diseases in the world. Mycobacterium tuberculosis β-ketoacyl-ACP Reductase (MabA) is a member of the fatty acid elongation system type II, providing precursors of mycolic acids that are essential to the bacterial cell growth and survival. MabA has been shown to be essential for M. tuberculosis survival and to play a role in intracellular signal transduction of bacilli. FINDINGS: Here we describe site-directed mutagenesis, recombinant protein expression and purification, steady-state kinetics, fluorescence spectroscopy, and molecular modeling for S140T and S140A mutant MabA enzymes. No enzyme activity could be detected for S140T and S140A. Although the S140T protein showed impaired NADPH binding, the S140A mutant could bind to NADPH. Computational predictions for NADPH binding affinity to WT, S140T and S140A MabA proteins were consistent with fluorescence spectroscopy data. CONCLUSIONS: The results suggest that the main role of the S140 side chain of MabA is in catalysis. The S140 side chain appears to also play an indirect role in NADPH binding. Interestingly, NADPH titrations curves shifted from sigmoidal for WT to hyperbolic for S140A, suggesting that the S140 residue may play a role in displacing the pre-existing equilibrium between two forms of MabA in solution. The results here reported provide a better understanding of the mode of action of MabA that should be useful to guide the rational (function-based) design of inhibitors of MabA enzyme activity which, hopefully, could be used as lead compounds with anti-TB action.

PMID: 23006410 PMCID: PMC3519566 DOI: 10.1186/1756-0500-5-526

Moraes FP, de Azevedo WF Jr.Targeting imidazoline site on monoamine oxidase B through molecular docking simulations. J Mol Model. 2012; 18(8):3877–3886.

 

Monoamine oxidase (MAO) is an enzyme of major importance in neurochemistry, because it catalyzes the inactivation pathway for the catecholamine neurotransmitters, noradrenaline, adrenaline and dopamine. In the last decade it was demonstrated that imidazoline derivatives were able to inhibit MAO activity. Furthermore, crystallographic studies identified the imidazoline-binding domain on monoamine oxidase B (MAO-B), which opens the possibility of molecular docking studies focused on this binding site. The goal of the present study is to identify new potential inhibitors for MAO-B. In addition, we are also interested in establishing a fast and reliable computation methodology to pave the way for future molecular docking simulations focused on the imidazoline-binding site of this enzyme. We used the program 'molegro virtual docker' (MVD) in all simulations described here. All results indicate that simplex evolution algorithm is able to succesfully simulate the protein-ligand interactions for MAO-B. In addition, a scoring function implemented in the program MVD presents high correlation coefficient with experimental activity of MAO-B inhibitors. Taken together, our results identified a new family of potential MAO-B inhibitors and mapped important residues for intermolecular interactions between this enzyme and ligands.

PMID: 22426510 DOI: 10.1007/s00894-012-1390-7

Soares MB, Silva CV, Bastos TM, Guimarães ET, Figueira CP, Smirlis D, Azevedo WF Jr. Anti-Trypanosoma cruzi activity of nicotinamide. Acta Trop. 2012; 12(2):224–229.

 

Inhibition of Trypanosoma brucei and Leishmania spp. sirtuins has shown promising antiparasitic activity, indicating that these enzymes may be used as targets for drug discovery against trypanosomatid infections. In the present work we carried out a virtual screening focused on the C pocket of Sir2 from Trypanosoma cruzi. Using this approach, the best ligand found was nicotinamide. In vitro tests confirmed the anti-T. cruzi activity of nicotinamide on epimastigote and trypomastigote forms. Moreover, treatment of T. cruzi-infected macrophages with nicotinamide caused a significant reduction in the number of amastigotes. In addition, alterations in the mitochondria and an increase in the vacuolization in the cytoplasm were observed in epimastigotes treated with nicotinamide. Analysis of the complex of Sir2 and nicotinamide revealed the details of the possible ligand-target interaction. Our data reveal a potential use of TcSir2 as a target for anti-T. cruzi drug discovery.

Copyright © 2012 Elsevier B.V. All rights reserved.

Vianna CP, de Azevedo WF Jr. Identification of new potential Mycobacterium tuberculosis shikimate kinase inhibitors through molecular docking simulations. J Mol Model. 2012; 18(2):755–764.

 

Tuberculosis (TB) is the major cause of human mortality from a curable infectious disease, attacking mainly in developing countries. Among targets identified in Mycobacterium tuberculosis genome, enzymes of the shikimate pathway deserve special attention, since they are essential to the survival of the microorganism and absent in mammals. The object of our study is shikimate kinase (SK), the fifth enzyme of this pathway. We applied virtual screening methods in order to identify new potential inhibitors for this enzyme. In this work we employed MOLDOCK program in all molecular docking simulations. Accuracy of enzyme-ligand docking was validated on a set of 12 SK-ligand complexes for which crystallographic structures were available, generating root-mean square deviations below 2.0 Å. Application of this protocol against a commercially available database allowed identification of new molecules with potential to become drugs against TB. Besides, we have identified the binding cavity residues that are essential to intermolecular interactions of this enzyme.

PMID: 21594693 DOI: 10.1007/s00894-011-1113-5

Timmers LF, Ducati RG, Sánchez-Quitian ZA, Basso LA, Santos DS, de Azevedo WF Jr. Combining molecular dynamics and docking simulations of the cytidine deaminase from Mycobacterium tuberculosis H37Rv. J Mol Model. 2012; 18(2):467–479.

 

Cytidine Deaminase (CD) is an evolutionarily conserved enzyme that participates in the pyrimidine salvage pathway recycling cytidine and deoxycytidine into uridine and deoxyuridine, respectively. Here, our goal is to apply computational techniques in the pursuit of potential inhibitors of Mycobacterium tuberculosis CD (MtCDA) enzyme activity. Molecular docking simulation was applied to find the possible hit compounds. Molecular dynamics simulations were also carried out to investigate the physically relevant motions involved in the protein-ligand recognition process, aiming at providing estimates for free energy of binding. The proposed approach was capable of identifying a potential inhibitor, which was experimentally confirmed by IC(50) evaluation. Our findings open up the possibility to extend this protocol to different databases in order to find new potential inhibitors for promising targets based on a rational drug design process.

PMID: 21541749 DOI: 10.1007/s00894-011-1045-0

Caceres RA, Timmers LF, Ducati RG, da Silva DO, Basso LA, de Azevedo WF Jr, Santos DS. Crystal structure and molecular dynamics studies of purine nucleoside phosphorylase from Mycobacterium tuberculosis associated with acyclovir. Biochimie. 2012; 94(1):155–165.

 

Consumption has been a scourge of mankind since ancient times. This illness has charged a high price to human lives. Many efforts have been made to defeat Mycobacterium tuberculosis (Mt). The M. tuberculosis purine nucleoside phosphorylase (MtPNP) is considered an interesting target to pursuit new potential inhibitors, inasmuch it belongs to the purine salvage pathway and its activity might be involved in the mycobacterial latency process. Here we present the MtPNP crystallographic structure associated with acyclovir and phosphate (MtPNP:ACY:PO(4)) at 2.10 Å resolution. Molecular dynamics simulations were carried out in order to dissect MtPNP:ACY:PO(4) structural features, and the influence of the ligand in the binding pocket stability. Our results revealed that the ligand leads to active site lost of stability, in agreement with experimental results, which demonstrate a considerable inhibitory activity against MtPNP (K(i) = 150 nM). Furthermore, we observed that some residues which are important in the proper ligand's anchor into the human homologous enzyme do not present the same importance to MtPNP. Therewithal, these findings contribute to the search of new specific inhibitors for MtPNP, since peculiarities between the mycobacterial and human enzyme binding sites have been identified, making a structural-based drug design feasible.

Copyright © 2011 Elsevier Masson SAS. All rights reserved.

PMID: 22033138 DOI: 10.1016/j.biochi.2011.10.003

Sá MS, de Menezes MN, Krettli AU, Ribeiro IM, Tomassini TC, Ribeiro dos Santos R, de Azevedo WF Jr, Soares MB. Antimalarial activity of physalins B, D, F, and G. J Nat Prod. 2011; 74(10):2269–2272.

 

The antimalarial activities of physalins B, D, F, and G (1-4), isolated from Physalis angulata, were investigated. In silico analysis using the similarity ensemble approach (SEA) database predicted the antimalarial activity of each of these compounds, which were shown using an in vitro assay against Plasmodium falciparum. However, treatment of P. berghei-infected mice with 3 increased parasitemia levels and mortality, whereas treatment with 2 was protective, causing a parasitemia reduction and a delay in mortality in P. berghei-infected mice. The exacerbation of in vivo infection by treatment with 3 is probably due to its potent immunosuppressive activity, which is not evident for 2.

PMID: 21954931 DOI: 10.1021/np200260f

Sánchez-Quitian ZA, Timmers LF, Caceres RA, Rehm JG, Thompson CE, Basso LA, de Azevedo WF Jr, Santos DS. Crystal structure determination and dynamic studies of Mycobacterium tuberculosis Cytidine deaminase in complex with products. Arch Biochem Biophys. 2011; 509(1):108–115.

 

Cytidine deaminase (CDA) is a key enzyme in the pyrimidine salvage pathway. It is involved in the hydrolytic deamination of cytidine or 2'-deoxycytidine to uridine or 2'-deoxyuridine, respectively. Here we report the crystal structures of Mycobacterium tuberculosis CDA (MtCDA) in complex with uridine (2.4 Å resolution) and deoxyuridine (1.9 Å resolution). Molecular dynamics (MD) simulation was performed to analyze the physically relevant motions involved in the protein-ligand recognition process, showing that structural flexibility of some protein regions are important to product binding. In addition, MD simulations allowed the analysis of the stability of tetrameric MtCDA structure. These findings open-up the possibility to use MtCDA as a target in future studies aiming to the rational design of new inhibitor of MtCDA-catalyzed chemical reaction with potential anti-proliferative activity on cell growth of M. tuberculosis, the major causative agent of tuberculosis.

Copyright © 2011 Elsevier Inc. All rights reserved.

PMID: 21295009 DOI: 10.1016/j.abb.2011.01.022

Rocha BA, Delatorre P, Oliveira TM, Benevides RG, Pires AF, Sousa AA, Souza LA, Assreuy AM, Debray H, de Azevedo WF Jr, Sampaio AH, Cavada BS. Structural basis for both pro- and anti-inflammatory response induced by mannose-specific legume lectin from Cymbosema roseum. Biochimie. 2011; 93(5):806–816.

 

Legume lectins, despite high sequence homology, express diverse biological activities that vary in potency and efficacy. In studies reported here, the mannose-specific lectin from Cymbosema roseum (CRLI), which binds N-glycoproteins, shows both pro-inflammatory effects when administered by local injection and anti-inflammatory effects when by systemic injection. Protein sequencing was obtained by Tandem Mass Spectrometry and the crystal structure was solved by X-ray crystallography using a Synchrotron radiation source. Molecular replacement and refinement were performed using CCP4 and the carbohydrate binding properties were described by affinity assays and computational docking. Biological assays were performed in order to evaluate the lectin edematogenic activity. The crystal structure of CRLI was established to a 1.8Å resolution in order to determine a structural basis for these differing activities. The structure of CRLI is closely homologous to those of other legume lectins at the monomer level and assembles into tetramers as do many of its homologues. The CRLI carbohydrate binding site was predicted by docking with a specific inhibitory trisaccharide. CRLI possesses a hydrophobic pocket for the binding of α-aminobutyric acid and that pocket is occupied in this structure as are the binding sites for calcium and manganese cations characteristic of legume lectins. CRLI route-dependent effects for acute inflammation are related to its carbohydrate binding domain (due to inhibition caused by the presence of α-methyl-mannoside), and are based on comparative analysis with ConA crystal structure. This may be due to carbohydrate binding site design, which differs at Tyr12 and Glu205 position.

Copyright © 2011 Elsevier Masson SAS. All rights reserved.

PMID: 21277932 DOI: 10.1016/j.biochi.2011.01.006

de Azevedo WF Jr. Protein targets for development of drugs against Mycobacterium tuberculosis. Curr Med Chem. 2011; 18(9):1255–1257.

 

PMID: 21366537

Heberlé G, de Azevedo WF Jr. Bio-inspired algorithms applied to molecular docking simulations. Curr Med Chem. 2011; 18(9):1339–1352.

 

Nature as a source of inspiration has been shown to have a great beneficial impact on the development of new computational methodologies. In this scenario, analyses of the interactions between a protein target and a ligand can be simulated by biologically inspired algorithms (BIAs). These algorithms mimic biological systems to create new paradigms for computation, such as neural networks, evolutionary computing, and swarm intelligence. This review provides a description of the main concepts behind BIAs applied to molecular docking simulations. Special attention is devoted to evolutionary algorithms, guided-directed evolutionary algorithms, and Lamarckian genetic algorithms. Recent applications of these methodologies to protein targets identified in the Mycobacterium tuberculosis genome are described.

PMID: 21366530

de Azevedo WF Jr. Molecular dynamics simulations of protein targets identified in Mycobacterium tuberculosis. Curr Med Chem. 2011; 18(9):1353–1366.

 

Application of molecular dynamics simulation technique has become a conventional computational methodology to calculate significant processes at the molecular level. This computational methodology is particularly useful for analyzing the dynamics of protein-ligand systems. Several uses of molecular dynamics simulation makes possible evaluation of important structural features found at interface between a ligand and a protein, such as intermolecular hydrogen bonds, contact area and binding energy. Considering structure-based virtual screening, molecular dynamics simulations play a pivotal role in understanding the features that are important for ligand-binding affinity. This information could be employed to select higher-affinity ligands obtained in screening processes. Many protein targets such as enoyl-[acyl-carrier-protein] reductase (InhA), purine nucleoside phosphorylase (PNP), and shikimate kinase have been submitted to these simulations and will be analyzed here. All command files used in this review are available for download at http://azevedolab.net/md_75.html.

PMID: 21366529

Ducati RG, Basso LA, Santos DS, de Azevedo WF Jr. Crystallographic and docking studies of purine nucleoside phosphorylase from Mycobacterium tuberculosis. Bioorg Med Chem. 2010; 18(13):4769–4774.

 

This work describes for the first time the structure of purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP) in complex with sulfate and its natural substrate, 2'-deoxyguanosine, and its application to virtual screening. We report docking studies of a set of molecules against this structure. Application of polynomial empirical scoring function was able to rank docking solutions with good predicting power which opens the possibility to apply this new criterion to analyze docking solutions and screen small-molecule databases for new chemical entities to inhibit MtPNP.

Copyright © 2010 Elsevier Ltd. All rights reserved.

PMID: 20570524 DOI: 10.1016/j.bmc.2010.05.009

De Azevedo WF Jr. Structure-based virtual screening. Curr Drug Targets. 2010; 11(3):261–263.

 

PMID: 20214598

De Azevedo WF Jr. MolDock applied to structure-based virtual screening. Curr Drug Targets. 2010; 11(3):327–334.

 

Molecular docking is a simulation process where the binding of a small molecule is identified in the structure of a protein target. There are several different computational approaches to solve this problem. Here it will be described recent developments in application of evolutionary algorithms to molecular docking simulations. Evolutionary algorithms are classified as a group of computational techniques based on the concepts of Darwin's theory of evolution that are designed to the best possible find solution to optimisation problems. A successfully implementation of this algorithm can be found in the program MolDock. The main features of MolDock are reviewed here we also describe application of MolDock to purine nucleoside phosphorylase, shikimate kinase and cyclin-dependent kinase 2.

PMID: 20210757

Hernandes MZ, Cavalcanti SM, Moreira DR, de Azevedo Junior WF, Leite AC. Halogen atoms in the modern medicinal chemistry: hints for the drug design. Curr Drug Targets. 2010; 11(3):303–314.

 

A significant number of drugs and drug candidates in clinical development are halogenated structures. For a long time, insertion of halogen atoms on hit or lead compounds was predominantly performed to exploit their steric effects, through the ability of these bulk atoms to occupy the binding site of molecular targets. However, halogens in drug - target complexes influence several processes rather than steric aspects alone. For example, the formation of halogen bonds in ligand-target complexes is now recognized as a kind of intermolecular interaction that favorably contributes to the stability of ligand-target complexes. This paper is aimed at introducing the fascinating versatility of halogen atoms. It starts summarizing the prevalence of halogenated drugs and their structural and pharmacological features. Next, we discuss the identification and prediction of halogen bonds in protein-ligand complexes, and how these bonds should be exploited. Interesting results of halogen insertions during the processes of hit-to-lead or lead-to-drug conversions are also detailed. Polyhalogenated anesthetics and protein kinase inhibitors that bear halogens are analyzed as cases studies. Thereby, this review serves as one guide for the virtual screening of libraries containing halogenated compounds and may be a source of inspiration for the medicinal chemists.

PMID: 20210755

Zanchi FB, Caceres RA, Stabeli RG, de Azevedo WF Jr. Molecular dynamics studies of a hexameric purine nucleoside phosphorylase. J Mol Model. 2010; 16(3):543–550.

 

Purine nucleoside phosphorylase (PNP) (EC.2.4.2.1) is an enzyme that catalyzes the cleavage of N-ribosidic bonds of the purine ribonucleosides and 2-deoxyribonucleosides in the presence of inorganic orthophosphate as a second substrate. This enzyme is involved in purine-salvage pathway and has been proposed as a promising target for design and development of antimalarial and antibacterial drugs. Recent elucidation of the three-dimensional structure of PNP by X-ray protein crystallography left open the possibility of structure-based virtual screening initiatives in combination with molecular dynamics simulations focused on identification of potential new antimalarial drugs. Most of the previously published molecular dynamics simulations of PNP were carried out on human PNP, a trimeric PNP. The present article describes for the first time molecular dynamics simulations of hexameric PNP from Plasmodium falciparum (PfPNP). Two systems were simulated in the present work, PfPNP in ligand free form, and in complex with immucillin and sulfate. Based on the dynamical behavior of both systems the main results related to structural stability and protein-drug interactions are discussed.

PMID: 19669809 DOI: 10.1007/s00894-009-0557-3

Sánchez-Quitian ZA, Schneider CZ, Ducati RG, de Azevedo WF Jr, Bloch C Jr, Basso LA, Santos DS. Structural and functional analyses of Mycobacterium tuberculosis Rv3315c-encoded metal-dependent homotetrameric cytidine deaminase. J Struct Biol. 2010; 169(3):413–423.

 

he emergence of drug-resistant strains of Mycobacterium tuberculosis, the causative agent of tuberculosis, has exacerbated the treatment and control of this disease. Cytidine deaminase (CDA) is a pyrimidine salvage pathway enzyme that recycles cytidine and 2'-deoxycytidine for uridine and 2'-deoxyuridine synthesis, respectively. A probable M. tuberculosis CDA-coding sequence (cdd, Rv3315c) was cloned, sequenced, expressed in Escherichia coli BL21(DE3), and purified to homogeneity. Mass spectrometry, N-terminal amino acid sequencing, gel filtration chromatography, and metal analysis of M. tuberculosis CDA (MtCDA) were carried out. These results and multiple sequence alignment demonstrate that MtCDA is a homotetrameric Zn(2+)-dependent metalloenzyme. Steady-state kinetic measurements yielded the following parameters: K(m)=1004 microM and k(cat)=4.8s(-1) for cytidine, and K(m)=1059 microM and k(cat)=3.5s(-1) for 2'-deoxycytidine. The pH dependence of k(cat) and k(cat)/K(M) for cytidine indicate that protonation of a single ionizable group with apparent pK(a) value of 4.3 abolishes activity, and protonation of a group with pK(a) value of 4.7 reduces binding. MtCDA was crystallized and crystal diffracted at 2.0 A resolution. Analysis of the crystallographic structure indicated the presence of a Zn(2+) coordinated by three conserved cysteines and the structure exhibits the canonical cytidine deaminase fold.

(c) 2009 Elsevier Inc. All rights reserved.

PMID: 20035876 DOI: 10.1016/j.jsb.2009.12.019

Caceres RA, Timmers LF, Pauli I, Gava LM, Ducati RG, Basso LA, Santos DS, de Azevedo WF Jr. Crystal structure and molecular dynamics studies of human purine nucleoside phosphorylase complexed with 7-deazaguanine. J Struct Biol. 2010; 169(3):379–388.

 

In humans, purine nucleoside phosphorylase (HsPNP) is responsible for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. HsPNP is a target for inhibitor development aiming at T-cell immune response modulation. Here we report the crystal structure of HsPNP in complex with 7-deazaguanine (HsPNP:7DG) at 2.75 A. Molecular dynamics simulations were employed to assess the structural features of HsPNP in both free form and in complex with 7DG. Our results show that some regions, responsible for entrance and exit of substrate, present a conformational variability, which is dissected by dynamics simulation analysis. Enzymatic assays were also carried out and revealed that 7-deazaguanine presents a lower inhibitory activity against HsPNP (K(i)=200 microM). The present structure may be employed in both structure-based design of PNP inhibitors and in development of specific empirical scoring functions.

(c) 2009 Elsevier Inc. All rights reserved.

PMID: 19932753 DOI: 10.1016/j.jsb.2009.11.010

Arcuri HA, Zafalon GF, Marucci EA, Bonalumi CE, da Silveira NJ, Machado JM, de Azevedo WF Jr, Palma MS. SKPDB: a structural database of shikimate pathway enzymes. BMC Bioinformatics. 2010; 11:12.

 

BACKGROUND: The functional and structural characterisation of enzymes that belong to microbial metabolic pathways is very important for structure-based drug design. The main interest in studying shikimate pathway enzymes involves the fact that they are essential for bacteria but do not occur in humans, making them selective targets for design of drugs that do not directly impact humans. DESCRIPTION: The ShiKimate Pathway DataBase (SKPDB) is a relational database applied to the study of shikimate pathway enzymes in microorganisms and plants. The current database is updated regularly with the addition of new data; there are currently 8902 enzymes of the shikimate pathway from different sources. The database contains extensive information on each enzyme, including detailed descriptions about sequence, references, and structural and functional studies. All files (primary sequence, atomic coordinates and quality scores) are available for downloading. The modeled structures can be viewed using the Jmol program. CONCLUSIONS: The SKPDB provides a large number of structural models to be used in docking simulations, virtual screening initiatives and drug design. It is freely accessible at http://lsbzix.rc.unesp.br/skpdb/.

PMID: 20055992 PMCID: PMC2824673 DOI: 10.1186/1471-2105-11-12

Ducati RG, Souto AA, Caceres RA, de Azevedo Jr. WF, Basso LA, Santos DS. Purine Nucleoside Phosphorylase as a Molecular Target to Develop Active Compounds Against Mycobacterium Tuberculosis. International Review of Biophysical Chemistry 2010; Vol. 1, N. 1, 34–40.

 

Despite the availability of chemotherapic and prophylactic approaches to fight consumption, human tuberculosis (TB) continues to claim millions of lives annually, mostly in developing nations, generally due to limited resources available to ensure proper treatment and where human immunodeficiency virus infections are common. Moreover, rising drug-resistant cases have worsened even further this picture. Accordingly, new therapeutic approaches have become necessary. The use of defined molecular targets could be explored as an attempt to develop new and selective chemical compounds to be employed in the treatment of TB. The present review highlights purine nucleoside phosphorylase, a component enzyme of the purine salvage pathway, as an attractive target for the development of new antimycobacterial agents, since this enzyme has been numbered among targets for Mycobacterium tuberculosis persistence in the human host. Enzyme kinetics and structural data are discussed to provide a basis to guide the rational anti-TB drug design. Copyright © 2010 Praise Worthy Prize S.r.l. - All rights reserved.

Keywords: Enzyme Kinetics and Structural Analysis, Human Tuberculosis, Mycobacterium Tuberculosis, Purine Nucleoside Phosphorylase, Rational Drug Development

Pauli I, Timmers LF, Caceres RA, Basso LA, Santos DS, de Azevedo WF Jr. Molecular modeling and dynamics studies of purine nucleoside phosphorylase from Bacteroides fragilis. J Mol Model. 2009; 15(8):913–922.

 

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides, except adenosine, to generate ribose 1-phosphate and the purine base. This work describes for the first time a structural model of PNP from Bacteroides fragilis (Bf). We modeled the complexes of BfPNP with six different ligands in order to determine the structural basis for specificity of these ligands against BfPNP. Comparative analysis of the model of BfPNP and the structure of HsPNP allowed identification of structural features responsible for differences in the computationally determined ligand affinities. The molecular dynamics (MD) simulation was assessed to evaluate the overall stability of the BfPNP model. The superposition of the final onto the initial minimized structure shows that there are no major conformational changes from the initial model, which is consistent with the relatively low root mean square deviation (RMSD). The results indicate that the structure of the model was stable during MD, and does not exhibit loosely structured loop regions or domain terminals.

PMID: 19172318 DOI: 10.1007/s00894-008-0445-2

Timmers LF, Caceres RA, Dias R, Basso LA, Santos DS, de Azevedo WF Jr. Molecular modeling, dynamics and docking studies of purine nucleoside phosphorylase from Streptococcus pyogenes. Biophys Chem. 2009; 142(1-3):7–16.

 

Purine Nucleoside Phosphorylase (PNP) catalyzes the reversible phosphorolysis of N-glycosidic bonds of purine nucleosides and deoxynucleosides, except for adenosine, to generate ribose 1-phosphate and the purine base. PNP has been submitted to intensive structural studies. This work describes for the first time a structural model of PNP from Streptococcus pyogenes (SpPNP). We modeled the complexes of SpPNP with six different ligands in order to determine the structural basis for specificity of these ligands against SpPNP. Molecular dynamics (MD) simulations were performed in order to evaluate the overall stability of SpPNP model. The analysis of the MD simulation was assessed mainly by principal component analysis (PCA) to explore the trimeric structure behavior. Structural comparison, between SpPNP and human PNP, was able to identify the main features responsible for differences in ligand-binding affinities, such as mutation in the purine-binding site and in the second phosphate-binding site. The PCA analysis suggests a different behavior for each subunit in the trimer structure.

PMID: 19282092 DOI: 10.1016/j.bpc.2009.02.006

Rocha BA, Moreno FB, Delatorre P, Souza EP, Marinho ES, Benevides RG, Rustiguel JK, Souza LA, Nagano CS, Debray H, Sampaio AH, de Azevedo WF Jr, Cavada BS. Purification, characterization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds. Appl Biochem Biotechnol. 2009; 152(3):383–393.

 

The unique carbohydrate-binding property of lectins makes them invaluable tools in biomedical research. Here, we report the purification, partial primary structure, carbohydrate affinity characterization, crystallization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds (CRLII). Isolation and purification of CRLII was performed by a single step using a Sepharose-4B-lactose affinity chromatography column. The carbohydrate affinity characterization was carried using assays for hemagglutination activity and inhibition. CRLII showed hemagglutinating activity toward rabbit erythrocytes. O-glycoproteins from mucine mucopolysaccharides showed the most potent inhibition capacity at a minimum concentration of 1.2 microg mL(-1). Protein sequencing by mass spectrometry was obtained by the digestion of CRLII with trypsin, Glu-C, and AspN. CRLII partial protein sequence exhibits 46% similarity with the ConA-like alpha chain precursor. Suitable protein crystals were obtained with the hanging-drop vapor-diffusion method with 8% ethylene glycol, 0.1 M Tris-HCl pH 8.5, and 11% PEG 8,000. The monoclinic crystals belong to space group P2(1) with unit cell parameters a = 49.4, b = 89.6, and c = 100.8 A.

PMID: 18712290 DOI: 10.1007/s12010-008-8334-9

de Azevedo WF Jr, Caceres RA, Pauli I, Timmers LF, Barcellos GB, Rocha KB, Soares MB. Protein-drug interaction studies for development of drugs against Plasmodium falciparum. Curr Drug Targets. 2009; 10(3):271–278.

 

The study of protein-drug interaction is of pivotal importance to understand the structural features essential for ligand affinity. The explosion of information about protein structures has paved the way to develop structure-based virtual screening approaches. Parasitic protein kinases have been pointed out as potential targets for antiparasitic development. The identification of protein kinases in the Plasmodium falciparum genome has opened the possibility to test new families of inhibitors as potential antimalarial drugs. In addition, other key enzymes which play roles in biosynthetic pathways, such as enoyl reductase and chorismate synthase, can be valuable targets for drug development. This review is focused on these protein targets that may help to materialize new generations of antimalarial drugs.

PMID: 19275563

Timmers LF, Pauli I, Barcellos GB, Rocha KB, Caceres RA, de Azevedo WF Jr, Soares MB. Genomic databases and the search of protein targets for protozoan parasites. Curr Drug Targets. 2009; 10(3):240–245.

 

The development of databases devoted to biological information opened the possibility to integrate, query and analyze biological data obtained from several sources that otherwise would be scattered through the web. Several issues arise in the handling of biological information, mainly due to the diversity of biological subject matter and the complexity of biological approaches towards phenomena of the living world. The integration of genomic data, three-dimensional structures of proteins, biological activity, and drugs availability allows a system approach to the study of the biology. Here we review the current status of these research efforts to develop genomic databases for protozoan parasites, such as the apicomplexan parasites, Trypanosoma cruzi and Leishmania spp. These databases may help in the discovery and development of new drugs against parasite-mediated diseases.

PMID: 19275560

de Azevedo WF Jr, Dias R, Timmers LF, Pauli I, Caceres RA, Soares MB. Bioinformatics tools for screening of antiparasitic drugs. Curr Drug Targets. 2009; 10(3):232–239.

 

Drug development has become the Holy Grail of many structural bioinformatics groups. The explosion of information about protein structures, ligand-binding affinity, parasite genome projects, and biological activity of millions of molecules opened the possibility to correlate this scattered information in order to generate reliable computational models to predict the likelihood of being able to modulate a target with a small-molecule drug. Computational methods have shown their potential in drug discovery and development allied with in vitro and in vivo methodologies. The present review discusses the main bioinformatics tools available for drug discovery and development.

PMID: 19275559

de Azevedo WF Jr, Soares MB. Selection of targets for drug development against protozoan parasites. Curr Drug Targets. 2009; 10(3):193–201.

 

Sequencing of parasite genomes opened the possibility to identify potential protein targets for drug development. Several protein targets have been found in the genome of Plasmodium falciparum, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major. Bioinformatics analysis is an important tool for the identification of protein targets for drug development against parasitic diseases. In this review we comment about three protein targets, identified in parasite genomes, and discuss the main features that may guide future efforts for virtual screening initiatives.

PMID: 19275556

Perez PC, Caceres RA, Canduri F, de Azevedo WF Jr. Molecular modeling and dynamics simulation of human cyclin-dependent kinase 3 complexed with inhibitors. Comput Biol Med. 2009; 39(2):130–140.

 

The complex CDK3-cyclin is involved in the control of the progression of G0. While the mechanisms governing early and late G1 progression are well understood, very little is known about the G0-G1 transition. Human CDK3 is closely related to cyclin-dependent kinase 2 (CDK2). Since there is no crystallographic structure of human CDK3, this work describes for the first time a molecular model of human CDK3 complexed with several inhibitors. Comparison of the binary complexes with different inhibitors strongly indicates that those inhibitors should inhibit CDK3 as well as CDK2.

PMID: 19152876 DOI: 10.1016/j.compbiomed.2008.11.004

Barcellos GB, Caceres RA, de Azevedo WF Jr. Structural studies of shikimate dehydrogenase from Bacillus anthracis complexed with cofactor NADP. J Mol Model. 2009; 15(2):147–155.

 

Bacillus anthracis has been employed as an agent of bioterrorism, with high mortality, despite anti-microbial treatment, which strongly indicates the need of new drugs to treat anthrax. Shikimate pathway is a seven step biosynthetic route which generates chorismic acid from phosphoenol pyruvate and erythrose-4-phosphate. Chorismic acid is the major branch point in the synthesis of aromatic amino acids, ubiquinone, and secondary metabolites. The shikimate pathway is essential for many pathological organisms, whereas it is absent in mammals. Therefore, these enzymes are potential targets for the development of nontoxic antimicrobial agents and herbicides and have been submitted to intensive structural studies. The forth enzyme of this pathway is responsible for the conversion of dehydroshikimate to shikimate in the presence of NADP. In order to pave the way for structural and functional efforts toward development of new antimicrobials we describe the molecular modeling of shikimate dehydrogenase from Bacillus anthracis complexed with the cofactor NADP. This study was able to identify the main residues of the NADP binding site responsible for ligand affinities. This structural study can be used in the design of more specific drugs against infectious diseases.

PMID: 19043750 DOI: 10.1007/s00894-008-0403-z

Vivan AL, Caceres RA, Basso LA, Santos DS; Walter F. de Azevedo Jr. Structural studies of PNP from Toxoplasma gondii. Int J Bioinform Res Appl. 2009; 5(2):154–162.

 

Toxoplasmosis is a chronic infection that affects approximately 30% of the human population and is caused by Toxoplasma gondii. Determination of the three dimensional structure of PNP from T. gondii could provide new insights into the purine binding site and sub-strate binding, and could be used for future rational design of new drugs against toxoplasmosis. This work describes the molecular model for three dimensional structure of PNP from T.gondii using, as a template, PNP from Plasmodium falciparum. Molecular dynamics showed that this model is stable during a trajectory of 3 ns.

PMID: 19324601 DOI: 10.1504/IJBRA.2009.024095

Timmers LF, Pauli I, Caceres RA, de Azevedo WF Jr. Drug-binding databases. Curr Drug Targets. 2008; 9(12):1092–1099.

 

Recent developments in computer power and chemoinformatics methodology make possible that a huge amount of data become available through internet. These databases are devoted to a wide spectrum of scientific fields. Here we are concerned with databases related to protein-drug interactions. More specifically, databases where potential new molecules could be accessed to be used in virtual screening initiatives. In the past decade several databases have been developed where molecules to be used in the virtual screening could be easily identified, downloaded and even purchased. This review describes and summarizes the recent advances in the development of these databases, and also the main applications related to virtual screening projects.

PMID: 19128220

Barcellos GB, Pauli I, Caceres RA, Timmers LF, Dias R, de Azevedo WF Jr. Molecular modeling as a tool for drug discovery. Curr Drug Targets. 2008; 9(12):1084–1091.

 

With the progression of structural genomics projects, comparative modeling remains an increasingly important method of choice to obtain 3D structure of proteins. It helps to bridge the gap between the available sequence and structure information by providing reliable and accurate protein models. Comparative modeling based on more than 30% sequence identity is now approaching its natural template-based limits and further improvements require the development of effective refinement techniques capable of driving models toward native structure. For difficult targets, for which the most significant progress in recent years has been observed, optimal template selection and alignment accuracy are still the major problems. The past year has seen a maturation of molecular modeling, with an increasing number of comparative studies between established methods becoming possible, together with an explosion of new works especially in the areas of combinatorial chemistry and molecular diversity. To achieve this, knowledge about three-dimensional protein structures is crucial for the understanding of their functional mechanisms, and for a rational drug design. This review described recent progress in molecular modeling methodology.

PMID: 19128219

Caceres RA, Pauli I, Timmers LF, de Azevedo WF Jr. Molecular recognition models: a challenge to overcome. Curr Drug Targets. 2008; 9(12):1077–1083.

 

Molecular recognition process describes the interaction involving two molecules. In the case of biomolecules, these pairs of molecules could be protein-protein, protein-ligand or protein-nucleic acid. The first model to capture the essential features, behind the molecular recognition problem, was the lock-and-key paradigm. The overall analysis protein-protein, protein-nucleic acid and protein-ligand interaction based on the three-dimensional structures and physicochemical parameters, such as binding affinity, opened the possibility to provide further insights in this basic phenomenon. The main ideas behind the molecular recognition are discussed in the present review.

PMID: 19128218

de Azevedo WF Jr, Dias R. Experimental approaches to evaluate the thermodynamics of protein-drug interactions. Curr Drug Targets. 2008; 9(12):1071–1076.

 

Precise experimental methods to determine ligand-binding affinity are needed to accelerate the discovery of new drugs. Assessing protein-ligand interaction is of great importance for drug development. One of the techniques that may be used to evaluate ligand-binding affinitty is isothermal titration calorimetry (ITC). This experimental methodology may be used to measure the heat of binding of a ligand to a protein. Furthermore, the development of new empirical scoring functions to assess evaluation protein-ligand interaction lack abundance of experimental information to be used to generate reliable scores. ITC technique may be used to fill this gap. Here we describe the application of this technique to ligand-binding affinity determination, and discuss the synergetic relationship between ITC data and the development of a new generation of empirical scoring functions.

PMID: 19128217

Dias R, Timmers LF, Caceres RA, de Azevedo WF Jr. Evaluation of molecular docking using polynomial empirical scoring functions. Curr Drug Targets. 2008; 9(12):1062–1070.

 

Molecular docking simulations are of pivotal importance for analysis of protein-ligand interactions and also an essential resource for virtual-screening initiatives. In molecular docking simulations several possible docked structures are generated, which create an ensemble of structures representing binary complexes. Therefore, it is crucial to find the best solution for the simulation. One approach to this problem is to employ empirical scoring function to identify the best docked structure. It is expected that scoring functions show a descriptive funnel-shaped energy surface without many false minima to impair the efficiency of conformational sampling. We employed this methodology against a test set with 300 docked structures. Docking simulations of these ligands against enzyme binding pocket indicated a funnel-shaped behavior of the complexation for this system. This review compares a set of recently proposed polynomial empirical scoring functions, implemented in a program called POLSCORE, with two popular scoring function programs (XSCORE and DrugScore). Overall comparison indicated that POLSCORE works better to predict the correct docked position, for the ensemble of docked structures analyzed in the present work.

PMID: 19128216

Pauli I, Timmers LF, Caceres RA, Soares MB, de Azevedo WF Jr. In silico and in vitro: identifying new drugs. Curr Drug Targets. 2008; 9(12):1054–1061.

 

Drug development is a high cost and laborious process, requiring a number of tests until a drug is made available in the market. Therefore, the use of methods to screen large number of molecules with less cost is crucial for faster identification of hits and leads. One strategy to identify drug-like molecules is the search for molecules able to interfere with a protein function, since protein interactions control most biological processes. Ideally the use of in silico screenings would make drug development faster and less expensive. Currently, however, the confirmation of biological activity is still needed. Due to the complexity of the task of drug discovery, an integrated and multi-disciplinary approach is ultimately required. Here we discuss examples of drugs developed through a combination of in silico and in vitro strategies. The potential use of these methodologies for the identification of active compounds as well as for early toxicity and bioavailability is also reviewed.

PMID: 19128215

Canduri F, de Azevedo WF. Protein crystallography in drug discovery. Curr Drug Targets. 2008; 9(12):1048–1053.

 

Protein crystallography is the main technique used to obtain three-dimensional information for binary complexes involving protein and drugs. Once a protein target has its three-dimensional structure elucidated, the next natural step is the solving of the structure complexed either with its natural substrate, or any ligand or even an inhibitor. Such information is of pivotal importance to understand the structural basis for inhibition of an enzyme. The relevant features, for application of protein crystallography to drug discovery, are discussed in this review.

PMID: 19128214

Dias R, de Azevedo WF Jr. Molecular docking algorithms. Curr Drug Targets. 2008; 9(12):1040–1047.

 

By means of virtual screening of small molecules databases it is possible to identify new potential inhibitors against a target of interest. Molecular docking is a computer simulation procedure to predict the conformation of a receptor-ligand complex. Each docking program makes use of one or more specific search algorithms, which are the methods used to predict the possible conformations of a binary complex. In the present review we describe several molecular-docking search algorithms, and the programs which apply such methodologies. We also discuss how virtual screening can be optimized, describing methods that may increase accuracy of the simulation process, with relatively fast docking algorithms.

PMID: 19128213

de Azevedo WF Jr, Dias R. Computational methods for calculation of ligand-binding affinity. Curr Drug Targets. 2008; 9(12):1031–1039.

 

Precise computational methods to determine ligand-binding affinity are needed to accelerate the discovery of new drugs. Assessing protein-ligand interaction is of great importance for virtual screening initiatives. The affinity may be computational evaluated using scoring functions involving terms for intermolecular hydrogen bonds, contact surface, hydrophobic contacts, electrostatic interactions and others. Empirical scoring functions have been developed to evaluate ligand-binding affinity very rapidly. In addition to predict affinity, these scoring functions have been employed to identify the best results obtained from docking simulations. This review describes several computational methods, employed to estimate ligand-binding affinity and discuss their development and main applications.

PMID: 19128212

de Azevedo WF Jr. Protein-drug interactions. Curr Drug Targets. 2008; 9(12):1030.

 

PMID: 19128211

Arcuri HA, Apponi LH, Valentini SR, Durigon EL, de Azevedo WF Jr, Fossey MA, Rahal P, de Souza FP. Expression and purification of human respiratory syncytial virus recombinant fusion protein. Protein Expr Purif. 2008; 62(2):146–152.

 

The Human Respiratory Syncytial Virus (HRSV) fusion protein (F) was expressed in Escherichia coli BL21A using the pET28a vector at 37 degrees C. The protein was purified from the soluble fraction using affinity resin. The structural quality of the recombinant fusion protein and the estimation of its secondary structure were obtained by circular dichroism. Structural models of the fusion protein presented 46% of the helices in agreement with the spectra by circular dichroism analysis. There are only few studies that succeeded in expressing the HRSV fusion protein in bacteria. This is a report on human fusion protein expression in E. coli and structure analysis, representing a step forward in the development of fusion protein F inhibitors and the production of antibodies.

PMID: 18786639 DOI: 10.1016/j.pep.2008.08.005

Timmers LF, Caceres RA, Vivan AL, Gava LM, Dias R, Ducati RG, Basso LA, Santos DS, de Azevedo WF Jr. Structural studies of human purine nucleoside phosphorylase: towards a new specific empirical scoring function. Arch Biochem Biophys. 2008 ; 479(1):28–38.

 

Human purine nucleoside phosphorylase (HsPNP) is a target for inhibitor development aiming at T-cell immune response modulation. In this work, we report the development of a new set of empirical scoring functions and its application to evaluate binding affinities and docking results. To test these new functions, we solved the structure of HsPNP and 2-mercapto-4(3H)-quinazolinone (HsPNP:MQU) binary complex at 2.7A resolution using synchrotron radiation, and used these functions to predict ligand position obtained in docking simulations. We also employed molecular dynamics simulations to analyze HsPNP in two conditions, as apoenzyme and in the binary complex form, in order to assess the structural features responsible for stability. Analysis of the structural differences between systems provides explanation for inhibitor binding. The use of these scoring functions to evaluate binding affinities and molecular docking results may be used to guide future efforts on virtual screening focused on HsPNP.

PMID: 18790691 DOI: 10.1016/j.abb.2008.08.015

de Oliveira TM, Delatorre P, da Rocha BA, de Souza EP, Nascimento KS, Bezerra GA, Moura TR, Benevides RG, Bezerra EH, Moreno FB, Freire VN, de Azevedo WF Jr, Cavada BS. Crystal structure of Dioclea rostrata lectin: insights into understanding the pH-dependent dimer-tetramer equilibrium and the structural basis for carbohydrate recognition in Diocleinae lectins. J Struct Biol. 2008; 164(2):177–182.

 

The legume lectins from the subtribe Diocleinae, often referred to as concanavalin A-like lectins, are a typical example of highly similar proteins that show distinct biological activities. The pH-dependent oligomerization that some of these lectins undergo and the relative position of amino acids within the carbohydrate-binding site are factors that have been reported to contribute to these differences in the activities of Diocleinae lectins. In the present work, we determined the amino acid sequence and the crystal structure of the lectin of Dioclea rostrata seeds (DRL), with the aim of investigating the structural bases of the different behavior displayed by this lectin in comparison to other Diocleinae lectins and determining the reason for the distinct pH-dependent dimer-tetramer equilibrium. In addition, we discovered a novel multimeric arrangement for this lectin.

PMID: 18682294 DOI: 10.1016/j.jsb.2008.05.012

de Azevedo WF Jr, Dias R. Evaluation of ligand-binding affinity using polynomial empirical scoring functions. Bioorg Med Chem. 2008; 16(20):9378–9382.

 

Assessing protein-ligand interaction is of great importance for virtual screening initiatives in order to discover new drugs. The present work describes a set of empirical scoring functions to assess the binding affinity, involving terms for intermolecular hydrogen bonds and contact surface. The results show that our methodology works better to predict protein-ligand affinity when compared with XSCORE, a popular empirical scoring function.

PMID: 18829335 DOI: 10.1016/j.bmc.2008.08.014

Pauli I, Caceres RA, de Azevedo WF Jr. Molecular modeling and dynamics studies of Shikimate Kinase from Bacillus anthracis. Bioorg Med Chem. 2008; 16(17):8098–8108.

 

Bacillus anthracis has been used as weapon in bioterrorist activities, with high mortality, despite anti-microbial treatment, which strongly indicates a need of new drugs to treat anthrax. Shikimate Pathway is a seven-step biosynthetic route which generates chorismic acid. The shikimate pathway is essential for many pathological organisms, whereas it is absent in mammals. Therefore, these enzymes are potential targets for the development of non-toxic anti-microbial agents and herbicides and have been submitted to intensive structural studies. Shikimate Kinase is the fifth enzyme of shikimate pathway and catalyzes the specific phosphorylation of the 3-hydroxyl group of shikimate using ATP as a co-substrate, resulting in shikimate-3-phosphate and ADP. The present work describes for the first time a structural model for the Shikimate Kinase from B. anthracis using molecular modeling approach and molecular dynamics simulations. This study was able to identify the main residues of the ATP-binding and the shikimate pockets responsible for ligand affinities. Analysis of the molecular dynamics simulations indicates the structural features responsible for the stability of the structure. This study may help in the identification of new inhibitors for this enzyme.

PMID: 18706819 DOI: 10.1016/j.bmc.2008.07.051

Vivan AL, Caceres RA, Abrego JR, Borges JC, Ruggiero Neto J, Ramos CH, de Azevedo WF Jr, Basso LA, Santos DS. Structural studies of prephenate dehydratase from Mycobacterium tuberculosis H37Rv by SAXS, ultracentrifugation, and computational analysis. Proteins. 2008; 72(4):1352–1362.

 

Tuberculosis (TB) is one of the most common infectious diseases known to man and responsible for millions of human deaths in the world. The increasing incidence of TB in developing countries, the proliferation of multidrug resistant strains, and the absence of resources for treatment have highlighted the need of developing new drugs against TB. The shikimate pathway leads to the biosynthesis of chorismate, a precursor of aromatic amino acids. This pathway is absent from mammals and shown to be essential for the survival of Mycobacterium tuberculosis, the causative agent of TB. Accordingly, enzymes of aromatic amino acid biosynthesis pathway represent promising targets for structure-based drug design. The first reaction in phenylalanine biosynthesis involves the conversion of chorismate to prephenate, catalyzed by chorismate mutase. The second reaction is catalyzed by prephenate dehydratase (PDT) and involves decarboxylation and dehydratation of prephenate to form phenylpyruvate, the precursor of phenylalanine. Here, we describe utilization of different techniques to infer the structure of M. tuberculosis PDT (MtbPDT) in solution. Small angle X-ray scattering and ultracentrifugation analysis showed that the protein oligomeric state is a tetramer and MtbPDT is a flat disk protein. Bioinformatics tools were used to infer the structure of MtbPDT. A molecular model for MtbPDT is presented and molecular dynamics simulations indicate that MtbPDT is stable. Experimental and molecular modeling results were in agreement and provide evidence for a tetrameric state of MtbPDT in solution.

2008 Wiley-Liss, Inc.

PMID: 18384085 DOI: 10.1002/prot.22034

Arcuri HA, Borges JC, Fonseca IO, Pereira JH, Neto JR, Basso LA, Santos DS, de Azevedo WF Jr. Structural studies of shikimate 5-dehydrogenase from Mycobacterium tuberculosis. Proteins. 2008; 72(2):720–730.

 

Tuberculosis (TB) remains the leading cause of mortality due to a single bacterial pathogen, Mycobacterium tuberculosis. The reemergence of TB as a potential public health threat, the high susceptibility of human immunodeficiency virus-infected persons to the disease, the proliferation of multi-drug-resistant strains (MDR-TB) and, more recently, of extensively drug resistant isolates (XDR-TB) have created a need for the development of new antimycobacterial agents. Amongst the several proteins and/or enzymes to be studied as potential targets to develop novel drugs against M. tuberculosis, the enzymes of the shikimate pathway are attractive targets because they are essential in algae, higher plants, bacteria, and fungi, but absent from mammals. The mycobacterial shikimate pathway leads to the biosynthesis of chorismate, which is a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Here we report the structural studies by homology modeling and circular dichroism spectroscopy of the shikimate dehydrogenase from M. tuberculosis (MtSDH), which catalyses the fourth step of the shikimate pathway. Our structural models show that the MtSDH has similar structure to other shikimate dehydrogenase structures previously reported either in presence or absence of NADP, despite the low amino acid sequence identity. The circular dichroism spectra corroborate the secondary structure content observed in the MtSDH models developed. The enzyme was stable up to 50 degrees C presenting a cooperative unfolding profile with the midpoint of the unfolding temperature value of approximately 63-64 degrees C, as observed in the unfolding experiment followed by circular dichroism. Our MtSDH structural models and circular dichroism data showed small conformational changes induced by NADP binding. We hope that the data presented here will assist the rational design of antitubercular agents.

(c) 2008 Wiley-Liss, Inc.

PMID: 18260104 DOI: 10.1002/prot.21953

Marques MR, Vaso A, Neto JR, Fossey MA, Oliveira JS, Basso LA, dos Santos DS, de Azevedo Junior WF, Palma MS. Dynamics of glyphosate-induced conformational changes of Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19) determined by hydrogen-deuterium exchange and electrospray mass spectrometry. Biochemistry. 2008; 47(28):7509–7522.

 

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the reaction between shikimate 3-phosphate and phosphoenolpyruvate to form 5-enolpyruvylshikimate 3-phosphate, an intermediate in the shikimate pathway, which leads to the biosynthesis of aromatic amino acids. EPSPS exists in an open conformation in the absence of substrates and/or inhibitors and in a closed conformation when bound to the substrate and/or inhibitor. In the present report, the H/D exchange properties of EPSPS from Mycobacterium tuberculosis ( Mt) were investigated for both enzyme conformations using ESI mass spectrometry and circular dichroism (CD). When the conformational changes identified by H/D exchanges were mapped on the 3-D structure, it was observed that the apoenzyme underwent extensive conformational changes due to glyphosate complexation, characterized by an increase in the content of alpha-helices from 40% to 57%, while the beta-sheet content decreased from 30% to 23%. These results indicate that the enzyme underwent a series of rearrangements of its secondary structure that were accompanied by a large decrease in solvent access to many different regions of the protein. This was attributed to the compaction of 71% of alpha-helices and 57% of beta-sheets as a consequence of glyphosate binding to the enzyme. Apparently, MtEPSPS undergoes a series of inhibitor-induced conformational changes, which seem to have caused synergistic effects in preventing solvent access to the core of molecule, especially in the cleft region. This may be part of the mechanism of inhibition of the enzyme, which is required to prevent the hydration of the substrate binding site and also to induce the cleft closure to avoid entrance of the substrates.

PMID: 18558720 DOI: 10.1021/bi800134y

Caceres RA, Saraiva Timmers LF, Dias R, Basso LA, Santos DS, de Azevedo WF Jr. Molecular modeling and dynamics simulations of PNP from Streptococcus agalactiae.Bioorg Med Chem. 2008; 16(9):4984–4993.

 

This work describes for the first time a structural model of purine nucleoside phosphorylase from Streptococcus agalactiae (SaPNP). PNP catalyzes the cleavage of N-ribosidic bonds of the purine ribonucleosides and 2-deoxyribonucleosides in the presence of inorganic orthophosphate as a second substrate. This enzyme is a potential target for the development of antibacterial drugs. We modeled the complexes of SaPNP with 15 different ligands in order to determine the structural basis for the specificity of these ligands against SaPNP. The application of a novel empirical scoring function to estimate the affinity of a ligand for a protein was able to identify the ligands with high affinity for PNPs. The analysis of molecular dynamics trajectory for SaPNP indicates that the functionally important motifs have a very stable structure. This new structural model together with a novel empirical scoring function opens the possibility to explorer larger library of compounds in order to identify the new inhibitors for PNPs in virtual screening projects.

PMID: 18381241 DOI: 10.1016/j.bmc.2008.03.044

Caceres RA, Macedo Timmers LF, Vivan AL, Schneider CZ, Basso LA, De Azevedo WF Jr, Santos DS. Molecular modeling and dynamics studies of cytidylate kinase from Mycobacterium tuberculosis H37Rv. J Mol Model.; 14(5): 427–34.

 

Bacterial cytidylate kinase or cytidine monophosphate kinase (CMP kinase) catalyses the phosphoryl transfer from ATP to CMP and dCMP, resulting in the formation nucleoside diphosphates. In eukaryotes, CMP/UMP kinase catalyses the conversion of UMP and CMP to, respectively, UDP and CDP with high efficiency. This work describes for the first time a model of bacterial cytidylate kinase or cytidine monophosphate kinase (CMP kinase) from mycobacterium tuberculosis (MtCMPK). We modeled MtPCMPK in apo form and in complex with cytidine 5'-monophosphate (CMP) to try to determine the structural basis for specificity. Comparative analysis of the model of MtCMPK allowed identification of structural features responsible for ligand affinities. Analysis of the molecular dynamics simulations of these two systems indicates the structural features responsible for the stability of the structure, and may help in the identification of new inhibitors for this enzyme.

PMID: 18343960 DOI: 10.1007/s00894-008-0291-2

Canduri F, Perez PC, Caceres RA, de Azevedo WF Jr. CDK9 a potential target for drug development. Med Chem. 2008; 4(3):210–218.

 

The family of Cyclin-Dependent Kinases (CDKs) can be subdivided into two major functional groups based on their roles in cell cycle and/or transcriptional control. CDK9 is the catalytic subunit of positive transcription elongation factor b (P-TEFb). CDK9 is the kinase of the TAK complex (Tat-associated kinase complex), and binds to Tat protein of HIV, suggesting a possible role for CDK9 in AIDS progression. CDK9 complexed with its regulatory partner cyclin T1, serves as a cellular mediator of the transactivation function of the HIV Tat protein. P-TEFb is responsible for the phosphorylation of the carboxyl-terminal domain of RNA Pol II, resulting in stimulation of transcription. Furthermore, the complexes containing CDK9 induce the differentiation in distinct tissue. The CDK9/cyclin T1 complex is expressed at higher level in more differentiated primary neuroectodermal and neuroblastoma tumors, showing a correlation between the kinase expression and tumor differentiation grade. This may have clinical and therapeutical implications for these tumor types. Among the CDK inhibitors two have shown to be effective against CDK9: Roscovitine and Flavopiridol. These two inhibitors prevented the replication of human immunodeficiency virus (HIV) type 1 by blocking Tat transactivation of the HIV type 1 promoter. These compounds inhibit CDKs by binding to the catalytic domain in place of ATP, preventing transfer of a phosphate group to the substrate. More sensitive therapeutic agents of CDK9 can be designed, and structural studies can add information in the understanding of this kinase. The major features related to CDK9 inhibition will be reviewed in this article.

PMID: 18473913

Moreno FB, de Oliveira TM, Martil DE, Viçoti MM, Bezerra GA, Abrego JR, Cavada BS, Filgueira de Azevedo W Jr. Identification of a new quaternary association for legume lectins. J Struct Biol. 2008; 161(2):133–143.

 

Lotus tetragonolobus lectin (LTA) is a fucose-specific legume lectin. Although several studies report a diverse combination of biological activities for LTA, little is known about the mechanisms involved in l-fucosyl oligosaccharide recognition. The crystal structure of LTA at 2.0A resolution reveals a different legume lectin tetramer. Its structure consists of a homotetramer composed of two back-to-back GS4-like dimers arranged in a new mode, resulting in a novel tetramer. The LTA N-linked carbohydrate at Asn4 and the unusual LTA dimer-dimer interaction are related to its particular mode of tetramerization. In addition, we used small angle X-ray scattering to investigate the quaternary structure of LTA in solution and to compare it to the crystalline structure. Although the crystal structure of LTA has revealed a conserved metal-binding site, its l-fucose-binding site presents some punctual differences. Our investigation of the new tetramer of LTA and its fucose-binding site is essential for further studies related to cross-linking between LTA and complex divalent l-fucosyl carbohydrates.

PMID: 18068379 DOI: 10.1016/j.jsb.2007.10.002

Timmers LF, Caceres RA, Basso LA, Santos DS, De Azevedo WF Jr. Structural bioinformatics study of PNP from Listeria monocytogenes. Protein Pept Lett. 2008;15(8):843–849.

 

This work describes for the first time a model of Purine Nucleoside Phosphorylase from Listeria monocytogenes (LmPNP). We modeled the complexes of LmPNP with ligands in order to determine the structural basis for specificity. Comparative analysis of the model of LmPNP allowed identification of structural features responsible for ligand affinities.

PMID: 18855758

Breda A, Basso LA, Santos DS, de Azevedo Jr WF. Virtual Screening of Drugs: Score Functions, Docking, and Drug Design. Current Computer Aided-Drug Design 2008; 4(4), 265–272.

 

The computational approach for new drug design and/or identification, was initially proposed in mid 70s. The virtual screening of chemical libraries against a biological target has proven its reliability on structure-based drug design, for instance, for many HIV virus protein inhibitors and for the development of Cyclin-Dependent Kinase inhibitors. Target- based virtual screening, allied to docking studies, enables searches on larger data set of probable ligands, with less costs than the traditional experimental screening. The increasing availability of small molecules databases and its free online distribution is now allowing not only pharmaceutical industries, but independent research labs as well, to apply this methodology on early stages of drug discovery. When the protein target structure is available, and a chemical virtual library is accessible, following questions need to be answered: how the target and the ligand interact and how these interactions may be evaluated? Several docking algorithms for the identification of the molecular features responsible for binding specificity are available. While such algorithms are very robust and accurate, the scoring functions remain more questionable in the sense of what parameters should be considered when defining protein-ligand binding affinity when ranking candidates pointed-out by the virtual screening to the next step on drug testing. Aside conformational and chemical information, pharmacokinetics properties should be considered as well when selecting potential new drugs. Along with structural well-match, appropriate molecular features that define desired kinetics characteristics should be consistently addressed for usefulness of virtual screening results. The present review is focused on these questions and their implication for virtual screening.

Keywords: Virtual screening, drug design, protein targets, filtering methods

Bezerra GA, Oliveira TM, Moreno FB, de Souza EP, da Rocha BA, Benevides RG, Delatorre P, de Azevedo WF Jr, Cavada BS. Structural analysis of Canavalia maritima and Canavalia gladiata lectins complexed with different dimannosides: new insights into the understanding of the structure-biological activity relationship in legume lectins. J Struct Biol. 2007; 160(2):168–176.

 

Plant lectins, especially those purified from species of the Leguminosae family, represent the best studied group of carbohydrate-binding proteins. The legume lectins from Diocleinae subtribe are highly similar proteins that present significant differences in the potency/efficacy of their biological activities. The structural studies of the interactions between lectins and sugars may clarify the origin of the distinct biological activities observed in this high similar class of proteins. In this way, this work presents a crystallographic study of the ConM and CGL (agglutinins from Canavalia maritima and Canavalia gladiata, respectively) in the following complexes: ConM/CGL:Man(alpha1-2)Man(alpha1-O)Me, ConM/CGL:Man(alpha1-3)Man(alpha1-O)Me and ConM/CGL:Man(alpha1-4)Man(alpha1-O)Me, which crystallized in different conditions and space group from the native proteins. The structures were solved by molecular replacement, presenting satisfactory values for R(factor) and R(free). Comparisons between ConM, CGL and ConA (Canavalia ensiformis lectin) binding mode with the dimannosides in subject, presented different interactions patterns, which may account for a structural explanation of the distincts biological properties observed in the lectins of Diocleinae subtribe.

PMID: 17881248 DOI: 10.1016/j.jsb.2007.07.012

Dias MV, Vasconcelos IB, Prado AM, Fadel V, Basso LA, de Azevedo WF Jr, Santos DS. Crystallographic studies on the binding of isonicotinyl-NAD adduct to wild-type and isoniazid resistant 2-trans-enoyl-ACP (CoA) reductase from Mycobacterium tuberculosis. J Struct Biol. 2007; 159(3):369–380.

 

The resumption of tuberculosis led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. Isoniazid (INH), the most prescribed drug to treat TB, inhibits an NADH-dependent enoyl-acyl carrier protein reductase (InhA) that provides precursors of mycolic acids, which are components of the mycobacterial cell wall. InhA is the major target of the mode of action of isoniazid. INH is a pro-drug that needs activation to form the inhibitory INH-NAD adduct. Missense mutations in the inhA structural gene have been identified in clinical isolates of Mycobacterium tuberculosis resistant to INH. To understand the mechanism of resistance to INH, we have solved the structure of two InhA mutants (I21V and S94A), identified in INH-resistant clinical isolates, and compare them to INH-sensitive WT InhA structure in complex with the INH-NAD adduct. We also solved the structure of unliganded INH-resistant S94A protein, which is the first report on apo form of InhA. The salient features of these structures are discussed and should provide structural information to improve our understanding of the mechanism of action of, and resistance to, INH in M. tuberculosis. The unliganded structure of InhA allows identification of conformational changes upon ligand binding and should help structure-based drug design of more potent antimycobacterial agents.

PMID: 17588773 DOI: 10.1016/j.jsb.2007.04.009

Delatorre P, Rocha BA, Souza EP, Oliveira TM, Bezerra GA, Moreno FB, Freitas BT, Santi-Gadelha T, Sampaio AH, Azevedo WF Jr, Cavada BS. Structure of a lectin from Canavalia gladiata seeds: new structural insights for old molecules. BMC Struct Biol. 2007; 7:52.

 

BACKGROUND: Lectins are mainly described as simple carbohydrate-binding proteins. Previous studies have tried to identify other binding sites, which possible recognize plant hormones, secondary metabolites, and isolated amino acid residues. We report the crystal structure of a lectin isolated from Canavalia gladiata seeds (CGL), describing a new binding pocket, which may be related to pathogen resistance activity in ConA-like lectins; a site where a non-protein amino-acid, alpha-aminobutyric acid (Abu), is bound. RESULTS: The overall structure of native CGL and complexed with alpha-methyl-mannoside and Abu have been refined at 2.3 A and 2.31 A resolution, respectively. Analysis of the electron density maps of the CGL structure shows clearly the presence of Abu, which was confirmed by mass spectrometry. CONCLUSION: The presence of Abu in a plant lectin structure strongly indicates the ability of lectins on carrying secondary metabolites. Comparison of the amino acids composing the site with other legume lectins revealed that this site is conserved, providing an evidence of the biological relevance of this site. This new action of lectins strengthens their role in defense mechanisms in plants.

PMID: 17683532 PMCID: PMC1955443 DOI: 10.1186/1472-6807-7-52

Pereira JH, Vasconcelos IB, Oliveira JS, Caceres RA, de Azevedo WF Jr, Basso LA, Santos DS. Shikimate kinase: a potential target for development of novel antitubercular agents. Curr Drug Targets. 2007; 8(3):459–468.

 

Tuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. However, no new classes of drugs for TB have been developed in the past 30 years. Therefore there is an urgent need to develop faster acting and effective new antitubercular agents, preferably belonging to new structural classes, to better combat TB, including MDR-TB, to shorten the duration of current treatment to improve patient compliance, and to provide effective treatment of latent tuberculosis infection. The enzymes in the shikimate pathway are potential targets for development of a new generation of antitubercular drugs. The shikimate pathway has been shown by disruption of aroK gene to be essential for the Mycobacterium tuberculosis. The shikimate kinase (SK) catalyses the phosphorylation of the 3-hydroxyl group of shikimic acid (shikimate) using ATP as a co-substrate. SK belongs to family of nucleoside monophosphate (NMP) kinases. The enzyme is an alpha/beta protein consisting of a central sheet of five parallel beta-strands flanked by alpha-helices. The shikimate kinases are composed of three domains: Core domain, Lid domain and Shikimate-binding domain. The Lid and Shikimate-binding domains are responsible for large conformational changes during catalysis. More recently, the precise interactions between SK and substrate have been elucidated, showing the binding of shikimate with three charged residues conserved among the SK sequences. The elucidation of interactions between MtSK and their substrates is crucial for the development of a new generation of drugs against tuberculosis through rational drug design.

PMID: 17348838

Marques MR, Pereira JH, Oliveira JS, Basso LA, de Azevedo WF Jr, Santos DS, Palma MS. The inhibition of 5-enolpyruvylshikimate-3-phosphate synthase as a model for development of novel antimicrobials. Curr Drug Targets. 2007; 8(3):445–457.

 

EPSP synthase (EPSPS) is an essential enzyme in the shikimate pathway, transferring the enolpyruvyl group of phosphoenolpyruvate to shikimate-3-phosphate to form 5-enolpyruvyl-3-shikimate phosphate and inorganic phosphate. This enzyme is composed of two domains, which are formed by three copies of betaalphabetaalphabetabeta-folding units; in between there are two crossover chain segments hinging the nearly topologically symmetrical domains together and allowing conformational changes necessary for substrate conversion. The reaction is ordered with shikimate-3-phosphate binding first, followed by phosphoenolpyruvate, and then by the subsequent release of phosphate and EPSP. N-[phosphomethyl]glycine (glyphosate) is the commercial inhibitor of this enzyme. Apparently, the binding of shikimate-3-phosphate is necessary for glyphosate binding, since it induces the closure of the two domains to form the active site in the interdomain cleft. However, it is somehow controversial whether binding of shikimate-3-phosphate alone is enough to induce the complete conversion to the closed state. The phosphoenolpyruvate binding site seems to be located mainly on the C-terminal domain, while the binding site of shikimate-3-phosphate is located primarily in the N-terminal domain residues. However, recent results demonstrate that the active site of the enzyme undergoes structural changes upon inhibitor binding on a scale that cannot be predicted by conventional computational methods. Studies of molecular docking based on the interaction of known EPSPS structures with (R)- phosphonate TI analogue reveal that more experimental data on the structure and dynamics of various EPSPS-ligand complexes are needed to more effectively apply structure-based drug design of this enzyme in the future.

PMID: 17348837

Dias MV, Ely F, Palma MS, de Azevedo WF Jr, Basso LA, Santos DS. Chorismate synthase: an attractive target for drug development against orphan diseases. Curr Drug Targets. 2007; 8(3):437–444.

 

The increase in incidence of infectious diseases worldwide, particularly in developing countries, is worrying. Each year, 14 million people are killed by infectious diseases, mainly HIV/AIDS, respiratory infections, malaria and tuberculosis.. Despite the great burden in the poor countries, drug discovery to treat tropical diseases has come to a standstill. There is no interest by the pharmaceutical industry in drug development against the major diseases of the poor countries, since the financial return cannot be guaranteed. This has created an urgent need for new therapeutics to neglected diseases. A possible approach has been the exploitation of the inhibition of unique targets, vital to the pathogen such as the shikimate pathway enzymes, which are present in bacteria, fungi and apicomplexan parasites but are absent in mammals. The chorismate synthase (CS) catalyses the seventh step in this pathway, the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. The strict requirement for a reduced flavin mononucleotide and the anti 1,4 elimination are both unusual aspects which make CS reaction unique among flavin-dependent enzymes, representing an important target for the chemotherapeutic agents development. In this review we present the main biochemical features of CS from bacterial and fungal sources and their difference from the apicomplexan CS. The CS mechanisms proposed are discussed and compared with structural data. The CS structures of some organisms are compared and their distinct features analyzed. Some known CS inhibitors are presented and the main characteristics are discussed. The structural and kinetics data reviewed here can be useful for the design of inhibitors.

PMID: 17348836

Canduri F, Perez PC, Caceres RA, de Azevedo WF Jr. Protein kinases as targets for antiparasitic chemotherapy drugs. Curr Drug Targets. 2007; 8(3):389–398.

 

Parasitic protozoa infecting humans have a great impact on public health, especially in the developing countries. In many instances, the parasites have developed resistance against available chemotherapeutic agents, making the search for alternative drugs a priority. In line with the current interest in Protein Kinase (PK) inhibitors as potential drugs against a variety of diseases, the possibility that PKs may represent targets for novel anti-parasitic agents is being explored. Research into parasite PKs has benefited greatly from genome and EST sequencing projects, with the genomes from a few species fully sequenced (notably that from the malaria parasite Plasmodium falciparum) and several more under way, the structural features that are important to design specific inhibitors against these PKs will be reviewed in the present work.

PMID: 17348832

Dias MV, Faím LM, Vasconcelos IB, de Oliveira JS, Basso LA, Santos DS, de Azevedo WF Jr. Effects of the magnesium and chloride ions and shikimate on the structure of shikimate kinase from Mycobacterium tuberculosis. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007; 63(Pt 1):1–6.

 

Bacteria, fungi and plants can convert carbohydrate and phosphoenolpyruvate into chorismate, which is the precursor of various aromatic compounds. The seven enzymes of the shikimate pathway are responsible for this conversion. Shikimate kinase (SK) is the fifth enzyme in this pathway and converts shikimate to shikimate-3-phosphate. In this work, the conformational changes that occur on binding of shikimate, magnesium and chloride ions to SK from Mycobacterium tuberculosis (MtSK) are described. It was observed that both ions and shikimate influence the conformation of residues of the active site of MtSK. Magnesium influences the conformation of the shikimate hydroxyl groups and the position of the side chains of some of the residues of the active site. Chloride seems to influence the affinity of ADP and its position in the active site and the opening length of the LID domain. Shikimate binding causes a closing of the LID domain and also seems to influence the crystallographic packing of SK. The results shown here could be useful for understanding the catalytic mechanism of SK and the role of ions in the activity of this protein.

PMID: 17183161 PMCID: PMC2330112 DOI: 10.1107/S1744309106046823

da Silveira NJF, Bonalumi, CE, Arcuri HA, de Azevedo Jr., WF. Molecular Modeling Databases: A New Way in the Search of Proteins Targets for Drug Development. Curr Bioinform 2007; 2(1): 1–10.

 

DBMODELING is a relational database of annotated comparative protein structure models and their metabolic pathway characterization. It is focused on enzymes identified in the genomes of Mycobacterium tuberculosis and Xylella fastidiosa. The main goal of the present database is to provide structural models to be used in docking simulations and drug design. However, since the accuracy of structural models is highly dependent on sequence identity between template and target, it is necessary to make clear to the user that only models which show high structural quality should be used in such efforts. Molecular modeling of these genomes generated a database, in which all structural models were built using alignments presenting more than 30% of sequence identity, generating models with medium and high accuracy. All models in the database are publicly accessible at http://www.biocristalografia.df.ibilce.unesp.br/tools. DBMODELING user interface provides users friendly menus, so that all information can be printed in one step from any web browser. Furthermore, DBMODELING also provides a docking interface, which allows the user to carry out geometric docking simulation, against the molecular models available in the database. There are three other important homology model databases: MODBASE, SWISSMODEL, and GTOP. The main applications of these databases are described in the present article.

Keywords: Structural bioinformatics; databases; drug design; molecular modeling; protein prediction servers

Moreno FB, Bezerra GA, de Oliveira TM, de Souza EP, da Rocha BA, Benevides RG, Delatorre P, Cavada BS, de Azevedo WF Jr. New crystal forms of Diocleinae lectins in the presence of different dimannosides. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006; 62(Pt 11):1100–1103.

 

Studying the interactions between lectins and sugars is important in order to explain the differences observed in the biological activities presented by the highly similar proteins of the Diocleinae subtribe. Here, the crystallization and preliminary X-ray data of Canavalia gladiata lectin (CGL) and C. maritima lectin (CML) complexed with Man(alpha1-2)Man(alpha1)OMe, Man(alpha1-3)Man(alpha1)OMe and Man(alpha1-4)Man(alpha1)OMe in two crystal forms [the complexes with Man(alpha1-3)Man(alpha1)OMe and Man(alpha1-4)Man(alpha1)OMe crystallized in space group P3(2) and those with Man(alpha1-2)Man(alpha1)OMe crystallized in space group I222], which differed from those of the native proteins (P2(1)2(1)2 for CML and C222 for CGL), are reported. The crystal complexes of ConA-like lectins with Man(alpha1-4)Man(alpha1)OMe are reported here for the first time.

PMID: 17077488 PMCID: PMC2225211 DOI: 10.1107/S1744309106038887

Krystof V, Cankar P, Frysová I, Slouka J, Kontopidis G, Dzubák P, Hajdúch M, Srovnal J, de Azevedo WF Jr, Orság M, Paprskárová M, Rolcík J, Látr A, Fischer PM, Strnad M. 4-arylazo-3,5-diamino-1H-pyrazole CDK inhibitors: SAR study, crystal structure in complex with CDK2, selectivity, and cellular effects. J Med Chem. 2006; 49(22):6500–6509.

 

In a routine screening of our small-molecule compound collection we recently identified 4-arylazo-3,5-diamino-1H-pyrazoles as a novel group of ATP antagonists with moderate potency against CDK2-cyclin E. A preliminary SAR study based on 35 analogues suggests ways in which the pharmacophore could be further optimized, for example, via substitutions in the 4-aryl ring. Enzyme kinetics studies with the lead compound and X-ray crystallography of an inhibitor-CDK2 complex demonstrated that its mode of inhibition is competitive. Functional kinase assays confirmed the selectivity toward CDKs, with a preference for CDK9-cyclin T1. The most potent inhibitor, 4-[(3,5-diamino-1H-pyrazol-4-yl)diazenyl]phenol 31b (CAN508), reduced the frequency of S-phase cells of the cancer cell line HT-29 in antiproliferation assays. Further observed cellular effects included decreased phosphorylation of the retinoblastoma protein and the C-terminal domain of RNA polymerase II, inhibition of mRNA synthesis, and induction of the tumor suppressor protein p53, all of which are consistent with inhibition of CDK9.

PMID: 17064068 DOI: 10.1021/jm0605740

Cavada BS, Moreno FB, da Rocha BA, de Azevedo WF Jr, Castellón RE, Goersch GV, Nagano CS, de Souza EP, Nascimento KS, Radis-Baptista G, Delatorre P, Leroy Y, Toyama MH, Pinto VP, Sampaio AH, Barettino D, Debray H, Calvete JJ, Sanz L. cDNA cloning and 1.75 A crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds. FEBS J. 2006; 273(17):3962–3974.

 

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407+/-15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed beta(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-beta-D-glucopyranose units in chitin. The full-length amino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 A resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (betaalpha)8 barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182.

PMID: 16934035 DOI: 10.1111/j.1742-4658.2006.05400.x

Borges JC, Pereira JH, Vasconcelos IB, dos Santos GC, Olivieri JR, Ramos CH, Palma MS, Basso LA, Santos DS, de Azevedo WF Jr. Phosphate closes the solution structure of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Mycobacterium tuberculosis. Arch Biochem Biophys. 2006; 452(2):156–164.

 

The 5-enolpyruvylshikimate-3-phosphate synthase catalyses the sixth step of the shikimate pathway that is responsible for synthesizing aromatic compounds and is absent in mammals, which makes it a potential target for drugs development against microbial diseases. Here, we report the phosphate binding effects at the structure of the 5-enolpyruvylshikimate-3-phosphate synthase from Mycobacterium tuberculosis. This enzyme is formed by two similar domains that close on each other induced by ligand binding, showing the occurrence of a large conformation change. We have monitored the phosphate binding effects using analytical ultracentrifugation, small angle X-ray scattering and, circular dichroism techniques. The low resolution results showed that the enzyme in the presence of phosphate clearly presented a more compact structure. Thermal-induced unfolding experiments followed by circular dichroism suggested that phosphate rigidified the enzyme. Summarizing, these data suggested that the phosphate itself is able to induce conformational change resulting in the closure movement in the M. tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase.

PMID: 16876105 DOI: 10.1016/j.abb.2006.05.008

Moreno FB, Martil DE, Cavada BS, de Azevedo WF Jr. Crystallization and preliminary X-ray diffraction analysis of an anti-H(O) lectin from Lotus tetragonolobus seeds. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006; 62(Pt 7):680–683.

 

The seed lectin from Lotus tetragonolobus (LTA) has been crystallized. The best crystals grew over several days and were obtained using the vapour-diffusion method at a constant temperature of 293 K. A complete structural data set was collected at 2.00 angstroms resolution using a synchrotron-radiation source. LTA crystals were found to be monoclinic, belonging to space group P2(1), with unit-cell parameters a = 68.89, b = 65.83, c = 102.53 angstroms, alpha = gamma = 90, beta = 92 degrees. Molecular replacement yielded a solution with a correlation coefficient and R factor of 34.4 and 51.6%, respectively. Preliminary analysis of the molecular-replacement solution indicates a new quaternary association in the LTA structure. Crystallographic refinement is under way.

PMID: 16820693 PMCID: PMC2242948 DOI: 10.1107/S1744309106021312

Lombardi FR, Anazetti MC, Santos GC, Olivieri JR, de Azevedo WF Jr, Bonilla-Rodriguez GO. Rattlesnake hemoglobins: Functional properties and tetrameric stability. Protein Pept Lett. 2006; 13(5):517–523.

 

The present work analyzed the tetrameric stability of the hemoglobins from the rattlesnake C. durissus terrificus using analytical gel filtration chromatography, SAXS and osmotic stress. We show that the dissociation mechanism proposed for L. miliaris hemoglobin does not apply for these hemoglobins, which constitute stable tetramers even at low concentrations.

PMID: 16800809

Rádis-Baptista G, Moreno FB, de Lima Nogueira L, Martins AM, de Oliveira Toyama D, Toyama MH, Cavada BS, de Azevedo WF Jr, Yamane T. Crotacetin, a novel snake venom C-type lectin homolog of convulxin, exhibits an unpredictable antimicrobial activity. Cell Biochem Biophys. 2006; 44(3):412–423.

 

Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins that are capable of interfering with blood stasis. A very well-studied svC-type lectin is the heterodimeric toxin, convulxin (CVX), from the venom of South American rattlesnake Crotalus durissus terrificus. CVX is able to activate platelets and induce their aggregation by acting via p62/GPVI collagen receptor. By using polymerase chain reaction homology screening, we have cloned several cDNA precursors of CVX subunit homologs. One of them, named crotacetin (CTC) beta-subunit, predicts a polypeptide with a topology very similar to the tridimensional conformations of other subunits of CVX-like snake toxins, as determined by computational analysis. Using gel permeation and reverse-phase high-performance liquid chromatography, CTC was purified from C. durissus venoms. CTC can be isolated from the venom of several C. durissus subspecies, but its quantitative predominance is in the venom of C. durissus cascavella. Functional analysis indicates that CTC induces platelet aggregation, and, importantly, exhibits an antimicrobial activity against Gram-positive and -negative bacteria, comparable with CVX.

PMID: 16679528

de Azevedo WF Jr, Canduri F, Basso LA, Palma MS, Santos DS. Determining the structural basis for specificity of ligands using crystallographic screening. Cell Biochem Biophys. 2006; 44(3):405–411.

 

Crystallographic screening has been used to identify new inhibitors for potential target for drug development. Here, we describe the application of the crystallographic screening to assess the structural basis of specificity of ligands against a protein target. The method is efficient and results in detailed crystallographic information. The utility of the method is demonstrated in the study of the structural basis for specificity of ligands for human purine nucleoside phosphorylase (PNP). Purine nucleoside phosphorylase catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. This enzyme is a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure- based drug design. This methodology may help in the future development of a new generation of PNP inhibitors.

PMID: 16679527 DOI: 10.1385/CBB:44:3:405

Dias MV, Canduri F, da Silveira NJ, Czekster CM, Basso LA, Palma MS, Santos DS, de Azevedo WF Jr. Molecular models of tryptophan synthase from mycobacterium tuberculosis complexed with inhibitors. Cell Biochem Biophys. 2006; 44(3):375–384.

 

The development of new therapies against infectious diseases is vital in developing countries. Among infectious diseases, tuberculosis is considered the leading cause of death. A target for development of new drugs is the tryptophan pathway. The last enzyme of this pathway, tryptophan synthase (TRPS), is responsible for conversion of the indole 3-glycerol phosphate into indol and the condensation of this molecule with serine-producing tryptophan. The present work describes the molecular models of TRPS from Mycobacterium tuberculosis (MtTRPS) complexed with six inhibitors, the indole 3-propanol phosphate and five arylthioalkyl-phosphonated analogs of substrate of the alpha-subunit. The molecular models of MtTRPS present good stereochemistry, and the binding of the inhibitors is favorable. Thus, the generated models can be used in the design of more specific drugs against tuberculosis and other infectious diseases.

PMID: 16679524 DOI: 10.1385/CBB:44:3:375

da Silveira NJ, Bonalumi CE, Uchõa HB, Pereira JH, Canduri F, de Azevedo WF. DBMODELING: a database applied to the study of protein targets from genome projects. Cell Biochem Biophys. 2006; 44(3):366–374.

 

Genome sequencing efforts are providing us with complete genetic blueprints for hundreds of organisms. We are now faced with assigning, understanding, and modifying the functions of proteins encoded by these genomes. DBMODELING is a relational database of annotated comparative protein structure models and their metabolic pathway characterization, when identified. This procedure was applied to complete genomes such as Mycobacterium tuberculosis and Xylella fastidiosa. The main interest in the study of metabolic pathways is that some of these pathways are not present in humans, which makes them selective targets for drug design, decreasing the impact of drugs in humans. In the database, there are currently 1,116 proteins from two genomes. It can be accessed by any researcher at http://www.biocristalografia.df.ibilce.unesp.br/tools/. This project confirms that homology modeling is a useful tool in structural bioinformatics and that it can be very valuable in annotating genome sequence information, contributing to structural and functional genomics, and analyzing protein-ligand docking.

PMID: 16679523

Delatorre P, Rocha BA, Gadelha CA, Santi-Gadelha T, Cajazeiras JB, Souza EP, Nascimento KS, Freire VN, Sampaio AH, Azevedo WF Jr, Cavada BS. Crystal structure of a lectin from Canavalia maritima (ConM) in complex with trehalose and maltose reveals relevant mutation in ConA-like lectins. J Struct Biol. 2006; 154(3):280–286.

 

The crystal structure of Canavalia maritima lectin (ConM) complexed with trehalose and maltose revealed relevant point mutations in ConA-like lectins. ConM with the disaccharides and other ConA-like lectins complexed with carbohydrates demonstrated significant differences in the position of H-bonds. The main difference in the ConM structure is the replacement of Pro202 by Ser202, a residue that promotes the approximation of Tyr12 to the carbohydrate-binding site. The O-6' of the second glucose ring in maltose interacts with Tyr12, while in trehalose the interaction is established by the O-2' and Tyr12, explaining the higher affinity of ConM for disaccharides compared to monosaccharides.

PMID: 16677825 DOI: 10.1016/j.jsb.2006.03.011

Oliveira JS, Pereira JH, Canduri F, Rodrigues NC, de Souza ON, de Azevedo WF Jr, Basso LA, Santos DS. Crystallographic and pre-steady-state kinetics studies on binding of NADH to wild-type and isoniazid-resistant enoyl-ACP(CoA) reductase enzymes from Mycobacterium tuberculosis. J Mol Biol. 2006; 359(3):646–666.

 

An understanding of isoniazid (INH) drug resistance mechanism in Mycobacterium tuberculosis should provide significant insight for the development of newer anti-tubercular agents able to control INH-resistant tuberculosis (TB). The inhA-encoded 2-trans enoyl-acyl carrier protein reductase enzyme (InhA) has been shown through biochemical and genetic studies to be the primary target for INH. In agreement with these results, mutations in the inhA structural gene have been found in INH-resistant clinical isolates of M.tuberculosis, the causative agent of TB. In addition, the InhA mutants were shown to have higher dissociation constant values for NADH and lower values for the apparent first-order rate constant for INH inactivation as compared to wild-type InhA. Here, in trying to identify structural changes between wild-type and INH-resistant InhA enzymes, we have solved the crystal structures of wild-type and of S94A, I47T and I21V InhA proteins in complex with NADH to resolutions of, respectively, 2.3A, 2.2A, 2.0 A, and 1.9A. The more prominent structural differences are located in, and appear to indirectly affect, the dinucleotide binding loop structure. Moreover, studies on pre-steady-state kinetics of NADH binding have been carried out. The results showed that the limiting rate constant values for NADH dissociation from the InhA-NADH binary complexes (k(off)) were eleven, five, and tenfold higher for, respectively, I21V, I47T, and S94A INH-resistant mutants of InhA as compared to INH-sensitive wild-type InhA. Accordingly, these results are proposed to be able to account for the reduction in affinity for NADH for the INH-resistant InhA enzymes.

PMID: 16647717 DOI: 10.1016/j.jmb.2006.03.055

Vivan AL, Dias MV, Schneider CZ, de Azevedo WF Jr, Basso LA, Santos DS. Crystallization and preliminary X-ray diffraction analysis of prephenate dehydratase from Mycobacterium tuberculosis H37Rv. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006; 62(Pt 4):357–360.

 

Tuberculosis remains the leading cause of mortality arising from a bacterial pathogen (Mycobacterium tuberculosis). There is an urgent need for the development of new antimycobacterial agents. The aromatic amino-acid pathway is essential for the survival of this pathogen and represents a target for structure-based drug design. Accordingly, the M. tuberculosis prephenate dehydratase has been cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 400 as a precipitant. The crystal belongs to the orthorhombic space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 98.26, b = 133.22, c = 225.01 angstroms, and contains four molecules in the asymmetric unit. A complete data set was collected to 3.2 angstroms resolution using a synchrotron-radiation source.

PMID: 16582484 PMCID: PMC2222585 DOI: 10.1107/S1744309106006385

Leopoldino AM, Canduri F, Cabral H, Junqueira M, de Marqui AB, Apponi LH, da Fonseca IO, Domont GB, Santos DS, Valentini S, Bonilla-Rodriguez GO, Fossey MA, de Azevedo WF Jr, Tajara EH. Expression, purification, and circular dichroism analysis of human CDK9. Protein Expr Purif. 2006; 47(2):614–620.

 

The human cyclin-dependent kinase 9 (CDK9) protein was expressed in E. coli BL21 using the pET23a vector at 30 degrees C. Several milligrams of protein were purified from soluble fraction using ionic exchange and ATP-affinity chromatography. The structural quality of recombinant CDK9 and the estimation of its secondary structure were obtained by circular dichroism. Structural models of CDK9 presented 26% of helices in agreement with the spectra by circular dichroism analysis. This is the first report on human CDK9 expression in Escherichia coli and structure analysis and provides the first step for the development of CDK9 inhibitors.

PMID: 16580843 DOI: 10.1016/j.pep.2006.02.012

Cavada BS, Marinho ES, Souza EP, Benevides RG, Delatorre P, Souza LA, Nascimento KS, Sampaio AH, Moreno FB, Rustiguel JK, Canduri F, de Azevedo WF Jr, Debray H. Purification, partial characterization and preliminary X-ray diffraction analysis of a mannose-specific lectin from Cymbosema roseum seeds. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006; 62(Pt 3):235–237.

 

A lectin from Cymbosema roseum seeds (CRL) was purified, characterized and crystallized. The best crystals grew in a month and were obtained by the vapour-diffusion method using a precipitant solution consisting of 0.1 M Tris-HCl pH 7.8, 8%(w/v) PEG 3350 and 0.2 M proline at a constant temperature of 293 K. A data set was collected to 1.77 A resolution at a synchrotron-radiation source. CRL crystals are orthorhombic, belonging to space group P2(1)2(1)2(1). Crystallographic refinement and full amino-acid sequence determination are in progress.

PMID: 16511310 PMCID: PMC2197170 DOI: 10.1107/S174430910600371X

Cavada BS, Castellón RE, Vasconcelos GG, Rocha BA, Bezerra GA, Debray H, Delatorre P, Nagano CS, Toyama M, Pinto VP, Moreno FB, Canduri F, Azevedo WF Jr. Crystallization and preliminary X-ray diffraction analysis of a new chitin-binding protein from Parkia platycephala seeds. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2005; 61(Pt 9):841–843.

 

A chitin-binding protein named PPL-2 was purified from Parkia platycephala seeds and crystallized. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 55.19, b = 59.95, c = 76.60 A, and grew over several days at 293 K using the hanging-drop method. Using synchrotron radiation, a complete structural data set was collected to 1.73 A resolution. The preliminary crystal structure of PPL-2, determined by molecular replacement, presents a correlation coefficient of 0.558 and an R factor of 0.439. Crystallographic refinement is in progress.

PMID: 16511174 PMCID: PMC1978108 DOI: 10.1107/S1744309105024462

Gadelha CA, Moreno FB, Santi-Gadelha T, Cajazeiras JB, Rocha BA, Rustiguel JK, Freitas BT, Canduri F, Delatorre P, Azevedo WF Jr, Cavada BS. Crystallization and preliminary X-ray diffraction analysis of a lectin from Canavalia maritima seeds. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2005; 61(Pt 1):87–89.

 

A lectin from Canavalia maritima seeds (ConM) was purified and submitted to crystallization experiments. The best crystals were obtained using the vapour-diffusion method at a constant temperature of 293 K and grew in 7 d. A complete structural data set was collected to 2.1 A resolution using a synchrotron-radiation source. The ConM crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 67.15, b = 70.90, c = 97.37 A. A molecular-replacement search found a solution with a correlation coefficient of 69.2% and an R factor of 42.5%. Crystallographic refinement is under way.

PMID: 16508099 PMCID: PMC1952371 DOI: 10.1107/S1744309104029197

Dias MV, Borges JC, Ely F, Pereira JH, Canduri F, Ramos CH, Frazzon J, Palma MS, Basso LA, Santos DS, de Azevedo WF Jr. Structure of chorismate synthase from Mycobacterium tuberculosis. J Struct Biol. 2006; 154(2):130–143.

 

In bacteria, fungi, plants, and apicomplexan parasites, the aromatics compounds, such as aromatics amino acids, are synthesized through seven enzymes from the shikimate pathway, which are absent in mammals. The absence of this pathway in mammals make them potential targets for development of new therapy against infectious diseases, such as tuberculosis, which is the world's second commonest cause of death from infectious disease. The last enzyme of shikimate pathway is the chorismate synthase (CS), which is responsible for conversion of the 5-enolpyruvylshikimate-3-phosphate to chorismate. Here, we report the crystallographic structure of CS from Mycobacterium tuberculosis (MtCS) at 2.65 A resolution. The MtCS structure is similar to other CS structures, presenting beta-alpha-beta sandwich structural topology, in which each monomer of MtCS consists of a central helical core. The MtCS can be described as a tetramer formed by a dimer of dimers. However, analytical ultracentrifugation studies suggest the MtCS is a dimer with a more asymmetric shape than observed on the crystallographic dimer and the existence of a low equilibrium between dimer and tetramer. Our results suggest that the MtCS oligomerization is concentration dependent and some conformational changes must be involved on that event.

PMID: 16459102 DOI: 10.1016/j.jsb.2005.12.008

Gadelha CA, Moreno FB, Santi-Gadelha T, Cajazeiras JB, Rocha BA, Assreuy AM, Lima Mota MR, Pinto NV, Passos Meireles AV, Borges JC, Freitas BT, Canduri F, Souza EP, Delatorre P, Criddle DN, de Azevedo WF Jr, Cavada BS. Native crystal structure of a nitric oxide-releasing lectin from the seeds of Canavalia maritima. J Struct Biol. 2005; 152(3):185–194.

 

Here, we report the crystallographic study of a lectin from Canavalia maritima seeds (ConM) and its relaxant activity on vascular smooth muscle, to provide new insights into the understanding of structure/function relationships of this class of proteins. ConM was crystallized and its structure determined by standard molecular replacement techniques. The amino acid residues, previously suggested incorrectly by manual sequencing, have now been determined as I17, I53, S129, S134, G144, S164, P165, S187, V190, S169, T196, and S202. Analysis of the structure indicated a dimer in the asymmetric unit, two metal binding sites per monomer, and loops involved in the molecular oligomerization. These confer 98% similarity between ConM and other previously described lectins, derived from Canavalia ensiformis and Canavalia brasiliensis. Our functional data indicate that ConM exerts a concentration-dependent relaxant action on isolated aortic rings that probably occurs via an interaction with a specific lectin-binding site on the endothelium, resulting in a release of nitric oxide.

PMID: 16337811 DOI: 10.1016/j.jsb.2005.07.012

Basso LA, da Silva LH, Fett-Neto AG, de Azevedo WF Jr, Moreira Ide S, Palma MS, Calixto JB, Astolfi Filho S, dos Santos RR, Soares MB, Santos DS. The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases--a review. Mem Inst Oswaldo Cruz. 2005; 100(6):475–506.

 

The modern approach to the development of new chemical entities against complex diseases, especially the neglected endemic diseases such as tuberculosis and malaria, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a defined target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, (iii) the development even in the initial stages of compounds with selective toxicity (the fundamental principle of chemotherapy), (iv) the evaluation of plant extracts as well as of pure substances. The current use of such technology, unfortunately, is concentrated in developed countries, especially in the big pharma. This fact contributes in a significant way to hamper the development of innovative new compounds to treat neglected diseases. The large biodiversity within the territory of Brazil puts the country in a strategic position to develop the rational and sustained exploration of new metabolites of therapeutic value. The extension of the country covers a wide range of climates, soil types, and altitudes, providing a unique set of selective pressures for the adaptation of plant life in these scenarios. Chemical diversity is also driven by these forces, in an attempt to best fit the plant communities to the particular abiotic stresses, fauna, and microbes that co-exist with them. Certain areas of vegetation (Amazonian Forest, Atlantic Forest, Araucaria Forest, Cerrado-Brazilian Savanna, and Caatinga) are rich in species and types of environments to be used to search for natural compounds active against tuberculosis, malaria, and chronic-degenerative diseases. The present review describes some strategies to search for natural compounds, whose choice can be based on ethnobotanical and chemotaxonomical studies, and screen for their ability to bind to immobilized drug targets and to inhibit their activities. Molecular cloning, gene knockout, protein expression and purification, N-terminal sequencing, and mass spectrometry are the methods of choice to provide homogeneous drug targets for immobilization by optimized chemical reactions. Plant extract preparations, fractionation of promising plant extracts, propagation protocols and definition of in planta studies to maximize product yield of plant species producing active compounds have to be performed to provide a continuing supply of bioactive materials. Chemical characterization of natural compounds, determination of mode of action by kinetics and other spectroscopic methods (MS, X-ray, NMR), as well as in vitro and in vivo biological assays, chemical derivatization, and structure-activity relationships have to be carried out to provide a thorough knowledge on which to base the search for natural compounds or their derivatives with biological activity.

PMID: 16302058 DOI: /S0074-02762005000600001

Fadel V, Bettendorff P, Herrmann T, de Azevedo WF Jr, Oliveira EB, Yamane T, Wüthrich K. Automated NMR structure determination and disulfide bond identification of the myotoxin crotamine from Crotalus durissus terrificus. Toxicon. 2005; 46(7):759–767.

 

Crotamine is one of four major components of the venom of the South American rattlesnake Crotalus durissus terrificus. Similar to its counterparts in the family of the myotoxins, it induces myonecrosis of skeletal muscle cells. This paper describes a new NMR structure determination of crotamine in aqueous solution at pH 5.8 and 20 degrees C, using standard homonuclear 1H NMR spectroscopy at 900MHz and the automated structure calculation software ATNOS/CANDID/DYANA. The automatic NOESY spectral analysis included the identification of a most likely combination of the six cysteines into three disulfide bonds, i.e. Cys4-Cys36, Cys11-Cys30 and Cys18-Cys37; thereby a generally applicable new computational protocol is introduced to determine unknown disulfide bond connectivities in globular proteins. A previous NMR structure determination was thus confirmed and the structure refined. Crotamine contains an alpha-helix with residues 1-7 and a two-stranded anti-parallel beta-sheet with residues 9-13 and 34-38 as the only regular secondary structures. These are connected with each other and the remainder of the polypeptide chain by the three disulfide bonds, which also form part of a central hydrophobic core. A single conformation was observed, with Pro13 and Pro21 in the trans and Pro20 in the cis-form. The global fold and the cysteine-pairing pattern of crotamine are similar to the beta-defensin fold, although the two proteins have low sequence homology, and display different biological activities.

PMID: 16185738 DOI: 10.1016/j.toxicon.2005.07.018

Silva RG, Pereira JH, Canduri F, de Azevedo WF Jr, Basso LA, Santos DS. Kinetics and crystal structure of human purine nucleoside phosphorylase in complex with 7-methyl-6-thio-guanosine. Arch Biochem Biophys. 2005; 442(1):49–58.

 

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and drugs that inhibit this enzyme may have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Here, we describe kinetics and crystal structure of human PNP in complex with 7-methyl-6-thio-guanosine, a synthetic substrate, which is largely used in activity assays. Analysis of the structure identifies different protein conformational changes upon ligand binding, and comparison of kinetic and structural data permits an understanding of the effects of atomic substitution on key positions of the synthetic substrate and their consequences to enzyme binding and catalysis. Such knowledge may be helpful in designing new PNP inhibitors.

PMID: 16154528 DOI: 10.1016/j.abb.2005.07.021

Manhani KK, Arcuri HA, da Silveira NJ, Uchôa HB, de Azevedo WF Jr, Canduri F. Molecular models of protein kinase 6 from Plasmodium falciparum. J Mol Model. 2005; 12(1):42–48.

 

Cyclin-dependent kinases (CDKs) have been identified as potential targets for development of drugs, mainly against cancer. These studies generated a vast library of chemical inhibitors of CDKs, and some of these molecules can also inhibit kinases identified in the Plasmodium falciparum genome. Here we describe structural models for Protein Kinase 6 from P. falciparum (PfPK6) complexed with Roscovitine and Olomoucine. These models show clear structural evidence for differences observed in the inhibition, and may help designing inhibitors for PfPK6 generating new potential drugs against malaria.

PMID: 16096806 DOI: 10.1007/s00894-005-0002-1

Canduri F, Silva RG, dos Santos DM, Palma MS, Basso LA, Santos DS, de Azevedo WF Jr. Structure of human PNP complexed with ligands. Acta Crystallogr D Biol Crystallogr. 2005; 61(Pt 7):856–862.

 

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine-salvage pathway, which allows cells to utilize preformed bases and nucleosides in order to synthesize nucleotides. PNP is specific for purine nucleosides in the beta-configuration and exhibits a strong preference for purines containing a 6-keto group and ribosyl-containing nucleosides relative to the corresponding analogues. PNP was crystallized in complex with ligands and data collection was performed using synchrotron radiation. This work reports the structure of human PNP in complex with guanosine (at 2.80 A resolution), 3'-deoxyguanosine (at 2.86 A resolution) and 8-azaguanine (at 2.85 A resolution). These structures were compared with the PNP-guanine, PNP-inosine and PNP-immucillin-H complexes solved previously.

PMID: 15983407 DOI: 10.1107/S0907444905005421

Silveira NJ, Uchôa HB, Pereira JH, Canduri F, Basso LA, Palma MS, Santos DS, de Azevedo WF Jr. Molecular models of protein targets from Mycobacterium tuberculosis. J Mol Model. 2005; 11(2):160–166.

 

Structural characterization of enzymes that belong to microbial metabolic pathways is very important for structure-based drug design since some of these proteins may be present in the bacterial genome, but absent in humans. Thus, metabolic pathways became potential targets for drug design. The motivation of this work is the fact that Mycobacterium tuberculosis is the cause of the deaths of millions of people in the world, so that the structural characterization of protein targets to propose new drugs has become essential. DBMODELING is a relational database, created to highlight the importance of methods of molecular modeling applied to the Mycobacterium tuberculosis genome with the aim of proposing protein-ligand docking analysis. There are currently more than 300 models for proteins from Mycobacterium tuberculosis genome in the database. The database contains a detailed description of the reaction catalyzed by each enzyme and their atomic coordinates. Information about structures, a tool for animated gif image, a table with a specification of the metabolic pathway, modeled protein, inputs used in modeling, and analysis methods used in this project are available in the database for download. The search tool can be used for researchers to find specific pathways or enzymes.

PMID: 15759144 DOI: 10.1007/s00894-005-0240-2

da Silveira NJ, Arcuri HA, Bonalumi CE, de Souza FP, Mello IM, Rahal P, Pinho JR, de Azevedo WF Jr. Molecular models of NS3 protease variants of the Hepatitis C virus. BMC Struct Biol. 2005; 5:1.

 

BACKGROUND: Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed. RESULTS: The atomic coordinates of crystallographic structure 1CU1 and 1DY9 were used as starting model for modeling of the NS3 protease variant structures. The NS3 protease variant structures are composed of six subdomains, which occur in sequence along the polypeptide chain. The protease domain exhibits the dual beta-barrel fold that is common among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related beta-alpha-beta subdomains and a third subdomain of seven helices and three short beta strands. The latter domain is usually referred to as the helicase alpha-helical subdomain. The rmsd value of bond lengths and bond angles, the average G-factor and Verify 3D values are presented for NS3 protease variant structures. CONCLUSIONS: This project increases the certainty that homology modeling is an useful tool in structural biology and that it can be very valuable in annotating genome sequence information and contributing to structural and functional genomics from virus. The structural models will be used to guide future efforts in the structure-based drug design of a new generation of NS3 protease variants inhibitors. All models in the database are publicly accessible via our interactive website, providing us with large amount of structural models for use in protein-ligand docking analysis.

PMID: 15663787 PMCID: PMC547903 DOI: 10.1186/1472-6807-5-1

Canduri F, Fadel V, Basso LA, Palma MS, Santos DS, de Azevedo WF Jr. New catalytic mechanism for human purine nucleoside phosphorylase. Biochem Biophys Res Commun. 2005; 327(3):646–649.

 

Human purine nucleoside phosphorylase has been submitted to intensive structure-based design of inhibitors, most of them using low-resolution structures of human PNP. Recently, several structures of human PNP have been reported, which allowed redefinition of the active site and understanding of the structural basis for inhibition of PNP by acyclovir and immucillin-H. Based on previously solved human PNP structures, we proposed here a new catalytic mechanism for human PNP, which is supported by crystallographic studies and explains previously determined kinetic data.

PMID: 15649395 DOI: 10.1016/j.bbrc.2004.12.052

Canduri F, Fadel V, Dias MV, Basso LA, Palma MS, Santos DS, de Azevedo WF Jr. Crystal structure of human PNP complexed with hypoxanthine and sulfate ion. Biochem Biophys Res Commun. 2005; 326(2):335–338.

 

Purine nucleoside phosphorylase (PNP) is a ubiquitous enzyme, which plays a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effects on B-cell function. Human PNP has been submitted to intensive structure-based design of inhibitors, most of them using low-resolution structures of human PNP. Here we report the crystal structure of human PNP in complex with hypoxanthine, refined to 2.6A resolution. The intermolecular interaction between ligand and PNP is discussed.

PMID: 15582582 DOI: 10.1016/j.bbrc.2004.11.038

Canduri F, de Azevedo Jr. WF. Structural Basis for Interaction of Inhibitors with Cyclin-Dependent Kinase 2. Curr Comput Aided Drug Des, 2005; 1(1): 53–64.

 

Cell cycle progression is tightly controlled by the activity of cyclin-dependent kinases (CDKs). CDKs are inactive as monomers, and activation requires binding to cyclins, a diverse family of proteins whose levels oscillate during the cell cycle, and phosphorylation by CDK-activating kinase (CAK) on a specific threonine residue. The central role of CDKs in cell cycle regulation makes them a promising target for studying inhibitory molecules that can modify the degree of cell proliferation, the discovery of specific inhibitors of CDKs such as polyhydroxylated flavones has opened the way to investigation and design of antimitotic compounds. A chlorinated form, flavopiridol, is currently in phase II clinical trials as a drug against breast tumors. The aromatic portion of the inhibitor binds to the adenine-binding pocket of CDK2, and the position of the phenyl group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP complex structure, the analysis of the position of this phenyl ring not only explains the great differences of kinase inhibition among the flavonoid inhibitors but also explains the specificity of roscovitine and olomoucine to inhibit CDK2. There is strong interest in CDKs inhibitors that could play an important role in the discovery of a new family of antitumor agents. The crystallographic analysis together with bioinformatics studies of CDKs are generating new information about the structural basis for inhibition of CDKs. The relevant structural features that may guide the structure based-design of a new generation of CDK inhibitors are discussed in this review.

Keywords: cdk2, drug design, flavopiridol, roscovitine, inhibitor, indirubin

Pereira JH, de Oliveira JS, Canduri F, Dias MV, Palma MS, Basso LA, Santos DS, de Azevedo WF Jr. Structure of shikimate kinase from Mycobacterium tuberculosis reveals the binding of shikimic acid. Acta Crystallogr D Biol Crystallogr. 2004; 60(Pt 12 Pt 2): 2310–2319.

 

Tuberculosis made a resurgence in the mid-1980s and now kills approximately 3 million people a year. The re-emergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons and the proliferation of multi-drug-resistant strains have created a need to develop new drugs. Shikimate kinase and other enzymes in the shikimate pathway are attractive targets for development of non-toxic antimicrobial agents, herbicides and anti-parasitic drugs, because the pathway is essential in these species whereas it is absent from mammals. The crystal structure of shikimate kinase from Mycobacterium tuberculosis (MtSK) complexed with MgADP and shikimic acid (shikimate) has been determined at 2.3 A resolution, clearly revealing the amino-acid residues involved in shikimate binding. This is the first three-dimensional structure of shikimate kinase complexed with shikimate. In MtSK, the Glu61 residue that is strictly conserved in shikimate kinases forms a hydrogen bond and salt bridge with Arg58 and assists in positioning the guanidinium group of Arg58 for shikimate binding. The carboxyl group of shikimate interacts with Arg58, Gly81 and Arg136 and the hydroxyl groups interact with Asp34 and Gly80. The crystal structure of MtSK-MgADP-shikimate will provide crucial information for the elucidation of the mechanism of the shikimate kinase-catalyzed reaction and for the development of a new generation of drugs against tuberculosis.

PMID: 15583379 DOI: 10.1107/S090744490402517X

Ribeiro SP, Mendes MA, Dos Santos LD, de Souza BM, Marques MR, de Azevedo WF Jr, Palma MS. Structural and functional characterization of N-terminally blocked peptides isolated from the venom of the social wasp Polybia paulista. Peptides. 2004; 25(12): 2069–2078.

 

Two novel peptides were isolated from the crude venom of the social wasp Polybia paulista, by using RP-HPLC under a gradient of MeCN from 5 to 60% (v/v) and named Polybine-I and -II. Further purification of these peptides under normal phase chromatography, rendered pure enough preparations to be sequenced by Edman degradation chemistry. However, both peptides did not interact with phenylisothiocyanate reagent, suggesting the existence of a chemically blocked N-terminus. Therefore, the sequences of both peptides were assigned by ESI-MS/MS under CID conditions, as follows: Polybine-I Ac-SADLVKKIWDNPAL-NH2 (Mr 1610 Da) and Polybine-II Ac-SVDMVMKGLKIWPL-NH2 (Mr 1657 Da). During the tandem mass spectrometry experiments, a loss of 43 a.m.u. was observed from the N-terminal residue of each peptide, suggesting the acetylation of the N-terminus. Subsequently, the peptides with and without acetylation were synthesized on solid phase and submitted to functional characterizations; the biological activities investigated were: hemolysis, chemotaxis of polymorphonucleated leukocytes (PMNL), mast cell degranulation and antibiosis. The results revealed that the acetylated peptides exhibited more pronounced chemotaxis of PMNL cells and mast cell degranulation than the respective non-acetylated congeners; no hemolytic and antibiotic activities were observed, irrespective to the blockage or not of the alpha-amino groups of the N-terminal residues of each peptide. Therefore, the N-terminal acetylation may be related to the increase of the inflammatory activity of both peptides.

PMID: 15572194 DOI: 10.1016/j.peptides.2004.08.019

Uchôa HB, Jorge GE, Freitas Da Silveira NJ, Camera JC Jr, Canduri F, De Azevedo WF Jr. Parmodel: a web server for automated comparative modeling of proteins. Biochem Biophys Res Commun. 2004; 325(4): 1481–1486.

 

Parmodel is a web server for automated comparative modeling and evaluation of protein structures. The aim of this tool is to help inexperienced users to perform modeling, assessment, visualization, and optimization of protein models as well as crystallographers to evaluate structures solved experimentally. It is subdivided in four modules: Parmodel Modeling, Parmodel Assessment, Parmodel Visualization, and Parmodel Optimization. The main module is the Parmodel Modeling that allows the building of several models for a same protein in a reduced time, through the distribution of modeling processes on a Beowulf cluster. Parmodel automates and integrates the main softwares used in comparative modeling as MODELLER, Whatcheck, Procheck, Raster3D, Molscript, and Gromacs. This web server is freely accessible at .

PMID: 15555595 DOI: 10.1016/j.bbrc.2004.10.192

Peres P, Lombardi FR, Dos Santos GC, Olivieri JR, Canduri F, Bonilla-Rodriguez GO, de Azevedo WF Jr. Molecular modeling and small angle X-ray scattering studies of Hoplosternum littorale cathodic haemoglobin. Biochem Biophys Res Commun. 2004; 325(2): 487–493.

 

Considerable interest is currently focused on fish haemoglobins in order to identify the structural basis for their diversity of functional behavior. Hoplosternum littorale is a catfish that presents bimodal gill (water)/gut (air)-breathing, which allows this species to survive in waters with low oxygen content. The hemolysate of this fish showed the presence of two main haemoglobins, cathodic and anodic. This work describes structural features analyzed here by integration of molecular modeling with small angle X-ray scattering. Here is described a molecular model for the cathodic haemoglobin in the unliganded and liganded states. The models were determined by molecular modeling based on the high-resolution crystal structure of fish haemoglobins. The structural models for both forms of H. littorale haemoglobin were compared to human haemoglobin.

PMID: 15530418 DOI: 10.1016/j.bbrc.2004.10.061

Peres P, de Azevedo Júnior WF, Bonilla-Rodriguez GO. Allosteric water and phosphate effects in Hoplosternum littorale hemoglobins. Eur J Biochem. 2004; 271(21): 4270–4274.

 

This paper reports the results obtained using the osmotic stress method applied to the purified cathodic and anodic hemoglobins (Hbs) from the catfish Hoplosternum littorale, a species that displays facultative accessorial air oxygenation. We demonstrate that water potential affects the oxygen affinity of H. littorale Hbs in the presence of an inert solute (sucrose). Oxygen affinity increases when water activity increases, indicating that water molecules stabilize the high-affinity state of the Hb. This effect is the same as that observed in tetrameric vertebrate Hbs. We show that both anodic and cathodic Hbs show conformational substrates similar to other vertebrate Hbs. For both Hbs, addition of anionic effectors, especially chloride, strongly increases the number of water molecules bound, although anodic Hb did not exhibit sensitivity to saturating levels of ATP. Accordingly, for both Hbs, we propose that the deoxy conformations coexist in at least two anion-dependent allosteric states, T(o) and T(x), as occurs for human Hb. We found a single phosphate binding site for the cathodic Hb.

PMID: 15511232 DOI: 10.1111/j.1432-1033.2004.04366.x

Dias MV, Ely F, Canduri F, Pereira JH, Frazzon J, Basso LA, Palma MS, de Azevedo WF Jr, Santos DS. Crystallization and preliminary X-ray crystallographic analysis of chorismate synthase from Mycobacterium tuberculosis. Acta Crystallogr D Biol Crystallogr. 2004; 60(Pt 11): 2003–2005.

 

The enzymes of the shikimate pathway are potential targets for the development of new therapies because they are essential for bacteria but absent from mammals. The last step in this pathway is performed by chorismate synthase (CS), which catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. Optimization of crystallization trials allowed the crystallization of homogeneous recombinant CS from Mycobacterium tuberculosis (MtCS). The crystals of MtCS belong to space group P6(4)22 (or P6(2)22) and diffract to 2.8 A resolution, with unit-cell parameters a = b = 129.7, c = 156.8 A. There are two molecules in the asymmetric unit. Molecular-replacement trials were not successful. Heavy-atom derivative screening is in progress.

PMID: 15502309 DOI: 10.1107/S0907444904019869

Nolasco DO, Canduri F, Pereira JH, Cortinóz JR, Palma MS, Oliveira JS, Basso LA, de Azevedo WF Jr, Santos DS. Crystallographic structure of PNP from Mycobacterium tuberculosis at 1.9A resolution. Biochem Biophys Res Commun. 2004; 324(2): 789–794.

 

Even being a bacterial purine nucleoside phosphorylase (PNP), which normally shows hexameric folding, the Mycobacterium tuberculosis PNP (MtPNP) resembles the mammalian trimeric structure. The crystal structure of the MtPNP apoenzyme was solved at 1.9 A resolution. The present work describes the first structure of MtPNP in complex with phosphate. In order to develop new insights into the rational drug design, conformational changes were profoundly analyzed and discussed. Comparisons over the binding sites were specially studied to improve the discussion about the selectivity of potential new drugs.

PMID: 15474496 DOI: 10.1016/j.bbrc.2004.09.137

Canduri F, Uchoa HB, de Azevedo WF Jr. Molecular models of cyclin-dependent kinase 1 complexed with inhibitors. Biochem Biophys Res Commun. 2004; 324(2): 661–666.

 

Roscovitine and flavopiridol have been shown to potently inhibit cyclin-dependent kinase 1 and 2 (CDK1 and 2). The structures of CDK2 complexed with roscovitine and deschoroflavopiridol have been reported, however no crystallographic structure is available for complexes of CDK1 with inhibitors. The present work describes two molecular models for the binary complexes CDK1:roscovitine and CDK1:flavopiridol. These structural models indicate that both inhibitors strongly bind to the ATP-binding pocket of CDK1 and structural comparison of the CDK complexes correlates the structures with differences in inhibition of these CDKs by flavopiridol and roscovitine. This article explains the structural basis for the observed differences in activity of these inhibitors.

PMID: 15474478 DOI: 10.1016/j.bbrc.2004.09.109

dos Santos Cabrera MP, de Souza BM, Fontana R, Konno K, Palma MS, de Azevedo WF Jr, Neto JR. Conformation and lytic activity of eumenine mastoparan: a new antimicrobial peptide from wasp venom. J Pept Res. 2004; 64(3): 95–103.

 

Eumenine mastoparan-AF (EMP-AF) is a novel membrane active tetradecapeptide recently isolated from the venom of solitary wasp, Anterhynchium flavomarginatum micado. It was reported previously that EMP-AF peptide presented low cytolytic activities in human erythrocytes and in RBL-2H3 mast cells. In the present work, we observed that this peptide is able to permeate anionic liposomes, and in less extension also the neutral ones. We present evidences showing that the permeation ability is well correlated with the amount of helical conformation assumed by the peptides in these environments. This peptide also showed a broad-spectrum inhibitory activity against Gram-positive and Gram-negative bacteria. The permeability of liposomes and the antibiotic effect showed a significant reduction when C-terminus was deamidated (in acidic form). The removal of the three first amino acid residues from the N-terminus rendered the peptide inactive both in liposomes and in bacteria. The results suggest that the mechanism of action involves a threshold in the accumulation of the peptide at level of cell membrane.

PMID: 15317499 DOI: 10.1111/j.1399-3011.2004.00173.x

Canduri F, dos Santos DM, Silva RG, Mendes MA, Basso LA, Palma MS, de Azevedo WF, Santos DS. Structures of human purine nucleoside phosphorylase complexed with inosine and ddI. Biochem Biophys Res Commun. 2004; 313(4): 907–914.

 

Human purine nucleoside phosphorylase (PNP) is a ubiquitous enzyme which plays a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effect on B-cell function. PNP is highly specific for 6-oxopurine nucleosides and exhibits negligible activity for 6-aminopurine nucleosides. The catalytic efficiency for inosine is 350,000-fold greater than for adenosine. Adenine nucleosides and nucleotides are deaminated by adenosine deaminase and AMP deaminase to their corresponding inosine derivatives which, in turn, may be further degraded. Here we report the crystal structures of human PNP in complex with inosine and 2('),3(')-dideoxyinosine, refined to 2.8A resolution using synchrotron radiation. The present structures provide explanation for ligand binding, refine the purine-binding site, and can be used for future inhibitor design.

PMID: 14706628

Sforça ML, Oyama S Jr, Canduri F, Lorenzi CC, Pertinhez TA, Konno K, Souza BM, Palma MS, Ruggiero Neto J, Azevedo WF Jr, Spisni A. How C-terminal carboxyamidation alters the biological activity of peptides from the venom of the eumenine solitary wasp. Biochemistry. 2004; 43(19): 5608–5617.

 

Inflammatory peptides display different types of post-transcriptional modifications, such as C-terminal amidation, that alter their biological activity. Here we describe the structural and molecular dynamics features of the mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF-NH(2)), found in the venom of the solitary wasp, and of its carboxyl-free C-terminal form (EMP-AF-COO(-)) characterized by a reduced activity. Circular dichroism indicates that both peptides switch from a random coil conformation in water to a helical structure in TFE and SDS micelles. NMR data, in 30% TFE, reveal that the two peptides fold into an alpha-helix spanning most of their length, while they differ in terms of molecular rigidity. To understand the origins of the conformational flexibility observed in the case of EMP-AF-COO(-), a 5 ns MD simulation was carried out for each peptide, in an explicit water/TFE environment. The results show that the two peptides differ in an H-bond between Leu14 NH(2) and the backbone carbonyl of Ile11. The loss of that H-bond in EMP-AF-COO(-) leads to a significant modification of its structural dynamics. In fact, as evidenced by essential dynamics analysis, while EMP-AF-NH(2) exists mainly as a rigid structure, EMP-AF-COO(-) presents two helical stretches that fluctuate in some sort of independent fashion. We conclude that the diverse biological activity of the two peptides is not simply due to the reduction of the net positive charge, as generally suggested, but also to a structural perturbation of the amphipathic alpha-helix that affects their ability to perturb the cell membrane.

PMID: 15134435 DOI: 10.1021/bi0360915

Arcuri HA, Canduri F, Pereira JH, da Silveira NJ, Camera Júnior JC, de Oliveira JS, Basso LA, Palma MS, Santos DS, de Azevedo Júnior WF. Molecular models for shikimate pathway enzymes of Xylella fastidiosa. Biochem Biophys Res Commun. 2004; 320(3): 979–991.

 

The Xylella fastidiosa is a bacterium that is the cause of citrus variegated chlorosis (CVC). The shikimate pathway is of pivotal importance for production of a plethora of aromatic compounds in plants, bacteria, and fungi. Putative structural differences in the enzymes from the shikimate pathway, between the proteins of bacterial origin and those of plants, could be used for the development of a drug for the control of CVC. However, inhibitors for shikimate pathway enzymes should have high specificity for X. fastidiosa enzymes, since they are also present in plants. In order to pave the way for structural and functional efforts towards antimicrobial agent development, here we describe the molecular modeling of seven enzymes of the shikimate pathway of X. fastidiosa. The structural models of shikimate pathway enzymes, complexed with inhibitors, strongly indicate that the previously identified inhibitors may also inhibit the X. fastidiosa enzymes.

PMID: 15240145 DOI: 10.1016/j.bbrc.2004.05.220

Moreno FB, Delatorre P, Freitas BT, Rocha BA, Souza EP, Facó F, Canduri F, Cardoso AL, Freire VN, Lima Filho JL, Sampaio AH, Calvete JJ, De Azevedo WF Jr, Cavada BS. Crystallization and preliminary X-ray diffraction analysis of the lectin from Canavalia gladiata seeds. Acta Crystallogr D Biol Crystallogr. 2004; 60(Pt 8): 1493–1495.

 

The seed lectin from Canavalia gladiata was purified and crystallized. Orthorhombic crystals belonging to space group C222(1) grew within three weeks at 293 K using the hanging-drop vapour-diffusion method. Using synchrotron X-ray radiation, a complete structural data set was collected at 2.3 A resolution. The preliminary crystal structure of the lectin, determined by molecular replacement, had a correlation coefficient of 0.569 and an R factor of 0.412.

PMID: 15272187 DOI: 10.1107/S0907444904014489

da Silveira NJ, Uchôa HB, Canduri F, Pereira JH, Camera JC Jr, Basso LA, Palma MS, Santos DS, de Azevedo WF Jr. Structural bioinformatics study of PNP from Schistosoma mansoni. Biochem Biophys Res Commun. 2004; 322(1): 100–104.

 

The parasite Schistosoma mansoni lacks the de novo pathway for purine biosynthesis and depends on salvage pathways for its purine requirements. Schistosomiasis is endemic in 76 countries and territories and amongst the parasitic diseases ranks second after malaria in terms of social and economic impact and public health importance. The PNP is an attractive target for drug design and it has been submitted to extensive structure-based design. The atomic coordinates of the complex of human PNP with inosine were used as template for starting the modeling of PNP from S. mansoni complexed with inosine. Here we describe the model for the complex SmPNP-inosine and correlate the structure with differences in the affinity for inosine presented by human and S. mansoni PNPs.

PMID: 15313179 DOI: 10.1016/j.bbrc.2004.07.088

de Azevedo WF Jr, Canduri F, dos Santos DM, Pereira JH, Bertacine Dias MV, Silva RG, Mendes MA, Basso LA, Palma MS, Santos DS. Crystal structure of human PNP complexed with guanine. Biochem Biophys Res Commun. 2003; 312(3): 767–772.

 

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. PNP is a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. More recently, the 3-D structure of human PNP has been refined to 2.3A resolution, which allowed a redefinition of the residues involved in the substrate-binding sites and provided a more reliable model for structure-based design of inhibitors. This work reports crystallographic study of the complex of Human PNP:guanine (HsPNP:Gua) solved at 2.7A resolution using synchrotron radiation. Analysis of the structural differences among the HsPNP:Gua complex, PNP apoenzyme, and HsPNP:immucillin-H provides explanation for inhibitor binding, refines the purine-binding site, and can be used for future inhibitor design.

PMID: 14680831 DOI: 10.1016/j.bbrc.2003.10.190

Pereira JH, Canduri F, de Oliveira JS, da Silveira NJ, Basso LA, Palma MS, de Azevedo WF Jr, Santos DS. Structural bioinformatics study of EPSP synthase from Mycobacterium tuberculosis. Biochem Biophys Res Commun. 2003; 312(3): 608–614.

 

The shikimate pathway is an attractive target for herbicides and antimicrobial agent development because it is essential in algae, higher plants, bacteria, and fungi, but absent from mammals. Homologues to enzymes in the shikimate pathway have been identified in the genome sequence of Mycobacterium tuberculosis. Among them, the EPSP synthase was proposed to be present by sequence homology. Accordingly, in order to pave the way for structural and functional efforts towards anti-mycobacterial agent development, here we describe the molecular modeling of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase isolated from M. tuberculosis that should provide a structural framework on which the design of specific inhibitors may be based on. Significant differences in the relative orientation of the domains in the two models result in "open" and "closed" conformations. The possible relevance of this structural transition in the ligand biding is discussed.

PMID: 14680808 DOI: 10.1016/j.bbrc.2003.10.175

Fadel V, Canduri F, Olivieri JR, Smarra AL, Colombo MF, Bonilla-Rodriguez GO, de Azevedo WF Jr. Crystal structure of hemoglobin from the maned wolf (Chrysocyon brachyurus) using synchrotron radiation. Protein Pept Lett. 2003; 10(6):551–559.

 

Crystal structure of hemoglobin isolated from the Brazilian maned wolf (Chrysocyon brachyurus) was determined using standard molecular replacement technique and refined using maximum-likelihood and simulated annealing protocols to 1.87A resolution. Structural and functional comparisons between hemoglobins from the Chrysocyon brachyurus and Homo sapiens are discussed, in order to provide further insights in the comparative biochemistry of vertebrate hemoglobins.

PMID: 14683506

Filgueira de Azevedo W Jr, dos Santos GC, dos Santos DM, Olivieri JR, Canduri F, Silva RG, Basso LA, Renard G, da Fonseca IO, Mendes MA, Palma MS, Santos DS. Docking and small angle X-ray scattering studies of purine nucleoside phosphorylase. Biochem Biophys Res Commun. 2003; 309(4): 923–928.

 

Docking simulations have been used to assess protein complexes with some success. Small angle X-ray scattering (SAXS) is a well-established technique to investigate protein spatial configuration. This work describes the integration of geometric docking with SAXS to investigate the quaternary structure of recombinant human purine nucleoside phosphorylase (PNP). This enzyme catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for PNP causes gradual decrease in T-cell immunity. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant rejection, rheumatoid arthritis, lupus, and T-cell lymphomas. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. The present analysis confirms the trimeric structure observed in the crystal. The potential application of the present procedure to other systems is discussed.

PMID: 13679062

Filgueira de Azevedo W Jr, Canduri F, Marangoni dos Santos D, Pereira JH, Dias MV, Silva RG, Mendes MA, Basso LA, Palma MS, Santos DS. Structural basis for inhibition of human PNP by immucillin-H. Biochem Biophys Res Commun. 2003; 309(4): 917–922.

 

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. PNP is a target for inhibitor development aiming at T-cell immune response modulation. This work reports on the crystallographic study of the complex of human PNP-immucillin-H (HsPNP-ImmH) solved at 2.6A resolution using synchrotron radiation. Immucillin-H (ImmH) inhibits the growth of malignant T-cell lines in the presence of deoxyguanosine without affecting non-T-cell tumor lines. ImmH inhibits activated normal human T cells after antigenic stimulation in vitro. These biological effects of ImmH suggest that this agent may have utility in the treatment of certain human diseases characterized by abnormal T-cell growth or activation. This is the first structural report of human PNP complexed with immucillin-H. The comparison of the complex HsPNP-ImmH with recent crystallographic structures of human PNP explains the high specificity of immucillin-H for human PNP.

PMID: 13679061

dos Santos DM, Canduri F, Pereira JH, Vinicius Bertacine Dias M, Silva RG, Mendes MA, Palma MS, Basso LA, de Azevedo WF Jr, Santos DS. Crystal structure of human purine nucleoside phosphorylase complexed with acyclovir. Biochem Biophys Res Commun. 2003; 308(3): 553–559.

 

In human, purine nucleoside phosphorylase (HsPNP) is responsible for degradation of deoxyguanosine and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. This work reports the first crystallographic study of human PNP complexed with acyclovir (HsPNP:Acy). Acyclovir is a potent clinically useful inhibitor of replicant herpes simplex virus that also inhibits human PNP but with a relatively lower inhibitory activity (K(i)=90 microM). Analysis of the structural differences among the HsPNP:Acy complex, PNP apoenzyme, and HsPNP:Immucillin-H provides explanation for inhibitor binding, refines the purine-binding site, and can be used for future inhibitor design.

PMID: 12914786

de Azevedo WF Jr, Canduri F, dos Santos DM, Silva RG, de Oliveira JS, de Carvalho LP, Basso LA, Mendes MA, Palma MS, Santos DS. Crystal structure of human purine nucleoside phosphorylase at 2.3A resolution. Biochem Biophys Res Commun. 2003; 308(3): 545–552.

 

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. In human, PNP is the only route for degradation of deoxyguanosine and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and its low resolution structure has been used for drug design. Here we report the structure of human PNP solved to 2.3A resolution using synchrotron radiation and cryocrystallographic techniques. This structure allowed a more precise analysis of the active site, generating a more reliable model for substrate binding. The higher resolution data allowed the identification of water molecules in the active site, which suggests binding partners for potential ligands. Furthermore, the present structure may be used in the new structure-based design of PNP inhibitors.

PMID: 12914785

Fonseca JC, Honda RT, Delatorre P, Fadel V, Bonilla-Rodriguez GO, de Azevedo WF Jr. Crystallization, preliminary X-ray analysis and molecular-replacement solution of haemoglobin-II from the fish matrinxã (Brycon cephalus). Acta Crystallogr D Biol Crystallogr. 2003; 59(Pt 4): 752–754.

 

Haemoglobins constitute a set of proteins with interesting structural and functional properties, especially when the two large animal groups reptiles and fishes are focused on. Here, the crystallization and preliminary X-ray analysis of haemoglobin-II from the South American fish matrinxã (Brycon cephalus) is reported. X-ray diffraction data have been collected to 3.0 A resolution using synchrotron radiation (LNLS). Crystals were determined to belong to space group P2(1) and preliminary structural analysis revealed the presence of two tetramers in the asymmetric unit. The structure was determined using the standard molecular-replacement technique.

PMID: 12657802

Filgueira de Azevedo W Jr, Gaspar RT, Canduri F, Camera JC Jr, Freitas da Silveira NJ. Molecular model of cyclin-dependent kinase 5 complexed with roscovitine. Biochem Biophys Res Commun. 2002; 297(5): 1154–1158.

 

Here is described a structural model for the binary complex CDK5-roscovitine. Roscovitine has been shown to potently inhibit cyclin-dependent kinases 1, 2 and 5 (CDK1, 2, and 5), and the structure of CDK2 complexed with roscovitine has been reported; however, no structural data are available for complexes of CDK5 with inhibitors. The structural model indicates that roscovitine strongly binds to the ATP-binding pocket of CDK5 and structural comparison of the CDK2-roscovitine complex correlates the structural differences with differences in inhibition of these CDKs by this inhibitor. This structure opens the possibility of testing new inhibitor families, in addition to new substituents for the already known lead structures of adenine derivatives.

PMID: 12372407

Filgueira de Azevedo W Jr, Canduri F, Simões de Oliveira J, Basso LA, Palma MS, Pereira JH, Santos DS. Molecular model of shikimate kinase from Mycobacterium tuberculosis. Biochem Biophys Res Commun. 2002; 295(1): 142–148.

 

Tuberculosis (TB) resurged in the late 1980s and now kills approximately 3 million people a year. The reemergence of tuberculosis as a public health threat has created a need to develop new anti-mycobacterial agents. The shikimate pathway is an attractive target for herbicides and anti-microbial agents development because it is essential in algae, higher plants, bacteria, and fungi, but absent from mammals. Homologs to enzymes in the shikimate pathway have been identified in the genome sequence of Mycobacterium tuberculosis. Among them, the shikimate kinase I encoding gene (aroK) was proposed to be present by sequence homology. Accordingly, to pave the way for structural and functional efforts towards anti-mycobacterial agents development, here we describe the molecular modeling of M. tuberculosis shikimate kinase that should provide a structural framework on which the design of specific inhibitors may be based.

(c) 2002 Elsevier Science (USA).

PMID: 12083781 DOI: 10.1016/S0006-291X(02)00632-0

de Sousa GF, Filgueiras CAL,Mascarenhas YP, de Azevedo Jr. WF. Synthesis, spectroscopic characterization and X-ray crystallographic studies of trans-[PtCl2(PEt3)(PySOR)] complexes, where PySOR = (2-methylsulphinyl)-pyridine and (2-n-propylsulphinyl)pyridine. Transition Met Chem. 2002; 27(4): 433–436

 

Alkylsulphinylpyridine ligands containing three potential donor centres: N, S and O atoms and two complexes of general formula trans-[PtCl2(PEt3)PySOR)] (R = Me and n Pr) were prepared and characterized by elemental analysis, i.r. spectroscopy, 1H- and 31P-n.m.r. and X-ray crystallography. The ambidentate ligands act in both situations as monodentate ligands, bonded to the metal exclusively through the nitrogen atom. The crystal structures revealed the occurrence of discrete molecules and, in both complexes, the Pt atoms are coordinated in square planar arrangements by two chloride ions, in a trans configuration, by the pyridine nitrogen atom, and by the phosphine P atom. The oxygen atoms do not take part in the complexation scheme.

Keywords: Pyridine Oxygen Atom Nitrogen Atom Phosphine Donor Centre

de Azevedo WF Jr, Canduri F, da Silveira NJ. Structural basis for inhibition of cyclin-dependent kinase 9 by flavopiridol. Biochem Biophys Res Commun. 2002; 293(1): 566–571.

 

Flavopiridol has been shown to potently inhibit CDK1 and 2 (cyclin-dependent kinases 1 and 2) and most recently it has been found that it also inhibits CDK9. The complex CDK9-cyclin T1 controls the elongation phase of transcription by RNA polymerase II. The present work describes a molecular model for the binary complex CDK9-flavopiridol. This structural model indicates that the inhibitor strongly binds to the ATP-binding pocket of CDK9 and the structural comparison of the complex CDK2-flavopiridol correlates the structural differences with differences in inhibition of these CDKs by flavopiridol. This structure opens the possibility of testing new inhibitor families, in addition to new substituents for the already known leading structures such as flavones and adenine derivatives.

PMID: 12054639 DOI: 10.1016/S0006-291X(02)00266-8

Konno K, Hisada M, Fontana R, Lorenzi CC, Naoki H, Itagaki Y, Miwa A, Kawai N, Nakata Y, Yasuhara T, Ruggiero Neto J, de Azevedo WF Jr, Palma MS, Nakajima T. Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis. Biochim Biophys Acta. 2001; 1550(1): 70–80.

 

A novel antimicrobial peptide, anoplin, was purified from the venom of the solitary wasp Anoplius samariensis. The sequence was mostly analyzed by mass spectrometry, which was corroborated by solid-phase synthesis. Anoplin, composed of 10 amino acid residues, Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-NH2, has a high homology to crabrolin and mastoparan-X, the mast cell degranulating peptides from social wasp venoms, and, therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the circular dichroism (CD) spectra of anoplin in the presence of trifluoroethanol or sodium dodecyl sulfate showed a high content, up to 55%, of the alpha-helical conformation. A modeling study of anoplin based on its homology to mastoparan-X supported the CD results. Biological evaluation using the synthetic peptide revealed that this peptide exhibited potent activity in stimulating degranulation from rat peritoneal mast cells and broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. Therefore, this is the first antimicrobial component to be found in the solitary wasp venom and it may play a key role in preventing potential infection by microorganisms during prey consumption by their larvae. Moreover, this peptide is the smallest among the linear alpha-helical antimicrobial peptides hitherto found in nature, which is advantageous for chemical manipulation and medical application.

PMID: 11738089

Canduri F, Teodoro LG, Fadel V, Lorenzi CC, Hial V, Gomes RA, Neto JR, de Azevedo WF Jr. Structure of human uropepsin at 2.45 A resolution. Acta Crystallogr D Biol Crystallogr. 2001; 57(Pt 11): 1560–1570.

 

The molecular structure of human uropepsin, an aspartic proteinase from the urine produced in the form of pepsinogen A in the gastric mucosa, has been determined by molecular replacement using human pepsin as the search model. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.99, b = 75.56, c = 89.90 A. Crystallographic refinement led to an R factor of 0.161 at 2.45 A resolution. The positions of 2437 non-H protein atoms in 326 residues have been determined and the model contains 143 water molecules. The structure is bilobal, consisting of two predominantly beta-sheet lobes which, as observed in other aspartic proteinases, are related by a pseudo-twofold axis. A model of the uropepsin-pepstatin complex has been constructed based on the high-resolution crystal structure of pepsin complexed with pepstatin.

PMID: 11679720

de Azevedo WF Jr, Canduri F, Fadel V, Teodoro LG, Hial V, Gomes RA. Molecular model for the binary complex of uropepsin and pepstatin. Biochem Biophys Res Commun. 2001; 287(1): 277–281.

 

The three-dimensional structure of human uropepsin complexed with pepstatin has been modelled using human pepsin as a template. Uropepsin is an aspartic proteinase from the urine, produced in the form of pepsinogen A in the gastric mucosa. The structure is bilobal, consisting of two predominantly beta-sheet lobes which, as observed in other aspartic proteinases, are related by a pseudo twofold axis. A structural comparison between binary complexes of pepsin:pepstatin and uropepsin:pepstatin is discussed.

Copyright 2001 Academic Press.

PMID: 11549287 DOI: 10.1006/bbrc.2001.5555

Delatorre P, Smarra AL, Fadel V, Canduri F, Dellamano M, Bonilla-Rodriguez GO, de Azevedo WF Jr. Purification, crystallization and Patterson search of haemoglobin IV from the armoured catfish Liposarcus anisitsi. Acta Crystallogr D Biol Crystallogr. 2001; 57(Pt 9): 1329–1331.

 

Considerable interest is currently focused on fish haemoglobins in order to identify the structural basis for their diversity of functional behaviour. The armored catfish Liposarcus anisitsi presents accessorial air breathing through a modified stomach, which allows this species to survive in waters with low oxygen content. The analysis of its haemolysate has shown the presence of four main haemoglobins, with this work focusing on haemoglobin IV (LaHb-IV). LaHb-IV was crystallized and X-ray diffraction data were collected to 2.4 A resolution using a synchrotron-radiation source. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 52.6 (1), b = 104.8 (2), c = 113.9 (2) A; preliminary structural analysis revealed the presence of one tetramer in the asymmetric unit. The structure was determined using the standard molecular-replacement technique.

PMID: 11526335

Delatorre P, Olivieri JR, Ruggiero Neto J, Lorenzi CC, Canduri F, Fadel V, Konno K, Palma MS, Yamane T, de Azevedo WF Jr. Preliminary cryocrystallography analysis of an eumenine mastoparan toxin isolated from the venom of the wasp Anterhynchium flavomarginatum micado. Biochim Biophys Acta. 2001; 1545(1-2): 372–376.

 

Mastoparans are tetradecapeptides found to be the major component of vespid venoms. These peptides present a wide spectrum of biological activities, such as mast cell degranulation, hemolytic activity and also reveals antimicrobial activity. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized. At room temperature these crystals diffracted to 2.8 A resolution. However, upon cooling to cryogenic temperature around 85 K, the original resolution limit could be improved to 2.0 A. Crystals were determined to belong to the space group P3(1) (P3(2)). This is the first mastoparan to be crystallized and it will provide further insights in the conformational significance of mastoparan toxins, with respect to their potency and activity in G protein regulation.

PMID: 11342062

Honda RT, Delatorre P, Fadel V, Canduri F, Dellamano M, de Azevedo WF Jr, Bonilla-Rodriguez GO. Crystallization, preliminary X-ray analysis and molecular-replacement solution of the carboxy form of haemoglobin I from the fish Brycon cephalus. Acta Crystallogr D Biol Crystallogr. 2000; 56(Pt 12): 1685–1687.

 

Haemoglobin, the 'honorary enzyme' [Brunori (1999), Trends Biochem. Sci. 24, 158-161], constitutes a prime prototype for allosteric models. Here, the crystallization and preliminary X-ray analysis of haemoglobin I from the South American fish Brycon cephalus are reported. X-ray diffraction data have been collected to 2.5 A resolution using synchrotron radiation (LNLS). Crystals were determined to belong to the space group P6(1)22 and preliminary structural analysis revealed the presence of one dimer (alphabeta) in the asymmetric unit. The structure was determined using standard molecular-replacement techniques.

PMID: 11092946

Canduri F, Delatorre P, Fadel V, Lorenzi CC, Pereira JH, Olivieri JR, Ruggiero Neto J, Konno K, Palma MS, Yamane T, de Azevedo WF Jr. Crystallization and preliminary X-ray diffraction analysis of a eumenine mastoparan toxin: a new class of mast-cell degranulating peptide in the wasp venom. Acta Crystallogr D Biol Crystallogr. 2000; 56(Pt 11): 1434–1436.

 

Mastoparans are tetradecapeptides found to be the major component of vespid venoms. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized and X-ray diffraction data collected to 2.7 A resolution using a synchrotron-radiation source. Crystals were determined to belong to the space group P6(2)22 (P6(4)22). This is the first mastoparan to be crystallized and will provide further insights into the conformational significance of mastoparan toxins with respect to their potency and activity in G-protein regulation.

PMID: 11053843

Smarra AL, de Azevedo WF Jr, Fadel V, Delatorre P, Dellamano M, Colombo MF, Bonilla-Rodriguez GO. Purification, crystallization and preliminary X-ray analysis of haemoglobin I from the armoured catfish Liposarcus anisitsi. Acta Crystallogr D Biol Crystallogr. 2000; 56(Pt 4): 495–497.

 

Liposarcus anisitsi is an armoured catfish that presents accessorial air oxygenation through a modified stomach, which allows this species to survive in waters with very low oxygen content. Analysis of its haemolysate has shown the presence of four haemoglobins; this work focuses on the main component, haemoglobin I. It has been crystallized in two different forms and X-ray diffraction data have been collected to 2.77 and 2.86 A resolution using synchrotron radiation. Crystals were determined to belong to the space groups C2 and P2(1) and preliminary structural analysis revealed the presence of one tetramer in the asymmetric unit in both crystal forms. The structure was determined using a standard molecular-replacement technique.

PMID: 10739931

Fadel V, Honda RT, Dellamano M, Smarra AL, Delatorre P, Olivieri JR, Bonilla-Rodriguez GO, de Azevedo WF Jr. Purification, crystallization and preliminary x-ray diffraction analysis of carboxyhaemoglobin-II from the fish Piaractus mesopotamicus (pacu). Acta Crystallogr D Biol Crystallogr. 2000; 56(Pt 3): 366–367.

 

Carboxyhaemoglobin-II isolated from the pacu (Piaractus mesopotamicus) has been crystallized and X-ray diffraction data were collected to 2.0 A resolution using synchrotron radiation. Crystals were characterized as belonging to the space group I23; preliminary structural analysis reveals the presence of one dimer in the asymmetric unit.

PMID: 10713529

Seixas FA, de Azevedo WF Jr, Colombo MF. Crystallization and x-ray diffraction data analysis of human deoxyhaemoglobin A(0) fully stripped of any anions. Acta Crystallogr D Biol Crystallogr. 1999; 55(Pt 11): 1914–1916.

 

In this work, initial crystallographic studies of human haemoglobin (Hb) crystallized in isoionic and oxygen-free PEG solution are presented. Under these conditions, functional measurements of the O(2)-linked binding of water molecules and release of protons have evidenced that Hb assumes an unforeseen new allosteric conformation. The determination of the high-resolution structure of the crystal of human deoxy-Hb fully stripped of anions may provide a structural explanation for the role of anions in the allosteric properties of Hb and, particularly, for the influence of chloride on the Bohr effect, the mechanism by which Hb oxygen affinity is regulated by pH. X-ray diffraction data were collected to 1.87 A resolution using a synchrotron-radiation source. Crystals belong to the space group P2(1)2(1)2 and preliminary analysis revealed the presence of one tetramer in the asymmetric unit. The structure is currently being refined using maximum-likelihood protocols.

PMID: 10531493

Smarra AL, Fadel V, Dellamano M, Olivieri JR, de Azevedo WF Jr, Bonilla-Rodriguez GO. Crystallization, preliminary X-ray diffraction analysis and Patterson search of oxyhaemoglobin I from the wolf (Chrysocyon brachiurus). Acta Crystallogr D Biol Crystallogr. 1999; 55(Pt 9): 1618–1619.

 

Oxyhaemoglobin I isolated from the Brazilian wolf Chrysocyon brachiurus has been crystallized and X-ray diffraction data has been collected to 2.06 A resolution using a synchrotron-radiation source. Crystals were determined to belong to the space group P2(1)2(1)2(1) and preliminary structural analysis revealed the presence of one tetramer in the asymmetric unit. The structure was determined using standard molecular-replacement techniques and is currently being refined using maximum-likelihood protocols. This is the first haemoglobin isolated from a member of the Canidae family to be crystallized and it will provide further insights in the comparative biochemistry of vertebrate haemoglobins.

PMID: 10489466

de Azevedo WF Jr, Ward RJ, Gutiérrez JM, Arni RK. Structure of a Lys49-phospholipase A2 homologue isolated from the venom of Bothrops nummifer (jumping viper). Toxicon. 1999; 37(2): 371–384.

 

Lys49-Phospholipase A2 (Lys49-PLA2) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity. We have solved the structure of myotoxin-I, a Lys49-PLA2 homologue isolated from the venom of Bothrops nummifer (jumping viper) at 2.4 A resolution using molecular replacement techniques. The final model has been refined to a final R-factor of 18.4% (R-free = 23.2%), and shows excellent geometry. The myotoxin-I from Bothrops nummifer is dimeric in the crystalline state as has been observed for other Lys49-PLA2 homologues. In addition, a continuous electron density in the active site and substrate binding channel could be successfully modeled as a fatty-acid molecule.

PMID: 10078866

Ward RJ, de Azevedo WF Jr, Arni RK. At the interface: crystal structures of phospholipases A2. Toxicon. 1998; 36(11): 1623–1633.

 

The protein content of many snake venoms often includes one or more phospholipases A2 (PLA2). In recent years a growing number of venoms from snakes of Agkistrodon, Bothrops and Trimeresurus species have been shown to contain a catalytically inactive PLA2-homologue in which the highly conserved aspartic acid at position 49 (Asp49) is substituted by lysine (Lys49). Although demonstrating little or no catalytic activity, these Lys49-PLA2s disrupt membranes by a Ca2+-independent mechanism of action. In addition, this family of PLA2s demonstrates myotoxic and cytolytic pharmacological activities, however the structural bases underlying these functional properties are poorly understood. Through the application of X-ray crystallography in combination with biophysical and bioinformatics techniques, we are studying structure/function relationships of Lys49-PLA2s. We here present results of a systematic X-ray crystallographic and amino acid sequence analysis study of Lys49 PLA2s and propose a model to explain the Ca2+-independent membrane damaging activity.

PMID: 9792179

Canduri F, Teodoro LG, Lorenzi CC, Gomes RA, Fontes MR, Arni RK, de Azevedo Júnior WF. Crystallization, preliminary X-ray analysis and Patterson search of a new aspartic protease isolated from human urine. Biochem Mol Biol Int. 1998; 46(2): 355–363.

 

Aspartic protease (EC 3.4.23) make up a widely distributed class of enzymes in animals, plants, microbes and, viruses. In animals these enzymes perform diverse functions, which range from digestion of food proteins to very specific regulatory roles. In contrast the information about the well-characterized aspartic proteases, very little is known about the corresponding enzyme in urine. A new aspartic protease isolated from human urine has been crystallized and X-ray diffraction data collected to 2.45 A resolution using a synchrotron radiation source. Crystals belong to the space group P2(1)2(1)2(1). The cell parameters obtained were a = 50.99, b = 75.56 and c = 89.90 A. Preliminary analysis revealed the presence of one molecule in the asymmetric unit. The structure was determined using the molecular replacement technique and is currently being refined using simulated annealing and conjugate gradient protocols.

PMID: 9801803

de Azevedo WF Jr, Ward RJ, Canduri F, Soares A, Giglio JR, Arni RK. Crystal structure of piratoxin-I: a calcium-independent, myotoxic phospholipase A2-homologue from Bothrops pirajai venom. Toxicon. 1998; 36(10): 1395–1406.

 

The crystal structure of Piratoxin-I (PrTX-I) a Lys49 homologue isolated from the venom of Bothrops pirajai has been determined and refined at 2.8 A to a crystallographic residual of 19.7% (Rfree = 29.7%). Amino-acid sequence differences between catalytically active phospholipases and PrTX-I in the putative Ca2+-binding loop, specifically the substitutions Tyr28 --> Asn, Gly32 --> Leu and Asp49 --> Lys, result in an altered conformation of this loop. The analysis of the position of the epsilon-amino group of Lys49 in the PrTX-I structure indicates that it fills the site normally occupied by the calcium ion in the catalytically active phospholipases. In contrast to the homologous monomeric Lys49 variant from Agkistrodon piscivorus piscivorus (App), PrTX-I is present as a dimer in the crystalline state, as observed in the structures of myotoxin II from Bothrops asper and Bothropstoxin I from Bothrops jararacussu. The two molecules in the asymmetric unit in the crystal structure of PrTX-I are related by a nearly perfect two-fold symmetry axis, yet the dimeric structure is radically different from the dimeric structure of the phospholipase from Crotalus atrox. In the C. atrox structure the dimer interface occludes the active sites, whereas in the PrTX-I structure they are exposed to solvent.

PMID: 9723838

Canduri F, Ward RJ, de Azevedo Júnior WF, Gomes RA, Arni RK. Purification and partial characterization of cathepsin D from porcine (Sus scrofa) liver using affinity chromatography. Biochem Mol Biol Int. 1998; 45(4): 797–803.

 

Cathepsin D, a lysosomal aspartic protease, has been purified from porcine liver using a combination of pepstatin-A agarose and Affi-Gel Blue affinity chromatography, followed by size-exclusion chromatography. The purified protein consists of two polypeptide chains of 15 and 30 kDa, and has an isoelectric point of 6.8. Porcine liver cathepsin D has maximum activity at pH 2.5-3.0 as determined by its activity against hemoglobin, with a Kcat of 14.3 s-1 and a kcat/KM of 2.70 x 10(6) s-1M-1 as determined by the hydrolysis of a fluorogenic peptide substrate.

PMID: 9713704

da Silva Giotto MT, Garratt RC, Oliva G, Mascarenhas YP, Giglio JR, Cintra AC, de Azevedo WF Jr, Arni RK, Ward RJ. Crystallographic and spectroscopic characterization of a molecular hinge: conformational changes in bothropstoxin I, a dimeric Lys49-phospholipase A2 homologue. Proteins. 1998; 30(4):442–454.

 

Bothropstoxin I (BthTX-I) from the venom of Bothrops jararacussu is a myotoxic phospholipase A2 (PLA2) homologue which, although catalytically inactive due to an Asp49-->Lys substitution, disrupts the integrity of lipid membranes by a Ca2+-independent mechanism. The crystal structures of two dimeric forms of BthTX-I which diffract X-rays to resolutions of 3.1 and 2.1 angstroms have been determined. The monomers in both structures are related by an almost perfect twofold axis of rotation and the dimer interfaces are defined by contacts between the N-terminal alpha-helical regions and the tips of the beta-wings of partner monomers. Significant differences in the relative orientation of the monomers in the two crystal forms results in "open" and "closed" dimer conformations. Spectroscopic investigations of BthTX-I in solution have correlated these conformational differences with changes in the intrinsic fluorescence emission of the single tryptophan residues located at the dimer interface. The possible relevance of this structural transition in the Ca2+-independent membrane damaging activity is discussed.

PMID: 9533628

De Azevedo WF, Leclerc S, Meijer L, Havlicek L, Strnad M, Kim SH. Inhibition of cyclin-dependent kinases by purine analogues: crystal structure of human cdk2 complexed with roscovitine. Eur J Biochem. 1997; 243(1-2): 518–526.

 

Cyclin-dependent kinases (cdk) control the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. A potent inhibitor of cdks, roscovitine [2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurin e], was identified by screening a series of C2,N6,N9-substituted adenines on purified cdc2/cyclin B. Roscovitine displays high efficiency and high selectivity (Meijer, L., Borgne, A., Mulner, O., Chong, J. P. J., Blow, J. J., Inagaki, N., Inagaki, M., Delcros, J.-G. & Moulinoux, J.-P. (1997) Eur. J. Biochem. 243, 527-536). It behaves as a competitive inhibitor for ATP binding to cdc2. We determined the crystal structure of a complex between cdk2 and roscovitine at 0.24-nm (2.4 A) resolution and refined to an Rfactor of 0.18. The purine portion of the inhibitor binds to the adenine binding pocket of cdk2. The position of the benzyl ring group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP-complex structure. Analysis of the position of this benzyl ring explains the specificity of roscovitine in inhibiting cdk2. The structure also reveals that the (R)-stereoisomer of roscovitine is bound to cdk2. The (R)-isomer is about twice as potent in inhibiting cdc2/cyclin B than the (S)-isomer. Results from structure/activity studies and from analysis of the cdk2/roscovitine complex crystal structure should allow the design of even more potent cdk inhibitors.

PMID: 9030780

De Azevedo WF Jr, Mueller-Dieckmann HJ, Schulze-Gahmen U, Worland PJ, Sausville E, Kim SH. Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase. Proc Natl Acad Sci U S A. 1996; 93(7): 2735–2740.

 

The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for studying inhibitory molecules that can modify the degree of cell proliferation. The discovery of specific inhibitors of CDKs such as polyhydroxylated flavones has opened the way to investigation and design of antimitotic compounds. A novel flavone, (-)-cis-5,7-dihydroxyphenyl-8-[4-(3-hydroxy-1-methyl)piperidinyl] -4H-1-benzopyran-4-one hydrochloride hemihydrate (L868276), is a potent inhibitor of CDKs. A chlorinated form, flavopiridol, is currently in phase I clinical trials as a drug against breast tumors. We determined the crystal structure of a complex between CDK2 and L868276 at 2.33 angstroms resolution and refined to an Rfactor 20.3%. The aromatic portion of the inhibitor binds to the adenine-binding pocket of CDK2, and the position of the phenyl group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP complex structure. The analysis of the position of this phenyl ring not only explains the great differences of kinase inhibition among the flavonoid inhibitors but also explains the specificity of L868276 to inhibit CDK2 and CDC2.

PMID: 8610110 PMCID: PMC39700

Kim SH, Schulze-Gahmen U, Brandsen J, de Azevedo Júnior WF. Structural basis for chemical inhibition of CDK2. Prog Cell Cycle Res. 1996; 2: 137–145.

 

The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for discovering small inhibitory molecules that can modify the degree of cell proliferation. The three-dimensional structure of CDK2 provides a structural foundation for understanding the mechanisms of activation and inhibition of CDK2 and for the discovery of inhibitors. In this article five structures of human CDK2 are summarised: apoprotein, ATP complex, olomoucine complex, isopentenyladenine complex, and des-chloro-flavopiridol complex.

PMID: 9552391

de Azevedo Júnior WF, Mascarenhas YP, De Sousa GF. Filgueiras CAL. cis-[1,2-Bis(propylsulfinyl)ethane-S,S']dichloroplatinum(II). Acta Cryst. 1995; C51: 619–621.