Publications by authors named "Walter F Azevedo"

33 Publications

Supervised Machine Learning Methods Applied to Predict Ligand- Binding Affinity.

Curr Med Chem 2017 ;24(23):2459-2470

Laboratory of Computational Systems Biology, Faculty of Biosciences - Pontifical Catholic University of Rio Grande do Sul (PUCRS), Av. Ipiranga, 6681, Porto Alegre-RS 90619-900. Brazil.

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.
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http://dx.doi.org/10.2174/0929867324666170623092503DOI Listing
September 2017

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 Jun 6;15:165. Epub 2015 Jun 6.

Department of Biological Sciences, Universidade Estadual de Santa Cruz (UESC), Ilheús, Bahia, Brazil.

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.
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http://dx.doi.org/10.1186/s12906-015-0681-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457080PMC
June 2015

Anti-Trypanosoma cruzi activity of nicotinamide.

Acta Trop 2012 May 18;122(2):224-9. Epub 2012 Jan 18.

Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Rua Waldemar Falcão, Salvador, BA, Brazil.

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.
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http://dx.doi.org/10.1016/j.actatropica.2012.01.001DOI Listing
May 2012

Crystal structure and molecular dynamics studies of purine nucleoside phosphorylase from Mycobacterium tuberculosis associated with acyclovir.

Biochimie 2012 Jan 20;94(1):155-65. Epub 2011 Oct 20.

Faculdade de Biociências, Laboratório de Bioquímica Estrutural, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre - RS, Brazil.

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.
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http://dx.doi.org/10.1016/j.biochi.2011.10.003DOI Listing
January 2012

Antimalarial activity of physalins B, D, F, and G.

J Nat Prod 2011 Oct 28;74(10):2269-72. Epub 2011 Sep 28.

Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia, Brazil.

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.
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http://dx.doi.org/10.1021/np200260fDOI Listing
October 2011

Identification of new potential Mycobacterium tuberculosis shikimate kinase inhibitors through molecular docking simulations.

J Mol Model 2012 Feb 19;18(2):755-64. Epub 2011 May 19.

Faculdade de Biociências, Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Laboratório de Bioquímica Estrutural (LaBioQuest), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Av. Ipiranga 6681, Porto Alegre, RS 90619-900, Brazil.

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.
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http://dx.doi.org/10.1007/s00894-011-1113-5DOI Listing
February 2012

Crystal structure determination and dynamic studies of Mycobacterium tuberculosis Cytidine deaminase in complex with products.

Arch Biochem Biophys 2011 May 2;509(1):108-15. Epub 2011 Feb 2.

Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Av. Ipiranga 6681, Porto Alegre, RS 90619-900, Brazil.

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.
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http://dx.doi.org/10.1016/j.abb.2011.01.022DOI Listing
May 2011

Structural basis for both pro- and anti-inflammatory response induced by mannose-specific legume lectin from Cymbosema roseum.

Biochimie 2011 May 26;93(5):806-16. Epub 2011 Jan 26.

BioMol-Lab, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, P. O. Box 6043, 60.455-970 Fortaleza, Ceará, Brazil.

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.
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http://dx.doi.org/10.1016/j.biochi.2011.01.006DOI Listing
May 2011

Crystallographic and docking studies of purine nucleoside phosphorylase from Mycobacterium tuberculosis.

Bioorg Med Chem 2010 Jul 10;18(13):4769-74. Epub 2010 May 10.

Centro de Pesquisas em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Avenida Ipiranga 6681/92-A, 90619-900 Porto Alegre, RS, Brazil.

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.
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http://dx.doi.org/10.1016/j.bmc.2010.05.009DOI Listing
July 2010

SKPDB: a structural database of shikimate pathway enzymes.

BMC Bioinformatics 2010 Jan 7;11:12. Epub 2010 Jan 7.

CEIS/Departamento de Biologia, Instituto de Biociências, UNESP, Rio Claro, São Paulo, Brasil.

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/.
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http://dx.doi.org/10.1186/1471-2105-11-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2824673PMC
January 2010

Structural and functional analyses of Mycobacterium tuberculosis Rv3315c-encoded metal-dependent homotetrameric cytidine deaminase.

J Struct Biol 2010 Mar 24;169(3):413-23. Epub 2009 Dec 24.

Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Av. Ipiranga, 6681, Porto Alegre, RS 90619-900, Brazil.

The 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.
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http://dx.doi.org/10.1016/j.jsb.2009.12.019DOI Listing
March 2010

Expression and purification of human respiratory syncytial virus recombinant fusion protein.

Protein Expr Purif 2008 Dec 26;62(2):146-52. Epub 2008 Aug 26.

Instituto de Biociências, Letras e Ciências Exatas, UNESP, Rua Cristóvão Colombo, São José do Rio Preto, SP, Brazil.

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.
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http://dx.doi.org/10.1016/j.pep.2008.08.005DOI Listing
December 2008

Purification, characterization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds.

Appl Biochem Biotechnol 2009 Mar 19;152(3):383-93. Epub 2008 Aug 19.

BioMol-Lab, Departmento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, P. O. Box 6043, 60.455-970, Fortaleza, Ceará, Brazil.

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.
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http://dx.doi.org/10.1007/s12010-008-8334-9DOI Listing
March 2009

Structural studies of shikimate 5-dehydrogenase from Mycobacterium tuberculosis.

Proteins 2008 Aug;72(2):720-30

Departamento de Física, UNESP, São José do Rio Preto, SP, 15054-000, Brasil.

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.
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http://dx.doi.org/10.1002/prot.21953DOI Listing
August 2008

Structure of a lectin from Canavalia gladiata seeds: new structural insights for old molecules.

BMC Struct Biol 2007 Aug 2;7:52. Epub 2007 Aug 2.

Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Ceará, Brazil.

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.
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http://dx.doi.org/10.1186/1472-6807-7-52DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1955443PMC
August 2007

Shikimate kinase: a potential target for development of novel antitubercular agents.

Curr Drug Targets 2007 Mar;8(3):459-68

Programa de Pós-Graduação em Biofísica Molecular, Departamento de Física, UNESP, São José do Rio Preto, SP, Brazil.

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.
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http://dx.doi.org/10.2174/138945007780059013DOI Listing
March 2007

Chorismate synthase: an attractive target for drug development against orphan diseases.

Curr Drug Targets 2007 Mar;8(3):437-44

Programa de Pós-Graduação em Biofísica Molecular, Departamento de Física, UNESP, São José do Rio Preto, SP 15054-000, Brasil.

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.
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http://dx.doi.org/10.2174/138945007780058924DOI Listing
March 2007

Protein kinases as targets for antiparasitic chemotherapy drugs.

Curr Drug Targets 2007 Mar;8(3):389-98

Universidade Federal de Mato Grosso do Sul, Centro de Ciências Biológicas e da Saúde, Departamento de Morfofisiologia-Laboratório de Bioquímica, Caixa Postal, Campo Grande-MS, Brazil.

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.
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http://dx.doi.org/10.2174/138945007780058979DOI Listing
March 2007

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 Nov;49(22):6500-9

Laboratory of Growth Regulators, Faculty of Science, Palacký University and Institute of Experimental Botany, Slechtitelů 11, 783 71 Olomouc, Czech Republic.

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.
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http://dx.doi.org/10.1021/jm0605740DOI Listing
November 2006

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 Sep;273(17):3962-74

BioMol-Laboratory, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil.

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.
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http://dx.doi.org/10.1111/j.1742-4658.2006.05400.xDOI Listing
September 2006

Phosphate closes the solution structure of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Mycobacterium tuberculosis.

Arch Biochem Biophys 2006 Aug 13;452(2):156-64. Epub 2006 Jun 13.

Departamento de Física, UNESP, São José do Rio Preto, SP 15054-000, Brazil.

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.
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http://dx.doi.org/10.1016/j.abb.2006.05.008DOI Listing
August 2006

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 Jun 21;154(3):280-6. Epub 2006 Apr 21.

Departamento de Bioquímica e Biologia Molecular--Universidade Federal do Ceará, Brazil.

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.
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http://dx.doi.org/10.1016/j.jsb.2006.03.011DOI Listing
June 2006

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 Jun 21;359(3):646-66. Epub 2006 Apr 21.

Centro de Pesquisas em Biologia Molecular e Funcional, Faculdade de Farmácia e Faculdade de Biociências, Instituto de Pesquisas Biomédicas, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS 90619-900, Brasil.

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.
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http://dx.doi.org/10.1016/j.jmb.2006.03.055DOI Listing
June 2006

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 Mar 10;62(Pt 3):235-7. Epub 2006 Feb 10.

Department of Biochemistry, Federal University of Ceará, Brazil.

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.
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http://dx.doi.org/10.1107/S174430910600371XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2197170PMC
March 2006

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 Sep 31;61(Pt 9):841-3. Epub 2005 Aug 31.

Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Biomol-Lab, CEP 60451-970, Caixa Postal 6043, Fortaleza-Ceará, Brazil.

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.
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http://dx.doi.org/10.1107/S1744309105024462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1978108PMC
September 2005

Structure of chorismate synthase from Mycobacterium tuberculosis.

J Struct Biol 2006 May 17;154(2):130-43. Epub 2006 Jan 17.

Programa de Pós-Graduação em Biofísica Molecular, Departamento de Física, UNESP, São José do Rio Preto, SP 15054-000, Brazil.

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.
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http://dx.doi.org/10.1016/j.jsb.2005.12.008DOI Listing
May 2006

Automated NMR structure determination and disulfide bond identification of the myotoxin crotamine from Crotalus durissus terrificus.

Toxicon 2005 Dec 26;46(7):759-67. Epub 2005 Sep 26.

Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Zürich, CH-8093 Zurich, Switzerland.

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.
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http://dx.doi.org/10.1016/j.toxicon.2005.07.018DOI Listing
December 2005

Kinetics and crystal structure of human purine nucleoside phosphorylase in complex with 7-methyl-6-thio-guanosine.

Arch Biochem Biophys 2005 Oct;442(1):49-58

Centro de Pesquisas em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas, PUCRS, Porto Alegre, RS, Brazil.

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.
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http://dx.doi.org/10.1016/j.abb.2005.07.021DOI Listing
October 2005

Molecular models of NS3 protease variants of the Hepatitis C virus.

BMC Struct Biol 2005 Jan 21;5. Epub 2005 Jan 21.

Department of Physics, IBILCE/UNESP, São José do Rio Preto, São Paulo, Brazil.

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.
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http://dx.doi.org/10.1186/1472-6807-5-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC547903PMC
January 2005

Crystallographic structure of PNP from Mycobacterium tuberculosis at 1.9A resolution.

Biochem Biophys Res Commun 2004 Nov;324(2):789-94

Departamento de Física, UNESP, São José do Rio Preto, SP 15054-000, Brazil.

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.
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http://dx.doi.org/10.1016/j.bbrc.2004.09.137DOI Listing
November 2004
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