Made available in DSpace on 2015-09-25T12:21:42Z (GMT). No. of bitstreams: 1 PDF - Patricia de Lima Martins.pdf: 1012001 bytes, checksum: ab70117935599ee610a03aabf8bc94dd (MD5) Previous issue date: 2013-03-19 / Peanut (Arachis hypogaea L.) is an oilseed very expressive worldwide, due to commercial value of its oil. In Brazil, crop is grown in all regions, in order to attend in natura and food markets. The post-harvest is serious problem that affect the final output of the crop, which in inappropriate conditions, lead to occurrence of pests in stored grains. Some legumes contain protease inhibitors (PI) in their seeds that act directly in defense against grain pests, causing damage in nutrient absorption and further death. The objective of this study was to detect the presence of trypsin inhibitors in peanut genotypes and to study the potential inhibition against digestive enzymes in Alphitobius diaperinus and other agricultural pests. PI detection was carried out in 10 peanut genotypes belonging to Improvement Program at Embrapa Algodão (Campina Grande, PB, Brazil). Total proteins from seeds were extracted in each accession, quantified, fractionated with ammonium sulfate, and submitted thermal stability and pH tests, for further assays. All peanut genotypes showed PI in seed-protein extracts, however the highest inhibition rate for digestive enzymes of A. diaperinus was found in L7 BRS 151, which also reduced the number of larvae, in bioassay. The CNPA AM 280 showed the highest inhibition rate in both assays, bovine trypsin and intestinal homogenate of A. diaperinus. This genotype was also highly stable in different pH and temperature assays and inhibited the intestinal serine proteases from Spodoptera frugiperda, Tenebrio molitor and Tribolium castaneum, in 16%, 42% and 82% respectively. The results obtained in this study are quite relevant due to contribute with the reduction of grain losses in post-harvest, considering that genotypes investigated here have interesting traits for further use as genetic resources with resistance to pests. / O amendoim (Arachis hypogaea L.) é uma oleaginosa expressiva no cenário mundial, devido ao valor comercial de seu óleo. No Brasil, é cultivado em todas as regiões, para atender aos mercados in natura e de alimentos. Um dos problemas detectado na lavoura é na fase de pós-colheita, que, em condições inapropriadas levam a ocorrência de pragas nos grãos armazenados. Sabe-se, contudo, que algumas leguminosas detêm em suas sementes os inibidores de proteases (IP) que atuam na defesa direta contra pragas de grãos, provocando prejuízos na absorção de nutrientes e até mesmo a sua morte. Objetivou-se com este trabalho detectar a presença de inibidores de tripsina em genótipos de amendoim e estudar o potencial de inibição contra as enzimas digestivas de Alphitobius diaperinus e outros insetos de interesse agronômico. Para tanto, procedeu-se a detecção de IPs em 10 genótipos de amendoim pertencentes ao Programa de Melhoramento da Embrapa Algodão. As proteínas totais de sementes de cada acesso foram extraídas, quantificadas, fracionadas com sulfato de amônio, e submetidas a testes de estabilidade térmica e de pH, para posteriores ensaios in vitro e bioensaios. Os resultados obtidos evidenciaram a presença de inibidores de tripsina nos extratos proteicos das sementes de amendoim dos dez genótipos, contudo o BRS 151 L7 apresentou a maior inibição in vitro para as enzimas do trato digestivo do A. diaperinus e o menor número de larvas no bioensaio. A F3 do genótipo CNPA 280 AM apresentou o maior percentual de inibição tanto nos ensaios com tripsina bovina como no homogenato intestinal de A. diaperinus, e foi altamente estável nos ensaios com diferentes pHs e temperaturas. Destacou-se ainda por inibir em 16%, 42% e 82% as tripsinas intestinais de Spodoptera frugiperda, Tenebrio molitor e Tribolium castaneum, respectivamente. Os resultados obtidos nesta pesquisa são bastante relevantes para contribuir com a redução de perdas de grãos no período de pós-colheita, tendo em vista que os genótipos investigados possuem características interessantes para posterior uso como recurso genético com fonte de resistência a insetos-praga.

Com a introdução de uma nova classe de antiretrovirais integrantes da terapia anti-retroviral altamente ativa (HAART) para o tratamento das infecções pelo vírus da imunodeficiência humana, começaram a ser descritos inúmeros efeitos secundários.Na tentativa de se estabelecer um modelo animal para o estudo destes efeitos buscou-se uma espécie com similaridade no perfil e metabolismo lipídico. Iniciou-se estudo em Mesocricetus auratus. Foram avaliados o perfil lipídico e glicêmico,função hepática e renal, níveis de auto-anticorpos anti ox-LDL, perfil eletrocardiográfico, alterações histopatológicas renais e cardíacas nos animais sob dieta hiperlipídica e normal,tratados com Indinavir, inibidor de protease utilizado na HAART. Observou-se uma diminuição da sobrevida nos animais tratados com indinavir, aumento do nível sérico de triglicérides e glicose, redução de auto-anticorpos anti ox-LDL,aumento do segmento QRS no eletrocardiograma, presença de fibrose renal e cardíaca, hipercelularidade glomerular nos animais tratados com a droga com ou sem dieta hiperlipídica quando comparados com os controles. Concluimos que Mesocricetus auratus se apresenta como um bom modelo para o desvendamento dos mecanismos patológicos observados na HAART. / With the introduction of a new antiretroviral class use, integrants of highly active anti-retroviral therapy (HAART) for the treatment of infections by human immunodeficiency virus, several side effects started to be described.To establish an animal model for the study of these side effects, was chosen specie that have similarities in the lipidic profile and metabolism. A study in Mesocricetus auratus was started. It was evaluated the lipidic and glicemic profile ,hepatic and renal function, the levels of auto-antibodies against ox-LDL, electrocardiographic profile and renal and cardiac histopathological alterations in these animals under hyperlipidic and normal diets,treated with Indinavir, a protease inhibitor used in HAART.It was observed a decrease in the survival rate in the animals treated with Indinavir; an increase of the triglycerides and glucose serum level; reduction of anti ox-LDL auto-antibodies; increased QRS segment in the electrocardiogram; presence of renal and cardiac fibrosis; glomerular hypercellularity in the animals treated with the drug, with or without hyperlipidic diet when compared with the controls. We conclude that the Mesocricetus auratus is a good model for disclosure of the pathological mechanisms generated by HAART.

>Magister Scientiae - MSc / This study investigated the effects that drought stress imposed on the growth and development of soybean plants. Soybeans were initially observed at the whole-plant level in order to identify the physical changes that had taken place in response to drought. Further investigation of the effects of drought stress on Soybean plants were quantified at the molecular level. Physical changes of soybeans in response to drought stress were typified by the change in leaf morphology and pigmentation. At the molecular level, it was observed that drought stress resulted in the accumulation of hydrogen peroxide in soybean leaves, which was met by elevated levels of lipid peroxidation. The effects of drought on the modulation of (and interplay between cystatins) cysteine protease (caspase-like) activity and programmed cell death (PCD) were also investigated. Total caspase-like activity and cell death were enhanced in response to water deficit despite the up-regulation in gene expression of the cystatin Glyma14g04250. The cystatin Glyma18g12240 was not expressed in soybean leaves, whilst the gene expression of the cystatin Glyma20g08800 remained unchanged in response to drought. This study was aimed at the characterization of two single domain soybean cystatins, namely, Glyma14g04250 and Glyma20g08800 which could potentially be overexpressed in transgenic soybean plants in an attempt to alleviate the effects of drought stress. / National Research Foundation (NRF)

<p>The following work describes the methodology and materials used to study three human protein complexes involved in the etiology and progression of cancer. The first, ubiquitin-specific protease 7 (USP7) is an isopeptidase that employs a unique auto-regulatory mechanism. The second is another ubiquitin-specific protease, USP28, which forms higher order states in solution. Lastly, the third case was a protein complex that utilizes an oxidation-sensitive dimeric protein, Keap1, and two components of an E3 ligase – Cul3-Rbx1. Each of these studies involved overcoming unique challenges for cryo-EM sample optimization. Not all yielded the quality of data that would result in high-resolution (< 6 Å) densities. Despite this, new information was discovered about each system.</p> <p>USP7 has a unique mechanism of intramolecular regulation that stems from a hypothesized tethered-rheostat, whereby the c-terminal distal domains activate the catalytic domain via a hypothetical wide degree of conformational movement. My cryo-EM work, done in collaboration with the Wen Jiang lab, is the first comprehensive structural data that provides structural evidence for the movement of the tethered-rheostat. The particle set showed a great degree of conformational heterogeneity, even after a strategy was employed with a chemically-modified ubiquitin substrate to ameliorate these issues. The data showed that during the ubiquitin-bound state, after the release of a hypothetical substrate, but prior to the release of mono-ubiquitin, the HUBL4-5 domains do not remain engaged with the catalytic domain. This information suggests a change to existing models of catalysis. </p> <p>Additionally, the structural model built from the cryo-EM density has revealed an interfacial region between domains that were previously not thought to interact. This interfacial region between the TRAF domain and HUBL1-3 represents a candidate location of binding for a mixed, non-competitive inhibitor of USP7 previously identified in the lab. Enzyme kinetics, DSF, and Glide molecular docking experiments all yielded data that corroborate this idea.</p> <p>Structural studies on USP28 have been difficult as the multi-domain enzyme adopts oligomers in solution and is generally not amenable to crystallographic analysis. Prior to the work described herein, the only structural data were a solution NMR structure describing a few alpha-helical motifs in the N-terminus. During my graduate studies, two articles were published of the USP28 catalytic domain crystallographic structure. Both corroborated the existence of a dimer. The USP28 catalytic domain migrates during analytical gel filtration assays with the apparent molecular weight of a tetramer. Furthermore, glutaraldehyde crosslinking experiments show the catalytic domain appears to adopt a tetrameric state, like the USP25 tetramer. The USP25 tetramer was published alongside the USP28 catalytic domain dimer, concluding that a USP28 tetrameric state was not observed. Upon cryo-EM data collection and single particle analysis, it was observed that the compositional heterogeneity of the dataset was too great for any meaningful reconstruction. Although, the dataset appeared to how the presence of the <i>E. coli</i> GroEL chaperone complex. Co-expression experiments confirmed that the GroEL chaperone complex migrates with USP28 throughout the purification and may be useful for purifying USPs for structural studies.</p> <p>Currently, our lab has a single-angle X-ray scattering (SAXS) model of the Keap1-Cul3 E3 ligase complex. But, the field does not fully agree on the molecular stoichiometry or the overall structure-function of this oxidation sensor – E3 ligase complex. It is hypothesized that Keap1 forms a dimer through its BTB domain, and a single Cul3 molecule then binds this dimer. The oxidation state of Keap1 cysteines appears to be critical to the interaction, but the field remains uncertain about which residues are responsible for the interaction with the Cul3-Rbx1 E3 ligase. To better understand this interaction and to obtain structural information to corroborate the SAXS model, recombinant Keap1 and Cul3-Rbx1 were purified and their interaction was tested by ITC, gel filtration assay, and a new technique called <i>mass photometry</i>. </p> <p>It was found that the Keap1 Cys151 residue is not the oxidation sensor critical to the interaction, contrary to what some in the field anticipated. Additionally, it was found that under oxidative conditions, WTKeap1 could not form a complex with Cul3-Rbx1. The complex was successfully purified and was measured by SDS-PAGE, gel filtration assay, and mass photometry, and then used for cryo-EM single particle analysis. Full data collection and analysis has not yet been completed. It is anticipated that like the data from mass photometry, analytical SEC, and cryo-EM single particle analysis will show the complex appears to show a 1:1 Keap1-Cul3 stoichiometry, as opposed to the anticipated 2:1 ratio.</p>

Antibiotics are an important resource in modern medicine used to treat serious infections and enable a wide array of vital medical interventions including surgery and cancer chemotherapy. However, due to the increasing prevalence of antibiotic resistant pathogens, many clinically useful antibiotics are being rendered ineffective with too few new antibiotics in development to combat them. With highly diverse chemistry and bioactivity exquisitely shaped by evolution, natural products provide an unrivaled source of antibiotic compounds that is impossible to reproduce instinctively in the laboratory. The armeniaspirols are polyketide natural products with a unique spiro-[4.4]non-8-ene core that were isolated from Streptomyces armeniacus and were shown to be active against drug-resistant Gram-positive bacteria. Promisingly, in vitro resistant Staphylococcus aureus strains could not be readily obtained even after thirty serial passages under sub-lethal doses. Herein, we decipher the mechanism of action for this structurally unprecedented natural product antibiotic in the Gram-positive model organism Bacillus subtilis. Through chemical proteomics with an armeniaspirol-inspired activity-based probe, quantitative proteomics, biochemical assays, and microscopy, we show that armeniaspirol is a competitive inhibitor of the AAA+ proteases ClpXP and ClpYQ. Armeniaspirol represents the first known natural product inhibitor of ClpP, a highly coveted target due to its prominent role in bacterial virulence. Using overlapping proteomic fingerprints of armeniaspirol-treatment with ΔclpQ and ΔclpP deletions in B. subtilis, inhibition or deletion of these proteases appears to dysregulate key proteins involved in cell division, including FtsZ, DivIVA, and MreB. The dual ClpXP and ClpYQ inhibition is responsible for armeniaspirol’s potent antibiotic activity and this unique pharmacology makes it a promising candidate for antibiotic development. Several armeniaspirol-inspired analogs were generated as part of a medicinal chemistry study and evaluated for antibiotic activity towards a panel of clinically relevant Gram-positive pathogens. As a result, we identify three exciting armeniaspirol analogs with improved antibiotic activity. Lastly, the foundation for elucidating the ClpYQ degradome is developed. Our proteomic fingerprint of the B. subtilis ΔclpQ deletion strain generated some of the first insights into potential substrates of the ClpYQ protease. As a largely uncharacterized AAA+ protease implicated in the mechanism of action of armeniaspirol, we pursued a previously established acyl-intermediate covalent trapping strategy to characterize the ClpYQ-substrate complexes in B. subtilis cell lysate. Through unnatural amino acid incorporation using an evolved tRNA/aminoacyl-tRNA synthetase pair, the N-terminal active site serine of ClpQ is substituted with a photocleavable precursor that generates 2,3-diaminopropionic acid. While we were successful in synthesizing the photocleavable precursor, initial experiments to incorporate this unnatural amino acid in ClpQ expression proved unsuccessful, leading us to outline necessary control experiments for future endeavours. Ultimately, the covalently trapped substrates will be identified by LC-MS/MS, where we expect to identify key divisome and elongasome proteins in corroboration with the armeniaspirol mechanism of action study.

In Caulobacter crescentus, progression through the cell cycle is regulated by the AAA+ protease ClpXP, and there are several classes of cell-cycle substrates that require adaptors in order to be degraded. CpdR, a single domain-response regulator, binds the N-terminal domain of ClpXP and primes the protease for degradation of downstream factors (Lau et al., 2015). The ability of CpdR to bind ClpX is regulated by its phosphorylation state. In the unphosphorylated state, CpdR binds ClpXP and guides its localization to the cell pole during the swarmer to stalked transition, where CpdR is mediates degradation of substrates such as PdeA. Phosphorylation of response regulator receiver domains requires magnesium as a cofactor to stabilize the phosphorylated aspartate and reciprocally, phosphorylated receiver domains bind magnesium more effectively. While it is understood that CpdR primers ClpX for substrate degradation, the mechanism by which it does so has remained unclear. Using CollabFold, we identified putative residues involved in CpdR-ClpX binding and validated them using a BACTH screening. In vitro, we characterized the role that magnesium plays in regulating CpdR binding to ClpX. In this work, we directly test the role of magnesium in CpdR priming of ClpXP to show that magnesium may play a regulatory role in CpdR-mediated degradation, and thus binding to ClpX. We identify residues in ClpX that seem to be important for CpdR binding, which prior to this work was not clear.

No description available.

Photorhabdus é um gênero de enterobactérias simbiontes de Heterorhabditis, um gênero de nematoides entomopatogênicos. Dentre as enzimas secretadas por P. luminescens TTO1, destaca-se PrtA, uma metaloprotease que pertence a subfamília das serralisinas. PrtS, uma protease capaz de induzir uma forte resposta de melanização no inseto, foi identificada em P. temperata Az29 mas não em P. luminescens TTO1. Neste trabalho foram detectadas e caracterizadas bioquimicamente algumas proteases secretadas pelo isolado MN7 de P. luminescens luminescens. Foram detectadas duas atividades mais proeminentes: uma de 50 kDa, e outra de 38 kDa. Com o sequenciamento do genoma desta bactéria, pudemos confirmar a presença no genoma de genes codificando proteínas de alta identidade com as descritas PrtA e PrtS, de massas similares às detectadas nas nossas zimografias. As proteases são inibidas por inibidores específicos de metaloproteases. P. luminescens MN7 apresenta secreção, portanto, de uma protease já descrita algumas vezes, e de outra presente apenas em P. temperata Az29. / Photorhabdus is a genus of Enterobacteriaceae, symbionts of Heterorhabditis, a genus of entomopathogenic nematodes. Among the enzymes secreted by P. luminescens TTO1 stands out PrtA, a metalloprotease that belongs to the subfamily of serralysins. PrtS, a protease capable of inducing a strong melanotic response from the insect, was identified in P. temperata Az29 but not in P. luminescens TTO1. In this work were detected and characterized biochemically few isolated proteases secreted by P. luminescens luminescens MN7. Two most prominent activities were detected: one with 50 kDa and one with 38 kDa. With the sequencing of the genome of MN7, we could confirm the presence in the genome of genes encoding proteins with high identity to PrtA and PrtS already described, with similar masses to those detected in our zimographies. These proteases are inhibited by specific inhibitors of metalloproteases. P. luminescens MN7 secretes a protease already described a few times, and other present only in P. temperata Az29.

Doenças causadas por agentes infecciosos e parasitários são chamadas negligenciadas por não despertarem interesse das indústrias farmacêuticas para o desenvolvimento de novas alternativas terapêuticas. Essas doenças são responsáveis por levar milhões de pessoas à morte todos os anos e afetam principalmente os países pobres e em desenvolvimento. Dentre estas, a doença de Chagas e as leishmanioses, parasitoses causadas por parasitas flagelados pertencentes à família Trypanosomatidae, T. cruzi e Leishmaina sp., respectivamente, se apresentam como um sério problema de saúde pública mundial. Endêmicas em vários países e causando milhões de mortes anualmente, ainda hoje não existem fármacos eficientes e seguros para o tratamento dessas doenças. Este panorama torna eminente a necessidade de pesquisa e desenvolvimento de novos fármacos para essas parasitoses. A busca por agentes quimioterápicos envolve a seleção de vias metabólicas essenciais à sobrevivência dos parasitas. Dentre estas, destacamse cisteíno-proteases presentes nesses tripanossomatídeos, deste modo a cruzaína no T. cruzi, e a CPB2.8 na Leishmania mexicana, se mostram como alvos bioquímicos promissores. A disponibilidade de estruturas cristalográficas da cruzaína e do sequenciamento genômico da CPB2.8, nos permite utilizar estratégias de planejamento de fármacos baseado no receptor (SBDD) na identificação de candidatos a fármacos para essas doenças. Entre as técnicas modernas de SBDD utilizadas, a triagem virtual possibilita identificar promissores candidatos a novos fármacos. Assim neste trabalho, obteve-se por meio da técnica de modelagem comparativa o modelo da enzima CPB2.8 de L. mexicana, visto a indisponibilidade da estrutura cristalográfica no Protein Data Bank (PDB). De modo a refinar o modelo construído realizou-se a simulação por dinâmica molecular de 100ns, apresentando estabilização a partir de 80ns. A simulação por dinâmica molecular foi validada por meio do gráfico de Ramachandran, gráfico de raio de giro, RMSD, gráfico de superfície hidrofóbica. Foram calculados os mapas de interação molecular no programa GRID das seguintes proteínas: cruzaína, CPB2.8, catepsina B e catepsina L, e, posteriormente, foi construído um modelo farmacofórico baseado no sítio ativo das enzimas cruzaína e CPB2.8. O modelo farmacofórico da cruzaína foi validado por curva ROC apresentando valor de AUC 61%. A triagem virtual foi realizada para ambas as proteínas e foram obtidos 369 compostos para a cuzaína e 225 compostos para a CPB2.8. Foi realizado o ancoramento molecular desses compostos obtidos pela triagem virtual a fim de diminuir a quantidade de compostos a serem avaliados experimentalmente. / Neglected diseases are caused by parasites and infectious agents and affect mainly people in poor areas being prevalent in 149 countries and causing 534,000 deaths per year. Among neglected diseases we can highlight Chagas Disease and Leishmaniasis, both have a high rate of morbidity and mortality and both are addressed in this project in the search of new drugs against a NTD. Nowadays, the search for new drugs involves the selection of biological pathways essential for parasite survival, in this class of parasites we can suggest the cysteine proteases, a proteases family present in Trypanosoma cruzi and and Leishmania ssp. In order to obtain a new agent against Neglected Disease in this work was obtained the model of the enzyme CPB2.8 of L. mexicana using the comparative modeling technique, due to the unavailability of the crystallographic structure in the Protein Data Bank (PDB). In order to refine the constructed model was performed the molecular dynamics simulation of 100ns, stabilization was achieved from 80ns. Molecular dynamics simulation was validated using the Ramachandran graph, radius of rotation graph, RMSD, hydrophobic surface area graph. The molecular interaction fields were calculated in the GRID program to cruzain, CPB2.8, cathepsin B and cathepsin L. Based on molecular interaction fields generated pharmacophoric models were constructed using information about the active site of the enzymes cruzain and CPB2.8. The pharmacophoric model of cruzain was validated by ROC curve presenting AUC value of 61%. Virtual screening was performed for both proteins and 369 compounds were obtained for cuzain and 225 compounds for CPB2.8. Docking studies of these compounds was performed in order to decrease the amount of compounds to be evaluated experimentally.

Doenças causadas por agentes infecciosos e parasitários são chamadas negligenciadas por não despertarem interesse das indústrias farmacêuticas para o desenvolvimento de novas alternativas terapêuticas. Essas doenças são responsáveis por levar milhões de pessoas à morte todos os anos e afetam principalmente os países pobres e em desenvolvimento. Dentre estas, a doença de Chagas e as leishmanioses, parasitoses causadas por parasitas flagelados pertencentes à família Trypanosomatidae, T. cruzi e Leishmaina sp., respectivamente, se apresentam como um sério problema de saúde pública mundial. Endêmicas em vários países e causando milhões de mortes anualmente, ainda hoje não existem fármacos eficientes e seguros para o tratamento dessas doenças. Este panorama torna eminente a necessidade de pesquisa e desenvolvimento de novos fármacos para essas parasitoses. A busca por agentes quimioterápicos envolve a seleção de vias metabólicas essenciais à sobrevivência dos parasitas. Dentre estas, destacamse cisteíno-proteases presentes nesses tripanossomatídeos, deste modo a cruzaína no T. cruzi, e a CPB2.8 na Leishmania mexicana, se mostram como alvos bioquímicos promissores. A disponibilidade de estruturas cristalográficas da cruzaína e do sequenciamento genômico da CPB2.8, nos permite utilizar estratégias de planejamento de fármacos baseado no receptor (SBDD) na identificação de candidatos a fármacos para essas doenças. Entre as técnicas modernas de SBDD utilizadas, a triagem virtual possibilita identificar promissores candidatos a novos fármacos. Assim neste trabalho, obteve-se por meio da técnica de modelagem comparativa o modelo da enzima CPB2.8 de L. mexicana, visto a indisponibilidade da estrutura cristalográfica no Protein Data Bank (PDB). De modo a refinar o modelo construído realizou-se a simulação por dinâmica molecular de 100ns, apresentando estabilização a partir de 80ns. A simulação por dinâmica molecular foi validada por meio do gráfico de Ramachandran, gráfico de raio de giro, RMSD, gráfico de superfície hidrofóbica. Foram calculados os mapas de interação molecular no programa GRID das seguintes proteínas: cruzaína, CPB2.8, catepsina B e catepsina L, e, posteriormente, foi construído um modelo farmacofórico baseado no sítio ativo das enzimas cruzaína e CPB2.8. O modelo farmacofórico da cruzaína foi validado por curva ROC apresentando valor de AUC 61%. A triagem virtual foi realizada para ambas as proteínas e foram obtidos 369 compostos para a cuzaína e 225 compostos para a CPB2.8. Foi realizado o ancoramento molecular desses compostos obtidos pela triagem virtual a fim de diminuir a quantidade de compostos a serem avaliados experimentalmente. / Neglected diseases are caused by parasites and infectious agents and affect mainly people in poor areas being prevalent in 149 countries and causing 534,000 deaths per year. Among neglected diseases we can highlight Chagas Disease and Leishmaniasis, both have a high rate of morbidity and mortality and both are addressed in this project in the search of new drugs against a NTD. Nowadays, the search for new drugs involves the selection of biological pathways essential for parasite survival, in this class of parasites we can suggest the cysteine proteases, a proteases family present in Trypanosoma cruzi and and Leishmania ssp. In order to obtain a new agent against Neglected Disease in this work was obtained the model of the enzyme CPB2.8 of L. mexicana using the comparative modeling technique, due to the unavailability of the crystallographic structure in the Protein Data Bank (PDB). In order to refine the constructed model was performed the molecular dynamics simulation of 100ns, stabilization was achieved from 80ns. Molecular dynamics simulation was validated using the Ramachandran graph, radius of rotation graph, RMSD, hydrophobic surface area graph. The molecular interaction fields were calculated in the GRID program to cruzain, CPB2.8, cathepsin B and cathepsin L. Based on molecular interaction fields generated pharmacophoric models were constructed using information about the active site of the enzymes cruzain and CPB2.8. The pharmacophoric model of cruzain was validated by ROC curve presenting AUC value of 61%. Virtual screening was performed for both proteins and 369 compounds were obtained for cuzain and 225 compounds for CPB2.8. Docking studies of these compounds was performed in order to decrease the amount of compounds to be evaluated experimentally.