Bacteria employ survival strategies to protect themselves against foreign invaders, including bacteriophages. The ‘immune system’ of bacteria relies mostly on restriction-modification (R-M) systems. The primary role of R-M systems is to protect the host from invading foreign DNA molecules. Three major types of R–M system are found in bacteria, viz.Types I, II and III. Type II R–M systems comprise a separate restriction endonuclease (REase) and a methyltransferase (MTase) that act independently of each other. Type II REases generally recognize palindromic sequences in DNA and cleave within or near their recognition sequences and produce DNA fragments of defined sizes. They have become indispensable tools in molecular biology and have been widely exploited for studying site-specific protein–DNA interactions. Surprisingly, these enzymes share little or no sequence homology among them, though the three-dimensional structures determined to date reveal a common-core motif (‘PD...D/EXK’ motif) with a central β-sheet that is flanked by α-helices on both sides. In the motif, two acidic residues (D and D/E) are important for the metal ion binding and catalysis. The work presented in this thesis deals with the determination of active site, elucidation of kinetic mechanism and study of evolution of sequence specificity using the well known, R.KpnI, from Klebsiella pneumoniae. The enzyme is a homodimer, which recognizes a palindromic double stranded DNA sequence, GGTAC↓C, and cleaves as shown. Unlike other REases, R.KpnI shows prolific promiscuous DNA cleavage in presence of Mg2+. Surprisingly, Ca2+ completely suppresses the Mg2+ mediated promiscuous activity and induces high fidelity cleavage at the recognition sequence. These unusual properties of R.KpnI led to the characterization of the active site of the enzyme. This thesis is divided into five chapters. Chapter 1 is a general introduction of R-M systems and an overview of the literature on active sites of Type II REases. It deals with discovery, nomenclature and classification followed by description of the enzymes diversity and general features of Type II REases. The different active site folds of the REases have been discussed in detail. The features of sequence specificity and the efforts undertaken to engineer the new specificity in the REases have been dealt at the end of the chapter. Chapter 2 describes identification and characterization of the R.KpnI active site by bioinformatics analyses, homology modeling and mutational studies. Bioinformatics analyses reveal that R.KpnI contains a ββα-Me-finger fold, which is a characteristic of many HNH-superfamily endonucleases. According to the homology model of R.KpnI, the putative active site residues correspond to the conserved residues present in HNH nucleases. Substitutions of these conserved residues in R.KpnI resulted in loss of the DNA cleavage activity, confirming their importance. This study provides the first experimental evidence for a Type IIP REase that is a member of the HNH superfamily and does not belong to the PD...D/EXK superfamily of nucleases. In Chapter 3 DNA binding and kinetic analysis of R.KpnI is presented. The metal ions which exhibit disparate pattern of DNA cleavage have no role in DNA recognition. The enzyme binds to both canonical and non-canonical DNA with comparable affinity irrespective of the metal ions used. Further, it was shown that Ca2+-imparted exquisite specificity of the enzyme is at the level of DNA cleavage and not at the binding step. The kinetic constants were obtained through steady-state kinetic analysis of R.KpnI in presence of different metal ions. With the canonical oligonucleotides, the cleavage rate of the enzyme was comparable for both Mg2+- and Mn2+-mediated reactions and was about three times slower with Ca2+. The enzyme discriminates non-canonical sequences poorly from the canonical sequence in Mg2+-mediated reactions unlike any other Type II REases, accounting for its promiscuous behavior. These studies suggest that R.KpnI displays properties akin to that of typical Type II REases and also endonucleases with degenerate specificity for DNA recognition and cleavage. In chapter 4, two uncommon roles for Zn2+ in R.KpnI are described. Examination of the sequence revealed the presence of a zinc finger (CCCH) motif rarely found in proteins of prokaryotic origin. Biophysical experiments and subsequent mutational analysis showed that the zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg2+- and Mn2+-mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in a REase. Chapter 5 describes generation of highly sequence specific R.KpnI. Towards this end, site-directed mutants were generated at the putative secondary metal binding site. The DNA binding and cleavage analyses of the mutants at putative secondary metal binding site revealed that the secondary site is not important for primary catalysis and have a role in sequence specificity. A single amino acid change at the D163 position abolished the promiscuous activity of the wt enzyme in the presence of Mg2+ and Mn2+. Thus, a single point mutation converts the promiscuous endonuclease to a high fidelity REase. In conclusion, the work described in the thesis reveals new information on the REases in general and R.KpnI in particular. Many of the properties of R.KpnI elucidated in this thesis represent hitherto unknown features amongst REases. The presence of an HNH catalytic motif in the enzyme indicates the diversity of active site fold in REases and their distinct origin. Similarly, the high degree of promiscuity exhibited by the enzyme may hint at the evolutionary link between non-specific and highly sequence specific nucleases. The present studies also provide an example for the role of mutations in the evolution of sequence specificity. The utilization of different metal ions for DNA cleavage and the architectural role for Zn2+ in maintaining the structural integrity are other unusual properties of the enzyme.

A tolerância imunológica é a propriedade essencial do sistema imune que controla as reações patológicas contra antígenos do próprio. O timo é visto como o principal órgão envolvido com a indução de tolerância aos antígenos próprios que são expressos pelas células tímicas (tolerância central), enquanto que a indução de tolerância aos antígenos relacionados a outros tecidos (TRAs) tem sido atribuída aos mecanismos de tolerância extratímica (tolerância periférica). Entretanto, a evidência da expressão de TRAs de órgãos e tecidos parenquimais no timo pelas células medulares epiteliais (mTECs) de camundongos e de humanos a qual foi referida como expressão gênica promíscua (PGE) reforçou a concepção de tolerância central de TRAs. O controle molecular dessa expressão tem sido atribuído ao gene Aire (Auto immune regulator) que é um regulador de transcrição. No presente estudo, procurou-se retratar a expressão gênica promíscua no timo de camundongos NOD (Non Obese Diabetic) por meio da análise da expressão gênica em grande escala, ou seja, descrevendo seu transcriptoma usando a tecnologia de cDNA microarrays. Para a análise dos dados utilizamos programas de bioinformática dedicados a microarrays bem como dados de bancos para a caracterização da PGE e susceptibilidade genética ao diabetes melitus do tipo 1 (DM-1). Três conjuntos de resultados puderam ser evidenciados. No primeiro conjunto observou-se a ocorrência da PGE de antígenos tecidos/órgãos parenquimatosos (TRAs) em timos recém removidos e em in vitro em cultura ATOC de camundogos NOD pré-autoimunes e autoimunes (diabéticos). O segundo conjunto de resultados consistiu na análise do efeito da inativação do transcrito de gene Aire na expressão gênica do timo de camundongos NOD in vitro em cultura ATOC. Finalmente, no último conjunto de dados, demonstrou-se que certos genes de TRAs com expressão promíscua, se encontram em regiões cromossômicas de susceptibilidade ao DM - 1 (idds). Como três deles (Il-4, Cd4 e Cdk4) são diretamente relacionados com a patogenia do DM-1 em camundongos foi possível estabelecer um paralelo entre PGE e susceptibilidade genética. / Immunologic tolerance is an essential property of the immune system, which controls immune reactions directed against the body self components. The thymus is seen as the main organ involved with the tolerance induction to self antigens, which are expressed by the thymic cells (central tolerance), while the tolerance induction to the diverse other peripheral tissues and organs is attributed to extra thymic mechanisms (peripheral tolerance). Nevertheless, the evidence for the expression of peripheral tissue related antigens (TRAs) in the thymus by the medullary thymic epithelial cells (mTECs) of mice and humans, which have been termed to as promiscuous gene expression (PGE), has contributed to the concept of central tolerance to TRAs. The molecular control of such gene expression has been attributed to the Aire (autoimmune regulator) gene, which plays a role as a transcriptional regulator. In the present study, we searched to picture PGE in the thymus of NOD (non obese diabetic) mice by means of high throughput gene expression, analyzing the transcriptome by the cDNA microarray method. To analyzing data we used bioinformatics programs dedicated to microarrays and specialized data banks to characterize PGE and genetic susceptibility to type 1 diabetes mellitus (DM-1). Studying pre and autoimmune NOD mice, we evidentiate three sets of results. In the first set, it was observed the occurrence of PGE of parenchymal tissue/organs antigens (TRAs) in fresh thymuses and in thymuses cultured in vitro in adult thymus organ cultures (ATOC). The second set of results consisted in the analysis of the effect of in vitro (ATOC) Aire gene silencing on PGE. Finally, in the third data set, we demonstrated that certain promiscuously expressed genes are positioned in DM-1 genetic susceptibility chromosomal regions (idds). As three of such genes (IL4, Cd4 and Cdk4) are directly associated to the DM-1 pathogenesis in mice, it was possible to establishing a parallel between PGE in the thymus and genetic susceptibility to this autoimmune disease.

O timo é um orgão linfóide primário, no qual ocorre a indução da tolerância imunológica central aos antígenos do próprio que são expressos pelos tecidos periféricos (PTAs). A medula tímica é formada por células tímicas medulares epiteliais (mTECs) que expressam centenas desses PTAs que representam virtualmente todos os órgãos e tecidos do corpo. Esse fenômeno foi denominado de expressão gênica promíscua (PGE) a qual é parcialmente regulada pelo modulador da transcrição Autoimmune regulator (Aire). Os precursores de células T oriundos da medula óssea migram para o timo (agora são denominados de timócitos) e na medula desse órgão, passam pela seleção negativa mediada pelas mTECs. As células sobreviventes evoluem para células T maduras e funcionais que migram para a periferia com capacidade de reconhecimento das moléculas de MHC e tolerantes aos PTAs. Além de controlar a transcrição de genes PTAs, Aire também controla a expressão de microRNAs (miRNAs), relacionados com a integridade e funcionalidade do microambiente tímico. A seleção negativa no timo é um processo essencial para a manutenção da autotolerância imunológica e o desbalanço desse processo está associado com o desenvolvimento de doenças autoimunes como, por exemplo, o diabetes mellitus do tipo 1 (DM1).Tendo em vista essas premissas, nosso trabalho se fundamentou em duas hipóteses: 1) Variações na expressão do gene Aire podem perturbar a expressão de genes PTAs e miRNAs no timo, causando alterações na PGE, 2) A expressão balanceada de genes como Aire e/ou PTAs nas mTECs, é fundamental para a integridade da tolerância central. O desbalanço na expressão desses genes, está associado com a emergência do diabetes mellitus tipo 1 no camundongo. Para testar nossa primeira hipótese efetuamos o silenciamento de Aire (Aire knockdown) por meio de eletrotransfeção de RNA interferente (siRNA) anti-Aire in vivo no timo de camundongos BALB/c. Análises do transcriptoma (mRNAs) e miRNoma (miRNAs) das mTECs, revelaram que silenciamento parcial e transitório de Aire foi suficiente para afetar a expressão de PTAs Aire dependentes bem como a de miRNAs. Redes de interação miRNA-mRNA, revelaram que o controle pós-transcricional da PGE também é afetado pelo silenciamento de Aire. Os resultados encontrados revelam que Aire e miRNAs podem formar uma via essencial durante a indução da tolerância central. Para testar nossa segunda hipótese comparamos o transcriptoma de mTECs de camundongos BALB/c (linhagem não-autoimune) com mTECs de camundongos non-obese diabetic NOD (modelo animal utilizado nos estudos de DM1 autoimune). Nossos resultados revelaram que a expressão transcricional de autoantígenos relacionados ao DM1 está desbalanceada em camundongos NOD já numa fase precoce, quando esses animais ainda não apresentavam a doença clínica (fase pré-diabética). Inesperadamente, os níveis transcrionais de Aire apresentaram-se equivalentes no timo dessas duas linhagens, porém os níveis da proteína AIRE estavam reduzidos no timo da linhagem NOD. Esses resultados sugerem a participação de algum mecanismo de atenuação póstrascricional de Aire nessa linhagem provavelmente envolvendo atuação de miRNAs. Isso poderia explicar o desbalanço de PTAs Aire-dependentes e a repressão autoantígenos relacionados ao DM1. Concluímos que nossos resultados, além de abrir novas perspectivas para pesquisas nesta área, contribuem com melhor compreensão dos mecanismos moleculares desencadeados por Aire e por miRNAs no controle da expressão de autoantígenos no timo o que é importante para a tolerância imunológica central. / The thymus is a primary lymphoid organ, in which occurs in the induction of central immune tolerance to self peripheral tissue antigens (PTAs). The thymic medulla is formed by medullary thymic epithelial cells (mTECs) expressing hundreds of such PTAs representing virtually all organs and tissues of the body. This phenomenon has been termed promiscuous gene expression (PGE), which is partially regulated by the Autoimmune regulator (Aire) gene. The T cell precursors derived from the bone marrow migrate to the thymus (now termed thymocytes). A part of these thymocytes are eliminated by negative selection mediated mTEC cells. The surviving cells to evolve and functional mature T cells that migrate to the periphery and are capable of recognizing MHC molecules and are tolerant to PTAs. In addition to controlling the transcription of PTA genes, Aire also controls the expression of microRNAs (miRNAs). The negative selection in the thymus is a process essential to the maintenance of immunologic self-tolerance and imbalance of this process is associated with the development of autoimmune diseases such as type 1 diabetes mellitus (DM1) . Given these assumptions, our work was based on two hypothesis: 1) Changes in the expression of the Aire gene can disrupt the expression of PTA genes and miRNAs in the thymus, causing changes in PGE, 2) The balanced expression of Aire / or PTA genes in mTECs is fundamental for central tolerance. The imbalance in the expression of these genes is associated with the emergence of type 1 diabetes in mice. To test our first hypothesis we made Aire silencing (Aire knockdown) through electrotransfection of anti - Aire interfering RNA (siRNA) in vivo in the thymus of BALB/c mice. Analysis of the transcriptome (mRNAs) and miRNome (miRNAs) of mTECs revealed that partial and transient silencing of Aire was enough to affect the expression of Aire - dependent PTAs as well as miRNAs. miRNA -mRNA interaction networks revealed that the posttranscriptional control of PGE is also affected by the silencing of Aire. The results show that Aire and can form an miRNA pathway essential for the induction of central tolerance. To test our second hypothesis we compared the transcriptome of mTECs of BALB/c mice (non-autoimmune strain) with mTECs from non - obese diabetic NOD (animal model used in studies of autoimmune DM1) . Our results indicate that the transcriptional expression of DM1-related autoantigens are unbalanced in NOD mice in an very early stage, when these animals have not had clinical disease (pre-diabetic period). Unexpectedly, the transcriptional levels of Aire in the thymus was equivalent in these two strains, but the AIRE protein levels were reduced in thymus of NOD strain. These results suggest that some mechanism of post-transcriptional attenuation of Aire is acting in this lineage probably involving action of miRNAs . This could explain the imbalance of Aire - dependent PTAs and repression autoantigens related to DM1. Our results open perspectives for research in this area, contributing to better understanding the molecular mechanisms triggered by Aire and miRNAs in control of the expression of autoantigens in the thymus, which is important for the central immune tolerance.

Today, enzymes are extensively used for many industrial applications, this includes bulk and fine-chemical synthesis, pharmaceuticals and consumer products. Though Nature has perfected enzymes for many millions of years, they seldom reach industrial performance targets. Natural enzymes could benefit from protein redesign experiments to gain novel functions or optimize existing functions. Glutathione transferases (GSTs) are detoxification enzymes, they also display disparate functions. Two Mu-class GSTs, M1-1 and M2-2, are closely related but display dissimilar substrate selectivity profiles. Saturation mutagenesis of a previously recognized hypervariable amino acid in GST M2-2, generated twenty enzyme variants with altered substrate selectivity profiles, as well as modified thermostabilities and expressivities. This indicates an evolutionary significance; GST Mu-class enzymes could easily alter functions in a duplicate gene by a single-point mutation. To further identify residues responsible for substrate selectivity in the GST M2-2 active site, three residues were chosen for iterative saturation mutagenesis. Mutations in position10, identified as highly conserved, rendered enzyme variants with substrate selectivity profiles resembling that of specialist enzymes. Ile10 could be conserved to sustain the broad substrate acceptance displayed by GST Mu-class enzymes. Enzymes are constructed from primarily twenty amino acids, it is a reasonable assumption that expansion of the amino acid repertoire could result in functional properties that cannot be accomplished with the natural set of building blocks. A combination approach of site-directed mutagenesis and chemical modifications in GST M2-2 and GST M1-1 resulted in novel enzyme variants that displayed altered substrate selectivity patterns as well as improved enantioselectivities. The results presented in this thesis demonstrate the use of different protein redesign techniques to modulate various functions in Mu-class GSTs. These techniques could be useful in search of optimized enzyme variants for industrial targets. / biokemi och organisk kemi

O timo é um orgão linfóide primário, no qual ocorre a indução da tolerância imunológica central aos antígenos do próprio que são expressos pelos tecidos periféricos (PTAs). A medula tímica é formada por células tímicas medulares epiteliais (mTECs) que expressam centenas desses PTAs que representam virtualmente todos os órgãos e tecidos do corpo. Esse fenômeno foi denominado de expressão gênica promíscua (PGE) a qual é parcialmente regulada pelo modulador da transcrição Autoimmune regulator (Aire). Os precursores de células T oriundos da medula óssea migram para o timo (agora são denominados de timócitos) e na medula desse órgão, passam pela seleção negativa mediada pelas mTECs. As células sobreviventes evoluem para células T maduras e funcionais que migram para a periferia com capacidade de reconhecimento das moléculas de MHC e tolerantes aos PTAs. Além de controlar a transcrição de genes PTAs, Aire também controla a expressão de microRNAs (miRNAs), relacionados com a integridade e funcionalidade do microambiente tímico. A seleção negativa no timo é um processo essencial para a manutenção da autotolerância imunológica e o desbalanço desse processo está associado com o desenvolvimento de doenças autoimunes como, por exemplo, o diabetes mellitus do tipo 1 (DM1).Tendo em vista essas premissas, nosso trabalho se fundamentou em duas hipóteses: 1) Variações na expressão do gene Aire podem perturbar a expressão de genes PTAs e miRNAs no timo, causando alterações na PGE, 2) A expressão balanceada de genes como Aire e/ou PTAs nas mTECs, é fundamental para a integridade da tolerância central. O desbalanço na expressão desses genes, está associado com a emergência do diabetes mellitus tipo 1 no camundongo. Para testar nossa primeira hipótese efetuamos o silenciamento de Aire (Aire knockdown) por meio de eletrotransfeção de RNA interferente (siRNA) anti-Aire in vivo no timo de camundongos BALB/c. Análises do transcriptoma (mRNAs) e miRNoma (miRNAs) das mTECs, revelaram que silenciamento parcial e transitório de Aire foi suficiente para afetar a expressão de PTAs Aire dependentes bem como a de miRNAs. Redes de interação miRNA-mRNA, revelaram que o controle pós-transcricional da PGE também é afetado pelo silenciamento de Aire. Os resultados encontrados revelam que Aire e miRNAs podem formar uma via essencial durante a indução da tolerância central. Para testar nossa segunda hipótese comparamos o transcriptoma de mTECs de camundongos BALB/c (linhagem não-autoimune) com mTECs de camundongos non-obese diabetic NOD (modelo animal utilizado nos estudos de DM1 autoimune). Nossos resultados revelaram que a expressão transcricional de autoantígenos relacionados ao DM1 está desbalanceada em camundongos NOD já numa fase precoce, quando esses animais ainda não apresentavam a doença clínica (fase pré-diabética). Inesperadamente, os níveis transcrionais de Aire apresentaram-se equivalentes no timo dessas duas linhagens, porém os níveis da proteína AIRE estavam reduzidos no timo da linhagem NOD. Esses resultados sugerem a participação de algum mecanismo de atenuação póstrascricional de Aire nessa linhagem provavelmente envolvendo atuação de miRNAs. Isso poderia explicar o desbalanço de PTAs Aire-dependentes e a repressão autoantígenos relacionados ao DM1. Concluímos que nossos resultados, além de abrir novas perspectivas para pesquisas nesta área, contribuem com melhor compreensão dos mecanismos moleculares desencadeados por Aire e por miRNAs no controle da expressão de autoantígenos no timo o que é importante para a tolerância imunológica central. / The thymus is a primary lymphoid organ, in which occurs in the induction of central immune tolerance to self peripheral tissue antigens (PTAs). The thymic medulla is formed by medullary thymic epithelial cells (mTECs) expressing hundreds of such PTAs representing virtually all organs and tissues of the body. This phenomenon has been termed promiscuous gene expression (PGE), which is partially regulated by the Autoimmune regulator (Aire) gene. The T cell precursors derived from the bone marrow migrate to the thymus (now termed thymocytes). A part of these thymocytes are eliminated by negative selection mediated mTEC cells. The surviving cells to evolve and functional mature T cells that migrate to the periphery and are capable of recognizing MHC molecules and are tolerant to PTAs. In addition to controlling the transcription of PTA genes, Aire also controls the expression of microRNAs (miRNAs). The negative selection in the thymus is a process essential to the maintenance of immunologic self-tolerance and imbalance of this process is associated with the development of autoimmune diseases such as type 1 diabetes mellitus (DM1) . Given these assumptions, our work was based on two hypothesis: 1) Changes in the expression of the Aire gene can disrupt the expression of PTA genes and miRNAs in the thymus, causing changes in PGE, 2) The balanced expression of Aire / or PTA genes in mTECs is fundamental for central tolerance. The imbalance in the expression of these genes is associated with the emergence of type 1 diabetes in mice. To test our first hypothesis we made Aire silencing (Aire knockdown) through electrotransfection of anti - Aire interfering RNA (siRNA) in vivo in the thymus of BALB/c mice. Analysis of the transcriptome (mRNAs) and miRNome (miRNAs) of mTECs revealed that partial and transient silencing of Aire was enough to affect the expression of Aire - dependent PTAs as well as miRNAs. miRNA -mRNA interaction networks revealed that the posttranscriptional control of PGE is also affected by the silencing of Aire. The results show that Aire and can form an miRNA pathway essential for the induction of central tolerance. To test our second hypothesis we compared the transcriptome of mTECs of BALB/c mice (non-autoimmune strain) with mTECs from non - obese diabetic NOD (animal model used in studies of autoimmune DM1) . Our results indicate that the transcriptional expression of DM1-related autoantigens are unbalanced in NOD mice in an very early stage, when these animals have not had clinical disease (pre-diabetic period). Unexpectedly, the transcriptional levels of Aire in the thymus was equivalent in these two strains, but the AIRE protein levels were reduced in thymus of NOD strain. These results suggest that some mechanism of post-transcriptional attenuation of Aire is acting in this lineage probably involving action of miRNAs . This could explain the imbalance of Aire - dependent PTAs and repression autoantigens related to DM1. Our results open perspectives for research in this area, contributing to better understanding the molecular mechanisms triggered by Aire and miRNAs in control of the expression of autoantigens in the thymus, which is important for the central immune tolerance.

No description available.

Com o objetivo de buscar e identificar novo(s) inibidor(es) da cruzaína uma cisteíno-protease do Trypanosoma cruzi, o agente etiológico da doença de Chagas foram propostos, validados e, a seguir, aplicados sobre a biblioteca de compostos ZINC (3.294.714 compostos), dois modelos de virtual screening (Modelos I e II). Os modelos de virtual screening propostos, contendo seqüências de filtros físicoquímicos, farmacofóricos, de docking e de seleção por inspeção visual, foram construídos a partir de informações de 13 complexos da cruzaína e de 20 complexos de outras cisteínoprotease, cujas estruturas estão disponíveis no PDB. Numa primeira etapa, o reconhecimento detalhado das características estruturais da cruzaína foi realizado por inspeção visual; pelos campos de interação molecular, gerados pelo programa GRID; pela identificação das propriedades de interação molecular na superfície da cavidade, geradas pelo programa CA VBASE e; por simulações de dinâmica molecular. O Modelo I de virtual screeníng - obtido a partir do reconhecimento das estruturas dos 13 complexos da cruzaína depositadas no PDB - foi aplicado sobre o ZINC, selecionando 10 compostos, dos quais 6 compostos foram adquiridos e submetidos ao teste de inibição enzimática da cruzaína, para a validação experimental do modelo. Observou-se que 3 destes compostos (ZINC02470662, ZINC02682879 e ZINC03192044, respectivamente) não mostraram inibição significativa da cruzaína, nas condições experimentais utilizadas, até a concentração de 7 mM, enquanto que os 3 restantes (ZINC02663001, ZINC01936854 e ZINC03326243, respectivamente) apresentaram inibição enzimática inespecífica, sugerindo que estes últimos agem pelo mecanismo promíscuo. O mecanismo promíscuo de inibição enzimática, foi verificado pela adição de 0,1% Triton X-100 no ensaio enzimático, observando-se a correspondente perda de inibição da cruzaína. Para estes compostos, a confirmação do mecanismo promíscuo foi feita observando-se a perda de inibição da enzima, após o aumento em dez vezes da concentração da cruzaína no ensaio enzimático. O Modelo II - obtido a partir do reconhecimento das estruturas dos 13 complexos da cruzaína e dos 20 complexos de outras cisteíno-proteases, identificadas na busca por cavidades similares à cruzaína - foi aplicado sobre o banco de dados ZINC,selecionando 55 compostos dos quais 19 foram adquiridos e submetidos ao teste de inibição enzimática da cruzaína, para validação experimental do modelo. Observou-se que o composto ZINC01794422 apresentou inibição específica da enzima com constante de inibição no valor de Ki = 21 µM, enquanto que os demais 18 compostos não mostraram inibição significativa, nas condições experimentais utilizadas, até a concentração de 592 µM. O mecanismo promíscuo de inibição enzimática não foi observado, uma vez que todos os testes foram realizados com 0,1% de Triton X-100. O Modelo II identificou, ainda, mais dois inibidores da cruzaína (ZINC04899534 e ZINC01547017) que, por serem estruturalmente semelhantes aos utilizados na construção do modelo e já terem sido descritos na literatura, não foram adquiridos ou testados nos ensaios enzimáticos. Considerando apenas o novo inibidor identificado, o Modelo II apresentou uma taxa de acerto de 5,3%. Este valor esta de acordo com as taxas de acerto encontradas na literatura que variam entre 1 a 50% . / In order to search and identify new cruzain inhibitor(s) - a cysteine-protease of Trypanosoma cruzi, the etiologic agent of Chagas disease - two virtual screening schemes(Models I and II) were proposed, validated- and applied to the ZINC database (3.294.714 compounds). The proposed virtual screening models, bearing a sequence of different physicalchemical, pharmacophore and docking filters, as well as a visual inspection filter, were built from information taken from 13 cruzain complexes and from 20 complexes of other cysteine proteases, having their structures available in PDB. In a first step, a detailed recognition of the cruzain structural features and characteristics was performed through visual inspection of the enzyme environment; followed by the analysis of GRID generated molecular interaction fields; through the identification of molecular interaction properties exposed at the enzyme cavity surface, generated by the CAVBASE program; and by molecular dynamics simulations. The virtual screening Model I, - generated from the structural characteristics recognized from 13 PDB cruzain complexes - when applied to the ZINC database selected 10 compounds. For the experimental validation ofthe model, six ofthese compounds have been acquired and were tested as cruzain inhibitors. It was observed that three of the tested compounds (ZINC02470662, ZINC02682879 and ZINC03192044, respectively) did not show any significant cruzain inhibition, up to 7 mM. Meanwhile the other three tested compounds (ZINC02663001, ZINC01936854 and ZINC03326243, respectively) showed an unspecific cruzain inhibition, suggesting that an enzyme inhibition by promiscuous mechanism occurred. This mechanism was verified by the addition of 0.1% Triton X-100 on the enzymatic assay with a concomitant loss of cruzain inhibition activity. For these compounds, the confirmation of the promiscuous mechanism was also done, observing the loss of enzyme inhibition, after a ten times increase in the cruzain concentration on the enzymatic assay. The virtual screenmg Model II - generated from the structural characteristics recognized from 13 cruzain complexes and 20 complexes of other cysteine proteases, that have been identified on a search for cavities similar to cruzain - selected 55 compounds, when applied to the ZINC database. In order to experimentally validate the model, nineteen compounds have been acquired and were tested as cruzain inhibitors. It has been observed that one compound, ZINC01794422, showed a specific cruzain inhibition (Ki = 21 µM), while the other eighteen showed no significant inhibition, up to 592 µM concentration. The promiscuous mechanism of enzymatic inhibition was not observed, since 0.1% of Triton X-100 was added in ali assays. Additionally, Model II identified two other cruzain inhibitors (ZINC04899534 and ZINC01547017). However, these compounds have not been acquired or tested, since they are known cruzain inhibitors - already described in the literature and are structurally similar to the inhibitors used in the construction of the mode!. Referring to new inhibitors found, Model II showed a hit rate of 5,3%. This value is in agreement with those found in the literature, which ranges from 1 to 50%.

Com o objetivo de buscar e identificar novo(s) inibidor(es) da cruzaína uma cisteíno-protease do Trypanosoma cruzi, o agente etiológico da doença de Chagas foram propostos, validados e, a seguir, aplicados sobre a biblioteca de compostos ZINC (3.294.714 compostos), dois modelos de virtual screening (Modelos I e II). Os modelos de virtual screening propostos, contendo seqüências de filtros físicoquímicos, farmacofóricos, de docking e de seleção por inspeção visual, foram construídos a partir de informações de 13 complexos da cruzaína e de 20 complexos de outras cisteínoprotease, cujas estruturas estão disponíveis no PDB. Numa primeira etapa, o reconhecimento detalhado das características estruturais da cruzaína foi realizado por inspeção visual; pelos campos de interação molecular, gerados pelo programa GRID; pela identificação das propriedades de interação molecular na superfície da cavidade, geradas pelo programa CA VBASE e; por simulações de dinâmica molecular. O Modelo I de virtual screeníng - obtido a partir do reconhecimento das estruturas dos 13 complexos da cruzaína depositadas no PDB - foi aplicado sobre o ZINC, selecionando 10 compostos, dos quais 6 compostos foram adquiridos e submetidos ao teste de inibição enzimática da cruzaína, para a validação experimental do modelo. Observou-se que 3 destes compostos (ZINC02470662, ZINC02682879 e ZINC03192044, respectivamente) não mostraram inibição significativa da cruzaína, nas condições experimentais utilizadas, até a concentração de 7 mM, enquanto que os 3 restantes (ZINC02663001, ZINC01936854 e ZINC03326243, respectivamente) apresentaram inibição enzimática inespecífica, sugerindo que estes últimos agem pelo mecanismo promíscuo. O mecanismo promíscuo de inibição enzimática, foi verificado pela adição de 0,1% Triton X-100 no ensaio enzimático, observando-se a correspondente perda de inibição da cruzaína. Para estes compostos, a confirmação do mecanismo promíscuo foi feita observando-se a perda de inibição da enzima, após o aumento em dez vezes da concentração da cruzaína no ensaio enzimático. O Modelo II - obtido a partir do reconhecimento das estruturas dos 13 complexos da cruzaína e dos 20 complexos de outras cisteíno-proteases, identificadas na busca por cavidades similares à cruzaína - foi aplicado sobre o banco de dados ZINC,selecionando 55 compostos dos quais 19 foram adquiridos e submetidos ao teste de inibição enzimática da cruzaína, para validação experimental do modelo. Observou-se que o composto ZINC01794422 apresentou inibição específica da enzima com constante de inibição no valor de Ki = 21 µM, enquanto que os demais 18 compostos não mostraram inibição significativa, nas condições experimentais utilizadas, até a concentração de 592 µM. O mecanismo promíscuo de inibição enzimática não foi observado, uma vez que todos os testes foram realizados com 0,1% de Triton X-100. O Modelo II identificou, ainda, mais dois inibidores da cruzaína (ZINC04899534 e ZINC01547017) que, por serem estruturalmente semelhantes aos utilizados na construção do modelo e já terem sido descritos na literatura, não foram adquiridos ou testados nos ensaios enzimáticos. Considerando apenas o novo inibidor identificado, o Modelo II apresentou uma taxa de acerto de 5,3%. Este valor esta de acordo com as taxas de acerto encontradas na literatura que variam entre 1 a 50% . / In order to search and identify new cruzain inhibitor(s) - a cysteine-protease of Trypanosoma cruzi, the etiologic agent of Chagas disease - two virtual screening schemes(Models I and II) were proposed, validated- and applied to the ZINC database (3.294.714 compounds). The proposed virtual screening models, bearing a sequence of different physicalchemical, pharmacophore and docking filters, as well as a visual inspection filter, were built from information taken from 13 cruzain complexes and from 20 complexes of other cysteine proteases, having their structures available in PDB. In a first step, a detailed recognition of the cruzain structural features and characteristics was performed through visual inspection of the enzyme environment; followed by the analysis of GRID generated molecular interaction fields; through the identification of molecular interaction properties exposed at the enzyme cavity surface, generated by the CAVBASE program; and by molecular dynamics simulations. The virtual screening Model I, - generated from the structural characteristics recognized from 13 PDB cruzain complexes - when applied to the ZINC database selected 10 compounds. For the experimental validation ofthe model, six ofthese compounds have been acquired and were tested as cruzain inhibitors. It was observed that three of the tested compounds (ZINC02470662, ZINC02682879 and ZINC03192044, respectively) did not show any significant cruzain inhibition, up to 7 mM. Meanwhile the other three tested compounds (ZINC02663001, ZINC01936854 and ZINC03326243, respectively) showed an unspecific cruzain inhibition, suggesting that an enzyme inhibition by promiscuous mechanism occurred. This mechanism was verified by the addition of 0.1% Triton X-100 on the enzymatic assay with a concomitant loss of cruzain inhibition activity. For these compounds, the confirmation of the promiscuous mechanism was also done, observing the loss of enzyme inhibition, after a ten times increase in the cruzain concentration on the enzymatic assay. The virtual screenmg Model II - generated from the structural characteristics recognized from 13 cruzain complexes and 20 complexes of other cysteine proteases, that have been identified on a search for cavities similar to cruzain - selected 55 compounds, when applied to the ZINC database. In order to experimentally validate the model, nineteen compounds have been acquired and were tested as cruzain inhibitors. It has been observed that one compound, ZINC01794422, showed a specific cruzain inhibition (Ki = 21 µM), while the other eighteen showed no significant inhibition, up to 592 µM concentration. The promiscuous mechanism of enzymatic inhibition was not observed, since 0.1% of Triton X-100 was added in ali assays. Additionally, Model II identified two other cruzain inhibitors (ZINC04899534 and ZINC01547017). However, these compounds have not been acquired or tested, since they are known cruzain inhibitors - already described in the literature and are structurally similar to the inhibitors used in the construction of the mode!. Referring to new inhibitors found, Model II showed a hit rate of 5,3%. This value is in agreement with those found in the literature, which ranges from 1 to 50%.