This thesis investigates the design, synthesis and biological assay of cysteine protease inhibitors within the papain superfamily of cysteine proteases. This is achieved by examining the effect of inhibitor design, especially warheads, on IC₅₀ values and structureactivity relationships between cysteine protease inhibitors of the papain superfamily. The representative proteases used are m-calpain, μ-calpain, cathepsin B and papain. Chapter One is an introductory chapter; Chapters Two-Four describe the design and synthesis of cysteine protease inhibitors; Chapter Five discusses assay protocol; and Chapter Six contains the assay results and structure-activity relationships of the synthesised inhibitors. Chapter One introduces cysteine proteases of the papain family and examines the structure, physiology and role in disease of papain, cathepsin B, m-calpain and μ-calpain. The close structural homology that exists between these members of the papain superfamily is identified, as well characteristics unique to each protease. Covalent reversible, covalent irreversible and non-covalent warheads are defined. The generic inhibitor scaffold of address region, recognition and warhead, upon which the inhibitors synthesised in this thesis are based, is also introduced. Chapter Two introduces reversible cysteine protease inhibitors found in the literature and that little is known about the effect of inhibitor warhead on selectivity within the papain superfamily. Oxidation of the dipeptidyl alcohols 2.6, 2.26, 2.29, 2.30, 2.35 and 2.36 utilising the sulfur trioxide-pyridine complex gave the aldehydes 2.3, 2.27, 2.19, 2.2, 2.21 and 2.22. Semicarbazones 2.37-2.40 were synthesised by a condensation reaction between the alcohol 2.3 and four available semicarbazides. The amidoximes 2.48 and 2.49 separately underwent thermal intramolecular cyclodehydration to give the 3-methyl-1,2,4- oxadiazoles 2.41 and 2.50. The aldehydes 2.3 and 2.27 were reacted with potassium cyanide to give the cyanohydrins 2.51 and 2.52. The cyanohydrins 2.51 and 2.52 were separately reacted to give 1) the α-ketotetrazoles 2.43 and 2.55; 2) the α-ketooxazolines 2.42 and 2.58; 3) the esterified cyanohydrins 2.60 and 2.61. A two step SN2 displacement reaction of the alcohol 2.6 to give the azide 2.62, an example of a non-covalent cysteine protease inhibitor. Chapter Three introduces inhibitors with irreversible warheads. The well-known examples of epoxysuccinic acids 3.1 and 3.5 are discussed in detail, highlighting the lack of irreversible cysteine protease specific inhibitors. The aldehydes 2.3 and 2.27 were reacted under Wittig conditions to give the α,β-unsaturated carbonyls 3.14-3.18. Horner- Emmons-Wadsworth methodology was utilised for the synthesis of the vinyl sulfones 3.20- 3.23. The dipeptidyl acids 2.24 and 2.28 were separately reacted with diazomethane to give the diazoketones 3.25 and 3.26. The diazoketones 3.25 and 3.26 were separately reacted with hydrogen bromide in acetic acid (33%) to give the α-bromomethyl ketones 3.27 and 3.28, which were subsequently reduced to give the α-bromomethyl alcohols 3.29-3.32. Under basic conditions the α-bromomethyl alcohols 3.29-3.32 ring-closed to form the peptidyl epoxides 3.33-3.36. Chapter Four introduces the disadvantages of peptide-based inhibitors. A discussion is given on the benefits of constraining inhibitors into the extended bioactive conformation known as a β-strand. Ring closing metathesis is utilised in the synthesis of the macrocyclic aldehyde 4.4, macrocyclic semicarbazone 4.15, the macrocyclic cyanohydrin 4.16, the macrocyclic α-ketotetrazole 4.18 and the macrocyclic azide 4.19. Chapter Five introduces enzyme inhibition studies. The BODIPY-casein fluorogenic assay used for establishing inhibitor potency against m-calpain and μ-calpain is validated. Assay protocols are also established and validated for cathepsin B, papain, pepsin and α- chymotrypsin. A discussion of the effect of solvent on enzyme activity is also included as part of this study. Chapter Six presents the assay results for all the inhibitors synthesised throughout this thesis and an extensive structure-activity relationship study between inhibitors is included. The alcohols 2.26 and 2.30 are unprecedented examples of non-covalent, potent, cathepsin B inhibitors (IC₅₀ = 0.075 μM selectivity 80-fold and 1.1 μM, selectivity 18-fold). The macrocyclic semicarbazone 4.15 is an unprecedented example of a potent macrocyclic cysteine protease inhibitor (m-calpain: IC₅₀ = 0.16 μM, selectivity 8-fold). The cyanohydrin 2.51 contains an unprecedented cysteine protease warhead and is a potent and selective inhibitor of papain (IC₅₀ = 0.030 μM, selectivity 3-fold). The O-protected cyanohydrin 2.61 is a potent and selective inhibitor of pepsin (IC₅₀ = 1.6 μM, selectivity 1.5-fold). The top ten warheads for potent, selective cathepsin B inhibition are: carboxylic acid, methyl ester, diazoketone, esterified cyanohydrin, α-bromomethyl ketone, α,β- unsaturated aldehyde, vinyl sulfones, α-bromomethyl-C₃-S,R-alcohol, alcohol and α,β- unsaturated ethyl ester. The selectivity of these warheads was between 5- and 130-fold for cathepsin B. The best inhibitors for cathepsin B were the α-bromomethyl ketone 3.26 (IC₅₀ = 0.075 μM, selectivity 16-fold), the α,β-unsaturated aldehyde 3.18 (IC₅₀ = 0.13 μM, selectivity 13-fold) and the esterified cyanohydrin 3.59 (IC₅₀ = 0.35 μM, selectivity 22- fold). Chapter Seven outlines the experimental details and synthesis of the compounds prepared in this thesis.

Orientador: Prof. Dr. Rodrigo L. O. R. Cunha. / Dissertação (mestrado) - Universidade Federal do ABC. Programa de Pós-Graduação em Ciência e Tecnologia/Química, 2017. / Nos últimos anos o rol de propriedades biológicas de compostos de selênio e de telúrio tem aumentado e outras atividades, além das bem descritas propriedades antioxidantes, também vêm sendo demonstradas. Neste contexto, a inibição enzimática promovida por espécies hipervalentes de selênio e telúrio (organocalcogenanas) foi relatada para cisteína proteases, tirosina fosfatases e o proteasoma, por exemplo. Esta inibição é resultado da particular reatividade que compostos hipervalentes de telúrio e selênio apresentam com tióis. Como a quantidade de biotióis reativos em sistemas celulares é vasta e nem todos estão disponíveis para ensaios in vitro com as organocalcogenanas, é possível vislumbrar a aplicação desses compostos como ligantes em cromatografia de afinidade para a identificação de proteínas reativas a esses compostos. Para atingir este objetivo foram utilizados organocalcogenetos derivados de benzaldeído. A estrutura dos ligantes é constituída por um grupo fenilcalcogenila ligado a um derivado de poliéter funcionalizado com um grupo amina que é ancorado à uma matriz de agarose. Foram preparadas duas classes de ligantes para a modificação de agarose que diferem na estrutura do espaçador, a primeira contém uma unidade de ácido succínico entre o organocalcogeneto e o derivado de poliéter (ligantes de Classe I) e a outra, isenta dessa unidade de ácido succínico, possui um grupo amino ionizável (ligantes catiônicos de Classe II). A rota de preparação dos ligantes de classe I envolveu uma sequencia sintética de seis etapas com rendimentos globais de 26% e 19% e os ligantes de Classe II foram preparados em uma sequencia de duas etapas com rendimentos globais de 43% e 22%, para os selenetos e teluretos, respectivamente. A oxido-redução do ligante de telúrio da classe II se mostrou rápido e satisfatório nos estudos preliminares, dando bons indícios para aplicação como ligante na cromatografia de afinidade. Por fim, os ligantes foram imobilizados na agarose funcionalizada com brometo de cianogênio e avaliados com papaína, utilizada como modelo de biotiol nesse trabalho. Notadamente os ligantes de telúrio apresentaram um comportamento distinto do que os de selênio quanto à eluição da papaína da matriz sólida, requerendo maior volume de solução com agente redutor para a eluição da proteína. Coletivamente os resultados apresentados nesse trabalho demonstram que matrizes sólidas funcionalizadas com organocalcogenetos são funcionais para a retenção e liberação de biomoléculas tioladas (funcionalidade "catch & release"), abrindo perspectivas para o estudo mais aprofundado da interação entre calcogenetos com tióis e, ainda para produção, caracterização e estudo de aplicações de diversas matrizes funcionalizadas com organocalcogenetos. / In recent years the role of biological properties of selenium compounds and tellurium compounds has increased, and other activities besides the well-described antioxidant properties have also been demonstrated. In this context, enzymatic inhibition promoted by hypervalent species of selenium and tellurium have been reported for cysteine proteases and tyrosine phosphatases. This activity is due to the particular reactivity of thiols with the chalcogenides atom in their hypervalent state. Apart from these enzymes, one can envisage a plenty of other potential reactive thiols in cellular systems that are prone to react with organocalcogenuranes which are still unknown. As the identification of these unknown targets would be helpful to understand therapeutical and toxicological aspects of these compounds, efforts in this direction are required. One strategy for this purpose consists in using the affinity purification technique using organochalcogenides as ligands, this is proposed in the present work. To achieve this goal, the use of organochalcogenides derived from benzylamines as ligands in affinity chromatography. The structure of this ligands contains a phenylchalcogenide moiety, a polyether spacer group functionalized with an amino group to attach it to an agarose substrate. Two different spacer classes were prepared, the first has a succinic acid spacer between the organochalcogenide and the polyether (Class I ligand); the second class has the polyether moiety directly linked to the phenylchalcogenide group and has an ionizable secondary amine (Class II cationic ligand). The preparation route of the class I ligands involved a six-step synthetic sequence in overall yields of 26% and 19%, and the Class II ligands were prepared in a two-step sequence in global yields of 43% and 22% of the selenides and tellurides, respectively. The studied oxidation and reduction reactions of Class II tellurium ligands revealed fast processes, suitable for a practical application of these novel organochalcogenides in affinity chromatography. Finally, ligands were immobilized in cyanogen bromide activated agarose and the modified matrices evaluated with papain as a model biothiol. Noteworthy, the selenide ligands released papain promptly by washing the matrix with dithiothreitol solution whereas telluride ligands showed the opposite behavior. Collectively, the results presented in this work demonstrate that organochalcogenide-modified agarose is a new class of functional materials for the retention and release of biothiols presenting a "catch & release" functionality. This work brings new perspectives for a more detailed study of the interaction between chalcogenides with thiols and also to the production, characterization and the study of novel applications of organochalcogenide-modified solid matrices.

IN ENGLISH Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Supervisors: prof. Dr. Michael Gütschow RNDr. Klára Konečná, Ph.D. Candidate: Michaela Hympánová Title of the diploma thesis: Enzyme kinetic evaluation of several potential inhibitors of certain human cysteine and serine proteases Background Cysteine and serine proteases are enzymes involved in many physiological processes. The imbalance between them and their endogenous inhibitors is associated with various diseases such as cancer and osteoporosis. Synthetic inactivators could be useful in the treatment of these enzyme-mediated pathological conditions. Therefore, there are ongoing attempts to develop low-molecular weight inactivators for therapeutically relevant cysteine and serine proteases. In the course of this thesis, compounds synthesized in prof. Gütschow's group were investigated as potential inhibitors of selected human proteases. They belong to imidazole compounds derived from N-protected cyclohexylalanine, 2-phenyl-7,8-dihydroimidazo[1,2- a]pyrazin-6(5H)-one derivatives, ,-unsaturated peptidomimetic compounds, carbamates, an N,N-dibenzylcrotonamide derivatives and peptoides. Aims This diploma thesis has been focused on the evaluation of new potential inhibitors against...

<p align=\"justify\">A necessidade urgente de descoberta de terapias mais seguras e eficazes para o tratamento da doença de Chagas e leishmanioses tem motivado a pesquisa por novos inibidores das enzimas cruzaína e CPB, as principais cisteíno proteases do T. cruzie e Leishmania spp., respectivamente. Uma série de 52 compostos nitrílicos que atuam como inibidores covalente-reversíveis de cisteíno proteases foi sintetizada no grupo NEQUIMED/IQSC/USP e avaliada quanto a sua atividade inibitória contra as enzimas cruzaína, CPB de Leishmania mexicana e catepsina L de humanos. Utilizando planejamento molecular baseado em hipótese, mapeamos as relações estrutura-atividade (SARs) desses inibidores através de variações nas posições P1, P2, P3 e P1\' do esqueleto dipeptidil nitrílico. A substituição do grupo eletrofílico (warhead) aminonitrila em P1 pelo grupo azanitrila melhorou a afinidade em duas ordens de magnitude para todos os alvos avaliados. Um dos mais potentes inibidores, o análogo azanitrila Neq0690 mostrou uma cinética de ligação lenta com valores de pKi de 8,8, 9,3 e 9,7 para cruzaína, catepsina L e LmCPB, respectivamente. A substituição bioisostérica da ligação amida entre as posições P2-P3 pelo grupo trifluoroetilamina resultou na síntese do Neq0659, um potente inibidor com um perfil de ação seletivo para as proteases de parasitos. A substituição do grupo metileno em P1 pelo ciclopropano aumentou a afinidade para todas as enzimas. Contudo, uma inibição seletiva da cruzaína e LmCPB foi associada à presença do grupo (R)-benzila como substituinte da posição P1 dos derivados CF3 substituídos. Embora os compostos substituídos com leucina, tirosina, triptofano e 3-cloro fenilalanina como substituintes da posição P2 foram relativamente bem tolerados pela cruzaína e catepsina L, uma restrita especificidade foi verificada para LmCPB com pequenos ganhos de afinidade para os inibidores que possuíam os grupos leucina e metil benzoato como substituintes dessa posição. Com relação à posição P3, a inserção do grupo 3-terc-butilpirazol e 3-bromo piridina aumentou a afinidade para todos os alvos avaliados enquanto que um ganho seletivo para a LmCPB foi observado para os compostos que possuíam o grupo bifenila nessa posição. Além disso, duas novas estruturas cristalográficas da LmCPB complexada com o Neq0690 e metil metanotiossulfonato (MMTS) foram determinadas com resoluções de 1,3 Å e 1,5 Å, respectivamente. As estruturas dos co-complexos revelaram os modos de interação (MoB) desses ligantes, bem como as principais características do processo de reconhecimento bimolecular. Isso permitirá o uso de estratégias de planejamento baseado na estrutura do alvo com translação natural para a pesquisa por novos inibidores de cisteíno proteases, com amplo espectro de ação na quimioterapia de doenças a elas relacionadas. / <p align=\"justify\">The urgent need for the discovery of safer and more effective therapies for the treatment of Chagas disease and leishmaniasis has motivated the search for new inhibitors of the enzymes cruzain and CPB, the major T. cruzi and Leishmania spp. cysteine proteases, respectively. A series of 52 nitrile-containing compounds acting as covalent-reversible inhibitors of cysteine proteases was synthesized at the NEQUIMED/IQSC/USP Medicinal Chemistry Group and evaluated for their inhibitory activity against the enzymes cruzain, Leishmania mexicana CPB and cathepsin L from humans. Using hypothesis-driven molecular design, we mapped the structure-activity relationships (SARs) of these inhibitors through variations in the P1, P2, P3 and P1\' positions of the dipeptidyl nitrile scaffold. The substitution of the aminonitrile by the azanitrile group improved the affinity by two orders of magnitude for all the evaluated targets. One of the most potent inhibitors, the azanitrile analogue dubbed Neq0690 showed a slow-binding kinetics with pKi values of 8.8, 9.3 and 9.7 for cruzain, cathepsin L and LmCPB, respectively. Bioisosteric substitution of the amide moiety between the P2-P3 positions by the trifluoroethylamine group resulted in the synthesis of Neq0659, a potent inhibitor with a selective action profile for parasite proteases. Substitution of the methylene group at P1 by cyclopropane increased the affinity for all enzymes. However, selective inhibition of cruzain and LmCPB was associated with the presence of the (R)-benzyl group as substituent of the P1 position of the substituted CF3 derivatives. Although leucine, tyrosine, tryptophan and 3-chloro phenylalanine substituted compounds as substituents of the P2 position were relatively well tolerated by cruzain and cathepsin L, a restricted specificity was verified for LmCPB with small affinity gains for the inhibitors possessing the leucine and methyl benzoate as substituents of that position. Regarding the P3 position, the insertion of the 3-tert-butylpyrazole and 3-bromo pyridine groups increased the affinity for all evaluated targets whereas a selective gain for LmCPB was observed for the compounds having the biphenyl moiety at that position. In addition, it is noteworthy that two new crystallographic structures of LmCPB complexed with Neq0690 and methyl methanethiosulfonate (MMTS) were determined with resolutions of 1.3 Å and 1.5 Å, respectively. The structures of the co-complexes revealed the modes of binding (MoB) of these ligands, as well as the main characteristics of the bimolecular recognition process. This will allow the natural translational target structure strategies for the search for new inhibitors of cysteine proteases with broad spectrum of action in the chemotherapy of related diseases.

<p align=\"justify\">A necessidade urgente de descoberta de terapias mais seguras e eficazes para o tratamento da doença de Chagas e leishmanioses tem motivado a pesquisa por novos inibidores das enzimas cruzaína e CPB, as principais cisteíno proteases do T. cruzie e Leishmania spp., respectivamente. Uma série de 52 compostos nitrílicos que atuam como inibidores covalente-reversíveis de cisteíno proteases foi sintetizada no grupo NEQUIMED/IQSC/USP e avaliada quanto a sua atividade inibitória contra as enzimas cruzaína, CPB de Leishmania mexicana e catepsina L de humanos. Utilizando planejamento molecular baseado em hipótese, mapeamos as relações estrutura-atividade (SARs) desses inibidores através de variações nas posições P1, P2, P3 e P1\' do esqueleto dipeptidil nitrílico. A substituição do grupo eletrofílico (warhead) aminonitrila em P1 pelo grupo azanitrila melhorou a afinidade em duas ordens de magnitude para todos os alvos avaliados. Um dos mais potentes inibidores, o análogo azanitrila Neq0690 mostrou uma cinética de ligação lenta com valores de pKi de 8,8, 9,3 e 9,7 para cruzaína, catepsina L e LmCPB, respectivamente. A substituição bioisostérica da ligação amida entre as posições P2-P3 pelo grupo trifluoroetilamina resultou na síntese do Neq0659, um potente inibidor com um perfil de ação seletivo para as proteases de parasitos. A substituição do grupo metileno em P1 pelo ciclopropano aumentou a afinidade para todas as enzimas. Contudo, uma inibição seletiva da cruzaína e LmCPB foi associada à presença do grupo (R)-benzila como substituinte da posição P1 dos derivados CF3 substituídos. Embora os compostos substituídos com leucina, tirosina, triptofano e 3-cloro fenilalanina como substituintes da posição P2 foram relativamente bem tolerados pela cruzaína e catepsina L, uma restrita especificidade foi verificada para LmCPB com pequenos ganhos de afinidade para os inibidores que possuíam os grupos leucina e metil benzoato como substituintes dessa posição. Com relação à posição P3, a inserção do grupo 3-terc-butilpirazol e 3-bromo piridina aumentou a afinidade para todos os alvos avaliados enquanto que um ganho seletivo para a LmCPB foi observado para os compostos que possuíam o grupo bifenila nessa posição. Além disso, duas novas estruturas cristalográficas da LmCPB complexada com o Neq0690 e metil metanotiossulfonato (MMTS) foram determinadas com resoluções de 1,3 Å e 1,5 Å, respectivamente. As estruturas dos co-complexos revelaram os modos de interação (MoB) desses ligantes, bem como as principais características do processo de reconhecimento bimolecular. Isso permitirá o uso de estratégias de planejamento baseado na estrutura do alvo com translação natural para a pesquisa por novos inibidores de cisteíno proteases, com amplo espectro de ação na quimioterapia de doenças a elas relacionadas. / <p align=\"justify\">The urgent need for the discovery of safer and more effective therapies for the treatment of Chagas disease and leishmaniasis has motivated the search for new inhibitors of the enzymes cruzain and CPB, the major T. cruzi and Leishmania spp. cysteine proteases, respectively. A series of 52 nitrile-containing compounds acting as covalent-reversible inhibitors of cysteine proteases was synthesized at the NEQUIMED/IQSC/USP Medicinal Chemistry Group and evaluated for their inhibitory activity against the enzymes cruzain, Leishmania mexicana CPB and cathepsin L from humans. Using hypothesis-driven molecular design, we mapped the structure-activity relationships (SARs) of these inhibitors through variations in the P1, P2, P3 and P1\' positions of the dipeptidyl nitrile scaffold. The substitution of the aminonitrile by the azanitrile group improved the affinity by two orders of magnitude for all the evaluated targets. One of the most potent inhibitors, the azanitrile analogue dubbed Neq0690 showed a slow-binding kinetics with pKi values of 8.8, 9.3 and 9.7 for cruzain, cathepsin L and LmCPB, respectively. Bioisosteric substitution of the amide moiety between the P2-P3 positions by the trifluoroethylamine group resulted in the synthesis of Neq0659, a potent inhibitor with a selective action profile for parasite proteases. Substitution of the methylene group at P1 by cyclopropane increased the affinity for all enzymes. However, selective inhibition of cruzain and LmCPB was associated with the presence of the (R)-benzyl group as substituent of the P1 position of the substituted CF3 derivatives. Although leucine, tyrosine, tryptophan and 3-chloro phenylalanine substituted compounds as substituents of the P2 position were relatively well tolerated by cruzain and cathepsin L, a restricted specificity was verified for LmCPB with small affinity gains for the inhibitors possessing the leucine and methyl benzoate as substituents of that position. Regarding the P3 position, the insertion of the 3-tert-butylpyrazole and 3-bromo pyridine groups increased the affinity for all evaluated targets whereas a selective gain for LmCPB was observed for the compounds having the biphenyl moiety at that position. In addition, it is noteworthy that two new crystallographic structures of LmCPB complexed with Neq0690 and methyl methanethiosulfonate (MMTS) were determined with resolutions of 1.3 Å and 1.5 Å, respectively. The structures of the co-complexes revealed the modes of binding (MoB) of these ligands, as well as the main characteristics of the bimolecular recognition process. This will allow the natural translational target structure strategies for the search for new inhibitors of cysteine proteases with broad spectrum of action in the chemotherapy of related diseases.

The hard tick Ixodes ricinus is an important blood-feeding parasite that transmits tick- borne diseases, such as tick-borne encephalitis and Lyme disease. Ticks employ a battery of proteolytic enzymes, including cathepsins, to digest their bloodmeal. These proteins are potential targets for the development of anti-tick vaccines. This work is focused on cathepsin L from I. ricinus (IrCL), namely its isoenzymes IrCL1 and IrCL3. IrCL1 was expressed in Pichia pastoris and chromatographically purified. Its substrate specifity was determined by the cleavage of (a) peptide fluorogenic substrates and (b) protein substrates analyzed by mass spectrometry. The proteolytic activity of IrCL1 was modulated by its interaction with glycosaminoglycans, which affected the pH optimum value. Futhermore, a proteolytically active mutant of IrCL1 with reduced number of N-glycosylation sites was prepared; this form will be used for crystallization experiments. IrCL3 was expressed in Escherichia coli, refolded and activated to its active form. The proteolytic activity of IrCL3 is in many ascpects similar to that of IrCL1, including substrate specifity, acidic pH optimum and activity modulation by glycosaminoglycans. Key words: cysteine proteases, cathepsin L, hard tick I. ricinus, substrate specifity, proteolytic activity...

Chez la drosophile, l’activation du récepteur Toll menant à une réponse antimicrobienne peut se faire par deux voies différentes. Ces deux voies sont activées soit par des récepteurs dédiés, les Pattern Recognition Receptors (PRRs) reconnaissant des motifs moléculaires microbiens, soit par la coupure d’une molécule circulante appelée Perséphone par des protéases microbiennes extrêmement diverses sécrétées pendant une infection. Cependant, le mécanisme par lequel Perséphone est activée demeurait ambigu. Nous avons identifié une région unique dans Perséphone fonctionnant comme un appât pour les protéases exogènes indépendamment de leur origine, type ou spécificité. Une coupure dans cette région constitue la première étape d’une activation séquentielle de Perséphone ; elle permet de recruter la cathepsine circulante 26-29-p, qui va générer la forme active de Perséphone.Ces travaux montrent comment un récepteur de l’immunité innée, Perséphone, peut être activé par un signal de danger, en l’occurrence des enzymes microbiennes, et non par la détection de motifs moléculaires qui peuvent être présents dans la flore microbienne hébergée par les animaux. / In Drosophila, the antimicrobial response against infections can be triggered by two different extracellular mechanisms that both lead to the activation of the Toll receptor. These two mechanisms are activated either by the recognition of specific microbial determinants by Pattern Recognition Receptors (PRRs), or by the cleavage of the circulating serine protease Persephone by a wide range of microbial proteases secreted during infections. However, the molecular mechanism underlying Persephone activation remained ambiguous. We identified a unique region in Persephone pro-domain that functions as a bait for exogenous proteases independently of their origin, type or specificity. Cleavage of Persephone in this bait region constitutes the first step of a sequential activation and licenses the subsequent maturation of Persephone to the endogenous circulating cysteine cathepsin 26-29-p. Our data establish Persephone itself as an immune receptor able to sense a broad spectrum of microbes through the recognition of danger signals rather than molecular patterns.

Inhibitors of proteases in tick saliva play an important role during tick feeding. Tick saliva contains a wide range of bioactive components which are able to modulate host imunity. Therefore, ticks are able to feed for a long time and transfer tick-borne diseases pathogens. The risk of transfer can be significantly reduced by deactivation of theese protease inhibitors. In this study I made monoclonal antibodies for detection and quantification of two serine protease inhibitors in tick saliva and other tick-body parts.

Made available in DSpace on 2014-12-17T14:16:35Z (GMT). No. of bitstreams: 1 YamaraASM_DISSERT_Parcial.pdf: 1235608 bytes, checksum: 64be8e29311055ebff593313fa2f1681 (MD5) Previous issue date: 2013-07-04 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The extraction, chemical and structural characterization of a wide variety of compounds derived from plants has been a major source of bioactive molecules. Several proteases have been isolated in the plant kingdom, with numerous pharmacological and biotechnological applications. Among the proteases isolated from plants, are the fibrinogenolytic, with relevant application in the treatment of disorders in the coagulation cascade, in addition to potential use as a tool in clinical laboratories. In this study, in addition to evaluating the effects of the protein extract of Cnidoscolus urens (L.) Arthur (Euphorbiaceae) in the coagulation cascade also investigates the presence of antimicrobial activity and characterizes the proteolytic activity detected in this extract, aiming to determine their potential pharmacological and biotechnological application. In this way, crude protein extracts obtained from the leaves of C. urens in Tris-HCl 0.05M, NaCl 0.15M, pH 7.5, were precipitated in different concentrations of acetone, and assessed for the presence of proteolytic activity in azocase?na and fibrinogen. The most active fraction (F1.0) in these tests was chosen for assessment of biological activity and biochemical characterization. The A? chain and B? of fibrinogen were completely cleaved at a concentration of 0.18 ?g/?L of protein fraction in 4 minutes. Fibrinogenolytic activity presented total inhibition in the presence of E-64 and partial in the presence of EDTA. The fraction demonstrated coagulant activity in plasm and reduced the APTT, demonstrating acting on the factors coagulation of the intrinsic pathway and common, not exerting effects on the PT. Fibrinolytic activity on plasma clot was detected only in SDS-PAGE in high concentrations of fraction, and there were no defibrinating. Although several proteases isolated from plants and venomous animals are classically toxic, the fraction F1.0 of C. urens not expressed hemorrhagic nor hemolytic activities. Fraction F1.0 also showed no antimicrobial activity. In proteolytic activity on the azocasein, the optimal pH was 5.0 and optimum temperature of 60?C. The enzyme activity has been shown to be sensitive to the presence of salts tested, with inhibition for all compounds. The surfactant triton did not influence the enzyme activity, but the tween-20 and SDS inhibited the activity. In the presence of reducing agents increase in enzyme activity occurred, a typical feature of enzymes belonging to the class of cysteine proteases. Several bands with proteolytic activity were detected in zymogram, in the region of high-molecular-weight, which were inhibited by E-64. In this study, we found that C. urens presents in its constitution cysteine proteases with fibrinogenolytic and procoagulant activity, which may be isolated, with potential application in treatment of bleeding disorders, thrombolytic and clinical laboratory / A extra??o, caracteriza??o qu?mica e estrutural de uma grande diversidade de compostos derivados de plantas tem sido uma fonte importante de mol?culas bioativas. Diversas proteases t?m sido isoladas no reino vegetal, com in?meras aplica??es farmacol?gicas e biotecnol?gicas. Dentre as proteases isoladas de plantas, est?o as fibrinogenol?ticas, com relevante aplica??o no tratamento de dist?rbios na cascata da coagula??o, al?m do uso em potencial como ferramenta em laborat?rios cl?nicos. Neste trabalho, al?m de avaliar os efeitos do extrato prot?ico de Cnidoscolus urens (L.) Arthur, pertencente ? fam?lia Euphorbiaceae, na cascata de coagula??o, tamb?m se investigou a presen?a de atividade antimicrobiana e caracterizou a atividade proteol?tica detectada neste extrato, tendo como objetivo determinar sua potencial aplica??o farmacol?gica e biotecnol?gica. Desse modo, extratos prot?icos brutos obtidos das folhas de C. urens em tamp?o Tris-HCl 0,05M, NaCl 0,15M, pH 7,5, foram precipitados em diferentes concentra??es de acetona, e avaliados quanto a presen?a de atividade proteol?tica em azocase?na e fibrinog?nio. A fra??o mais ativa (F1.0) nestes testes foi escolhida para realiza??o de avalia??o de atividade biol?gica e caracteriza??o bioqu?mica. As cadeias A? e B? do fibrinog?nio foram completamente clivadas na concentra??o de 0.18 ?g/?L de prote?na da fra??o em 4 minutos. A atividade fibrinogenol?tica apresentou inibi??o total em presen?a de E-64 e parcial em presen?a de EDTA. A fra??o demonstrou atividade coagulante sobre o plasma e reduziu o tempo de tromboplastina parcial ativada, indicando atuar sobre os fatores da via intr?nseca e comum da coagula??o, n?o exercendo efeitos sobre o tempo de protrombina. A atividade fibrinol?tica sobre o co?gulo de plasma foi detectado apenas em SDS-PAGE em concentra??es elevadas da fra??o, e apesar da atividade fibrin(ogen)ol?tica, n?o foi observada atividade defibrinogenante in vivo. Apesar de v?rias proteases de plantas e animais pe?onhentos serem classicamente t?xicas, a frac??o F1.0 n?o expressou atividade hemorr?gica nem hemol?tica. A fra??o F1.0 tamb?m n?o demonstrou atividade antimicrobiana. Na avalia??o da atividade proteol?tica sobre a azocase?na, o pH ?timo de rea??o foi 5.0, e a temperatura ?tima igual a 60?C. A atividade enzim?tica demonstrou ser sens?vel ? presen?a dos sais testados, com inibi??o para todos os compostos. O tensoativo triton n?o influenciou a atividade enzim?tica, por?m o tween-20 e SDS inibiram tal atividade. Em presen?a de agentes redutores ocorreu aumento da atividade enzim?tica, caracter?stica t?pica de enzimas pertencentes ? classe das ciste?no proteases. Diversas bandas prot?icas com atividade proteol?tica foram detectadas em zimograma, na regi?o de elevada massa molecular, que foram inibidas por E-64. Neste trabalho, foi revelado que C. urens apresenta fra??o enriquecida com ciste?no-proteases que apresentam atividade fibrinogenol?tica e procoagulante, que podem ser isoladas, com potencial aplica??o no tratamento de dist?rbios hemorr?gicos, como trombol?tico e em laborat?rio cl?nico / 2020-01-01