The 1,2,5-Thiadiazolidin-3-one 1,1-dioxide scaffold was used in the design and synthesis of inhibitors of Dengue Virus and West Nile Virus proteases and human tryptase. The scaffold was successfully used in the synthesis of potential inhibitors of Dengue Virus and West Nile Virus proteases. Inhibitors of Human tryptase synthesized based on the 1,2,5-Thiadiazolidin-3-one 1,1- dioxide scaffold were shown to be effective mechanism-based inhibitors of the enzyme. / Thesis (M.S.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry. / "July 2006." / Includes bibliographic references (leaves 64-66).

Engineering protease substrate specificity and selectivity has the potential to yield entirely new possibilities in the analytical, biotechnological, and therapeutic domains. For example, therapeutic applications can be envisioned in which engineered proteases could replace antibodies by irreversibly inactivating a large excess of disease-associated target proteins in a catalytic fashion. Technological advances in molecular biology have made laboratory-based evolution techniques for protein engineering readily accessible. However, the ability to interrogate the activities and substrate preference of large numbers of protease variants is predicated on the availability of quantitative high-throughput assays that maintain the essential link between genotype and phenotype. In this work we have investigated a variety of novel single cell fluorescence assays and selections for engineering protease substrate specificity and selectivity, and demonstrated the utility of some of these systems for the engineering of novel enzymes. The second chapter of this dissertation reports the isolation of a highly active ([chemical formula]) variant of the Escherichia coli endopeptidase OmpT that selectively hydrolyzes peptides after 3-nitrotyrosine while effectively discriminating against similar peptides containing unmodified tyrosine, sulfotyrosine, phosphotyrosine and phosphoserine. The isolation of protease variants that can discriminate between substrates based on the posttranslational modification of Tyr was made possible by implementing a multi-color flow cytometric assay using multiple simultaneous counter-selection substrates for the screening of large mutant libraries. While primary sequence recognition may suffice for proteomic applications, many therapeutic applications of engineered proteases will require the cleavage of folded protein targets. Unfortunately, we have found that engineered proteases that can cleave peptides very efficiently are often unable to digest the same sequences inserted into the loop regions of a folded protein. The logical conclusion, then, is that an entire target protein or at least a protein domain, rather than peptide segments, must be incorporated into protease engineering screening assays. As a critical first step toward the development of next generation, single cell screening systems for therapeutic protease engineering we have developed novel assays that exploit cell surface capture of exogenous protein substrates. One assay (Chapter 3) relies on an autoinhibited protein fusion that capitalizes on the p53 antagonist MDM2 as a detector of protease activity in addition to its utility as a counter-selection substrate. Using this system we successfully isolated OmpT variants that selectively cleave a designed site within our autoinhibited substrate. A second high-throughput screen (Chapter 4) monitors native protein cleavage. Target proteins are captured at the cell surface using a polycationic tail, incorporating counter-selection, and the proteolytic state of the substrate can be monitored using epitope tags fused to the N-and C-termini and fluorescently labeled anti-epitope tag antibodies. / text

Calpains are intracellular enzymes that merge cysteine protease and calcium sensing activities together in one molecule. They respond to Ca2+ signals and modify the activity of their targets by selective proteolysis. Calpains are involved in normal cellular process like cell migration and apoptosis. The over-activation of calpain due to disturbances in Ca2+ homeostasis or inactivation due to mutations, contribute to diseases like ischemic injury and muscular dystrophy. The classical calpains 1 and 2 are heterodimeric enzymes containing a large (80 kDa) subunit and a small subunit (28 kDa). Dimerization occurs through the 5th EF-hand of penta-EF-hand (PEF) domains present in both large and small subunits. In this study, I have used structural genomics approaches to explore the PEF and C2-like regulatory domains of some of the other 12 human calpain isoforms. I have shown that recombinant PEF domain of skeletal muscle-specific calpain 3 exists as a stable homodimer when produced alone. Modelling studies suggest that there would be no barriers for dimerization of the full-length enzyme through the PEF domains which would place the protease cores at opposite ends of the dimer. Co-expression studies using small subunit were performed with PEF domains of calpains 1, 3, 8, 9, 11, 12 and 13. A differential tagging system was devised to differentiate heterodimers from homodimers. The PEF domains of calpains 1, 3, 9 and 13 co-expressed with the small subunit, while the others failed to express. The PEF domains of calpains 1 and 9 formed heterodimers. Conversely, the PEF domain of calpain 3 formed a homodimer and that of calpain 13 predominantly formed a homodimer with a small amount of heterodimer. Homodimerization of calpains implies they are less-likely to be inhibited by the endogenous calpain inhibitor, calpastatin. C2-like regulatory domains of calpains 5-13 were also studied. The structure of the distal C2-like domain of calpain 7 was solved. It is markedly different from canonical C2 domains and may not bind Ca2+. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2009-02-11 12:30:29.18

Of the organisms with sequenced genomes, plants appear to possess the most rhomboid protease-encoding genes. However, our knowledge of processes in plants that involve Regulated Intramembrane Proteolysis (RIP) and rhomboid proteases remains low. As expressed recently by other researchers, finding a natural substrate for a rhomboid protease represents the biggest experimental challenge. Using yeast mitochondria-based assays, a potential link between the plastid translocon component Tic40 and organellar rhomboid proteases was recently uncovered. In this particular link, rhomboid proteases appear capable of influencing the pattern of imported Tic40 in yeast mitochondria. Tic40 may thus represent a natural plant target of organellar rhomboid proteases. Here, we obtained further motif-oriented evidence supporting Tic40 as a natural plant rhomboid substrate. A comparative analysis of sequences revealed that Tic40 may also possess similar TMD motifs found in the model substrate, Spitz. Rhomboid proteases often require these motifs to cleave substrates within intramembrane environments. Using site-directed mutagenesis and yeast mitochondria assays, the impact of mutations occurring in the motifs ASISS, GV, QP, and GVGVG of Tic40 was assessed. In terms of cleavage and changing the pattern of imported Tic40, some of the mutations showed decreased activities and a few showed enhancements. More importantly, the overall observed pattern associated with select Tic40 mutations resembled the characteristics reported for the model substrates. In particular, mutations in the Tic40 GV motif produced similar results as that observed with Spitz, by drastically decreasing or increasing cleavage as a function of amino acid sequence. / Thesis (Master, Biology) -- Queen's University, 2010-05-30 10:22:07.72

No description available.

Viral proteases are often considered to be attractive drug targets because of their crucial function in the viral replication machinery. In order to increase our knowledge of these important targets and to contribute to the discovery and development of new antiviral drugs, the proteases from hepatitis C virus (HCV) and human cytomegalovirus (HCMV) have been produced and their interactions with inhibitors and fragments have been characterized, using enzyme inhibition and SPR biosensor based interaction assay. The structure activity relationships and the resistance profiles of a series of HCV NS3 protease inhibitors based on either P2 proline or phenylglycine residues were analyzed using wild type genotype 1a and the major resistant variants A156T and D168V. The observed susceptibility to substitutions associated with these resistance variants was concluded to depend on the P2 and the P1 residue, and not only on the P2 residue as previously had been suggested. In order to be able to evaluate how the potency of inhibitors is affected by genetic variation, their effect was evaluated on wild type NS3 from genotype 1a, 1b and 3a as well as on the resistant variant R155K from genotype 1a. To enable a comparison of the inhibitory effect on the enzyme variants, the compounds were analyzed under conditions optimized for each variant. VX-950 was found to be the least susceptible compound to resistance and genetic variation. A more detailed analysis showed that the kinetic and mechanistic features of the inhibitors were significantly different for the different genotypes. The reversible non covalent macrocyclic inhibitor ITMN 191 was revealed to have favorable kinetics for all three genotypes. This is an advantage for the design of broad spectrum drugs. A fragment based procedure for identifying and validating novel scaffolds for inhibitors of HCMV protease was established. It identified fragments that may serve as starting points for the discovery of effective inhibitors against this challenging target.   The procedures developed for the evaluation and identification of novel HCV NS3 and HCMV protease inhibitors have contributed to a deeper understanding of protease-inhibitor interactions that is expected to have an impact on the design of novel antiviral drugs.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2010 / Made available in DSpace on 2012-10-25T04:42:11Z (GMT). No. of bitstreams: 1 285029.pdf: 700907 bytes, checksum: 0b15ab2a05efe94828d644e1103f043c (MD5) / Nas larvas de Tenebrio molitor (verme do trigo) são encontrados variados tipos de proteinases digestivas serínicas e cisteínicas, quando alimentados com uma dieta controle de farinha de trigo. Quando alimentados com farinha do feijão comum (Phaseolus vulgaris), pelo menos cinco proteinases apresentam atividade mais elevadas, enquanto a expressão de três enzimas pré-existentes foi reprimida. A indução dessas proteinases ocorreu entre 30 min e 1 h após a alimentação. Aqui nós relatamos o fracionamento das proteinases envolvidas na adaptação de T. molitor a inibidores de proteinases em sementes de leguminosas incorporadas em uma dieta controle usando cromatografia de troca iônica e filtração em gel, seguida pela caracterização das enzimas com substratos e inibidores naturais e sintéticos. Os inibidores utilizados neste estudo (todos incorporados na concentração de 0,4% no farelo de trigo) foram: inibidor de tripsina de soja (STI), inibidor de tripsina e quimotripsina de soja (SBI), inibidor de tripsina de Adenanttera pavonina (ApTI) e o inibidor de tripsina de P. vulgaris (BTI). As atividades proteolíticas foram ensaiadas para azoalbumina, para os substratos fluorogênicos z-PR-MCA, z-RR-MCA e succinil-GGR-MCA e os substratos cromogênicos suc-AAPF-pNA e benzoil-R-pNA. As atividades também foram monitoradas através de zimogramas e de fracionamento em géis bidimensionais. Dos quatro inibidores testados, o ApTI foi capaz de inibir as enzimas proteolíticas majoritárias sem induzir enzimas insensíveis a este inibidor, enquanto os outros inibidores além de inibir as proteinases constitutivas causaram indução de um conjunto diferente de proteinases serínicas. Entre as proteinases induzidas, uma era de interesse particular, pois foi induzida após sessenta minutos após a ingestão de SBI, STI e BTI. Esta proteinase teve sua atividade caracterizada como uma quimotripsina através de análise eletroforética bidimensional seguida de sequenciamento por espectrometria de massas.

Com o crescimento populacional, a indústria avícola tem se desenvolvido rapidamente, devido à demanda de alimentos. O seu produto possui grande aceitação no mercado mundial, em função do seu valor nutricional e por não existirem restrições culturais. Entretanto, este alimento gera grande quantidade de resíduos, dentre eles, as penas. Elas são compostas principalmente por queratina, substância de difícil degradação. Neste trabalho foram utilizadas duas bactérias queratinolíticas de resíduos da indústria avícola, para se avaliar a capacidade de degradação das mesmas. Foram produzidos hidrolisados de penas por proteólise bacteriana com ambos microrganismos: Bacillus cereus (KR16) e Chryseobacterium sp. (KR6). O crescimento das bactérias em diferentes quantidades de penas, e o fator de degradação das penas comprovaram que em até 5 % obteve-se degradação para KR6, enquanto, para a KR16, obteve-se degradação até 1%. A digestibilidade in vitro dos hidrolisados foi avaliada. Observou-se que o hidrolisado da bactéria KR6 apresentou maior digestibilidade, enquanto o de farinha de penas apresentou o menor valor. A composição de aminoácidos dos hidrolisados foi determinada, sendo observadas baixas concentrações de metionina, histidina e lisina. A partir da digestibilidade in vitro e da composição de aminoácidos, foram calculados o escore de aminoácidos corrigidos (PDCAAs), o coeficiente de eficiência protéica (PER) e o valor biológico (BV). O tratamento das penas com KR6 resultou em hidrolisados com os valores mais altos de PDCAAs, PER e BV, sugerindo que este microrganismo produz hidrolisados com propriedades nutricionais superiores aos demais.

Tese (doutorado) - Pontifícia Universidade Católica do Paraná, Curitiba, 2011 / Bibliografia: f. 37-40 / Introdução: Apesar do aumento no número de transplantes de órgãos sólidos e no aumento da sobrevida do paciente, as infecções fúngicas, principalmente, a candidose, ainda representam uma grande ameaça aos pacientes imunocomprometidos. A modulação da virul / Introduction: In spite of the growing number of solid organ transplantations and the increase in patient survival, fungal infections, especially candidiasis, still poses a significant threat to immunocompromised patients. Modulation of Candida albicans vi

VASCONCELOS, Eliane Silva Araújo de. Proteases do látex de Calotropis procera: purificação, caracterização bioquímica, enzimática e molecular e atividades biológicas. 2013. 140 f. Tese (Doutorado em bioquímica)- Universidade Federal do Ceará, Fortaleza-CE, 2013. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-09-02T11:59:28Z No. of bitstreams: 1 2013_tese_esaraujo.pdf: 2565494 bytes, checksum: 4d162fd198a10a8d88c2c3e0811e739d (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-09-05T20:30:20Z (GMT) No. of bitstreams: 1 2013_tese_esaraujo.pdf: 2565494 bytes, checksum: 4d162fd198a10a8d88c2c3e0811e739d (MD5) / Made available in DSpace on 2016-09-05T20:30:20Z (GMT). No. of bitstreams: 1 2013_tese_esaraujo.pdf: 2565494 bytes, checksum: 4d162fd198a10a8d88c2c3e0811e739d (MD5) Previous issue date: 2013 / Studies have shown that latex of plants is a rich source of enzymes with proteolytic activities. Isolation and characterization of cysteine proteases of latex have recently been reported. In this work we report the purification and characterization of three new cysteine proteases of laticifer fluid of Calotropis procera, as well as its activity in plasma coagulation assays. The three proteases, termed CpCP-1, 2-CpCP and CpCP-3 are isoforms of cysteine proteases and were purified using two sequential steps of ion exchange chromatography on CM-Sepharose and Resource S columns, coupled to FPLC system. Their molecular masses were determined by ESI-Q-TOF mass spectrometry: CPCP-1 had mass = 26.213, CPCP-2 = 26.133 and CPCP-3 = 25.086 Da. The amino acid sequences of the N-terminal region was identical for all three enzymes, being composed of 30 amino acid residues. Analysis revealed high sequence identity with others cysteine proteases. The proteolytic activity of these enzymes was tested against different substrates (azocasein, BANA and BApNA) and at different pH and temperature. The three enzymes are capable of degrading azocasein and BANA, substrates nonspecific and specific for cysteine proteases, respectively. CPCP-1 showed proteolytic activity twice that CPCP-3, and this, a little bigger than CPCP-2. Enzymes maintained 60-80% of their activities even when tested at 60 °C temperature, and the optimum pH for these activities was 6.0. Circular Dichroism Analysis showed that the secondary structure of the proteases was composed of 15.1 to 19.9% of alpha-helices and 20.6 to 21.3% of beta-sheets. The spectra deconvolution of proteases showed that their structures were altered in the presence of the reducing agent DTT, suggesting the presence of disulfide bridges stabilizing the three dimensional structures. In biological tests proteases were able to strongly inhibit the germination of spores of the fungus Colletotrichum gloeosporioides and also exhibited plasma coagulation activity by thrombin-like mechanism. / Estudos têm demonstrado que látex de plantas é uma rica fonte de enzimas com atividades proteolíticas. O isolamento e a caracterização de proteases cisteínicas de látex têm sido recentemente relatados. Neste trabalho nós reportamos a purificação e caracterização de três novas proteases cisteínicas do fluido laticífero de Calotropis procera, bem como sua atividade em ensaios de coagulação plasmática. As três proteases, denominadas CpCP-1, CpCP-2 e CpCP-3 são isoformas de proteases cisteínicas e foram purificadas utilizando dois passos sequenciais de cromatografias de troca iônica em colunas de CM-Sepharose e Resource S, acoplada a sistema FPLC. Suas massas moleculares foram determinadas por espectrometria de massas em aparelho do tipo ESI-Q-TOF, onde: CpCP-1 apresentou massa=26,213, CpCP-2=26,133 e CpCP-3=25,086. A sequência de aminoácidos da região N-terminal foi idêntica para as três enzimas, sendo constituída de 30 resíduos de aminoácidos. Análises de sequências revelaram alto nível de identidade (88%) com proteases cisteínicas A atividade proteolítica dessas enzimas foi testada frente a diferentes substratos (Azocaseína, BANA e BApNA) e em diferentes valores de pH e temperatura. As três enzimas foram capazes de degradar Azocaseína e BANA, substratos inespecífico e específico para proteases cisteínicas, respectivamente. CpCP-1 apresentou atividade proteolítica duas vezes maior que CpCP-3, e esta, um pouco maior que CpCP-2. As enzimas mantiveram 60-80% de suas atividades mesmo quando ensaiadas a 60ºC de temperatura, e o pH ótimo para essas atividades foi 6,0. Análises de Dicroísmo Circular revelaram que a estrutura secundária das proteases era composta de 15,1-19,9% de alfa-hélices e 20,6-21,3% de folhas-beta. Os espectros de desconvolução das proteases mostrou que suas estruturas foram alteradas na presença do agente redutor DTT, sugerindo a presença de pontes dissulfeto na estabilização das estruturas tridimensionais. Em testes biológicos as proteases foram capazes de inibir fortemente a germinação de esporos do fungo Colletotrichum gloesporioides e também exibiram atividade de coagulação plasmática por um mecanismo do tipo trombina.