Activity and transcription profiles of two classes of cysteine proteases, papain- and legumain-like cysteine proteases, as well as their potential inhibitors, cysteine protease inhibitors (cystatins), were investigated in soybean nodules during nodule development and after drought inducing premature senescence. During nodule development total protease activity increased with major activity bands detected protease zymography in older nodules. Expressed cysteine proteases during nodule development were detected by tagging proteases with the cysteine protease inhibitor DCG-04 with major DCG-04 tagged bands found in both young and old nodules. Increase in protease activity was associated with a significant decrease in nitrogenase activity of nodules measured as acetylene reduction. Semi-quantitative RT-PCR for cysteine protease and cystatin transcription profiling showed a decrease in transcription during development and also after drought treatment of several papain-like cysteine proteases (Glyma04g04400, Glyma17g05670, Glyma10g35100, and Glyma04g03090). In contrast, transcription of three legumain-like cysteine proteases (Glyma17g14680, Glyma05g04230 and Glyma14g10620) increased during nodule development and also after drought treatment. Transcription of two cystatins (Glyma13g27980 and Glyma05g28250) increased during nodule development with Glyma13g27980 strongly up-regulated after drought treatment and Glyma05g28250 constitutively strongly expressed in both well-watered and drought treated nodules. Overall, the study has contributed in establishing an expression profile of cysteine proteases and cystatins in soybean nodules. This knowledge provides a basis which can be used to determine the importance of the individual components of the cysteine protease – cystatin system, during soybean nodule development and during stress-induced premature nodule senescence. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Plant Science / unrestricted

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

Blastocystis spp. est un Straménopile parasite anaérobie fréquemment rencontré dans le tractus gastro-intestinal de l’homme et de divers animaux. Ce parasite est associé à des troubles gastro‐intestinaux aspécifiques, et semble impliqué dans des désordres fonctionnels tels que le syndrome de l’intestin irritable (IBS). Ce travail de thèse s’appuie sur le séquençage du génome de Blastocystis sp. ST7 réalisé en collaboration avec le Génoscope d’Evry, l’Université Nationale de Singapour, l’Institut Pasteur de Lille et l’Université de Provence. Ce génome est constitué d’un génome nucléaire de 18,8 Mpb pour 6020 gènes, et d’un génome mitochondrial de 29 kpb localisé dans des organites apparentés aux mitochondries. L’analyse de ce génome apporte des informations au niveau de l’évolution de ce microorganisme, de son adaptation à l’environnement intestinal et de ces facteurs de virulence potentiels. En effet, les analyses in silico de ce génome ont montré que Blastocystis sp. ST7 possède plusieurs gènes codant des protéines pouvant agir à l’interface entre l’hôte et le parasite et connues chez d’autres protozoaires pour être impliquées dans des phénomènes de pathogénie. Ce sont en particulier des PKS, des NRPS, et des hydrolases dont des protéases. D’autre part, des activités protéolytiques ont été mises en évidence expérimentalement dans les surnageants de culture du parasite. Deux protéases à cystéines (une cathepsine B et une légumaïne) pouvant être impliquées dans la physiopathologie du parasite, ont été identifiées et caractérisées dans les surnageants, confirmant ainsi nos analyses in silico. Ce travail ouvre de nombreuses pistes intéressantes à explorer pour évaluer l’impact de ce parasite en santé humaine. / Blastocystis spp. is a highly prevalent anaerobic Stramenopile parasite found in the intestinal tract of humans and various animals. This parasite is associated with non specific intestinal disorders, and could be involved in functional disorders such as the irritable bowel syndrome (IBS). In this work, the Blastocystis sp. ST7 genome sequencing project was carried out in collaboration with the Génoscope of Evry, the National University of Singapore, the Pasteur Institute of Lille and the University of Provence. This genome consists in a nuclear genome of 18,8 Mpb encoding 6020 genes, and a mitochondria‐like genome of 29 kpb localised in the mitochondrion‐like organelles. The analysis of this genome brings information about the evolution of this micro‐organism, its adaptation to the intestinal environment and its potential virulence factors. Blastocystis sp. ST7 was predicted to harbor several genes coding proteins that could act at the parasite‐host interface, and that are known to be involved in the pathogeny of many protozoa. They are PKS, NRPS, and hydrolases among them proteases. In addition, proteolytic activities were highlighted in the parasite culture supernatants. Two cysteine proteases (a cathepsin B and a legumain) were identified and characterized from the supernatants and could play a role in the physiopathology of the parasite, that confirm our in silico analyses. This work opens new ways to evaluate the impact of this parasite in human health.

nÃo hà / 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 ÂC 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 tÃm demonstrado que lÃtex de plantas à uma rica fonte de enzimas com atividades proteolÃticas. O isolamento e a caracterizaÃÃo de proteases cisteÃnicas de lÃtex tÃm sido recentemente relatados. Neste trabalho nÃs reportamos a purificaÃÃo e caracterizaÃÃo de trÃs novas proteases cisteÃnicas do fluido laticÃfero de Calotropis procera, bem como sua atividade em ensaios de coagulaÃÃo plasmÃtica. As trÃs proteases, denominadas CpCP-1, CpCP-2 e CpCP-3 sÃo isoformas de proteases cisteÃnicas e foram purificadas utilizando dois passos sequenciais de cromatografias de troca iÃnica 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 sequÃncia de aminoÃcidos da regiÃo N-terminal foi idÃntica para as trÃs enzimas, sendo constituÃda de 30 resÃduos de aminoÃcidos. AnÃlises de sequÃncias revelaram alto nÃvel de identidade (88%) com proteases cisteÃnicas A atividade proteolÃtica dessas enzimas foi testada frente a diferentes substratos (AzocaseÃna, BANA e BApNA) e em diferentes valores de pH e temperatura. As trÃs enzimas foram capazes de degradar AzocaseÃna e BANA, substratos inespecÃfico e especÃfico para proteases cisteÃnicas, respectivamente. CpCP-1 apresentou atividade proteolÃtica 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 60ÂC de temperatura, e o pH Ãtimo para essas atividades foi 6,0. AnÃlises de DicroÃsmo Circular revelaram que a estrutura secundÃria das proteases era composta de 15,1-19,9% de alfa-hÃlices e 20,6-21,3% de folhas-beta. Os espectros de desconvoluÃÃo das proteases mostrou que suas estruturas foram alteradas na presenÃa do agente redutor DTT, sugerindo a presenÃa de pontes dissulfeto na estabilizaÃÃo das estruturas tridimensionais. Em testes biolÃgicos as proteases foram capazes de inibir fortemente a germinaÃÃo de esporos do fungo Colletotrichum gloesporioides e tambÃm exibiram atividade de coagulaÃÃo plasmÃtica por um mecanismo do tipo trombina.

Made available in DSpace on 2014-07-29T15:16:27Z (GMT). No. of bitstreams: 1 dissertacao adriane g gamboa.pdf: 1009208 bytes, checksum: bb95e46e7798790f86bb339947708a21 (MD5) Previous issue date: 2010-03-25 / In this work papain inhibitor from A. pavonina was immobilized by covalent bond in polyaniline (PANI) modified with glutaraldehyde (PANIG) for be used as stationary phase for affinity chromatography and then applied in the purification of cysteine proteases bromelain and ficin. The extraction and purification of inhibitors protease from A. pavonina resulted in a yield of 3.9% in the last step of purification. Gel filtration chromatography performed in Sephadex G-75 resin as a purification step resulted in three protein peaks (F1, F2 and F3), but only F1 was used in the experiments of immobilization because of higher specific activity. Immobilization was performed using PANIG. To optimize the immobilization conditions the amount of PANIG in the reaction (5, 10 and 15mg), time (30, 60 and 90 min) and pH (5.0 to 8.0) were varied. The best conditions for immobilization of A. pavonina inhibitor, according to tests performed were 5mg PANIG, reaction time of 30min and pH 7.0. PANIG-I was used as bio-affinity stationary phase for separation of bromelain and ficin. Electrophoresis (SDS-PAGE) performed after the separation process revealed the presence of a single band for both bromelain and ficina, with 28 and 25 kDa, respectively. In this process, ficin was purified 2,60 fold and bromelain 0,89 fold, showing that the use of inhibitors of A. pavonina immobilized in PANIG were efficient in the purification of cysteine proteases. / No presente trabalho um inibidor de papaína extraído de sementes de A. pavonina foi imobilizado por ligação covalente em polianilina (PANI) modificada com glutaraldeído (PANIG), visando a aplicação desse material como fase estacionária para cromatografia de afinidade e sua aplicação na purificação das proteases cisteínicas bromelaína e ficina. A extração e purificação dos inibidores de A. pavonina resultou num rendimento de 3,9% no último passo de purificação. A cromatografia de gel filtração em resina Sephadex G-75 como passo para purificação dos inibidores resultou em três picos protéicos (F1, F2 e F3) dos quais F1 foi utilizado nos experimentos de imobilização por ter apresentado atividade específica mais alta. A imobilização foi feita utilizando-se PANIG. Para otimização das condições de imobilização foram variados na reação a quantidade de PANIG (5, 10 e 15mg), tempo (30, 60 e 90 min) e pH (5,0 a 8,0). As melhores condições para imobilização dos inibidores de A. pavonina, de acordo com os ensaios realizados, foram 5mg de PANIG, tempo de reação de 30min e pH 7,0. PANIG-I foi utilizada como fase estacionária de bioafinidade para separação de bromelaína e ficina. Eletroforese (SDS-PAGE) após o processo de separação revelou a presença de uma única banda, tanto para bromelaína como para ficina, de 28 e 25 kDa, respectivamente. Nesse processo, a ficina foi purificada 2,60 vezes e a bromelaína 0,89 vezes, mostrando que o uso dos inibidores de A. pavonina imobilizados em PANIG foram eficientes na purificação de proteases cisteínicas

Proteases are important enzymes in the biotechnology due to their specific cleavage of substrates. HRV 3C, sortase A and TEV are some examples of cysteine proteases which become more of use lately in applications as removal of affinity tags (3C/TEV) and labelling of proteins (sortase). Here an investigation was made on the proteases by displaying them on two different prokaryotic hosts; E. coli and S. carnosus and to use these to cleave away affinity proteins (Affibody molecule) from other cells with an incorporated cleavage site. Constructs were cloned and incorporated into expressing strains which were then cultivated and induced. Analysis of surface expression was done by flow cytometer. Cleavage was made by cultivating combinations with cleavable bacteria and bacteria displaying proteases. A functional protease would lead to the presence of Affibody molecules in the supernatant. Flow cytomtery analysis was first made to inevstigate signal difference in Affibody binding by the addition of flurophores. Secondly SDS-PAGE was made on the centrifuged supernatant to investigate the presence of a product. Finally analysis of the bacteria was made by examining the reaction with soluble substrate and comparing activity with soluble enzyme. All of the enzymes were able to be displayed on the surface of bacteria with a clear separation from control. The cleavage analysis showed however varying results yet no clear evidence of product. Best flow cytometer results were seen for 3C but SDS-PAGE/MS did not show any cleaved product. For Sortase SDS-PAGE showed positive result but analysis with MS showed no product. TEV was concluded not to be funcional at all hence the failing to cleave soluble substrate  when condition seemed near optimal and faulty flow cytometer data. Even though the lack of success there is still many further studies that can be done on the proteases in order to prove its absence/presence  of activity.

Trichomonas vagina/is, one of the most common sexually transmitted diseases, has been shown to increase patients' susceptibility to HIV infection and cervical cancer; moreover, resistance to metronidazole is increasing, and new drug targets must be identified in order to combat resistant strains. T vagina/is expresses cysteine proteases that have been implicated in vaginal epithelial apoptosis as well as immune system evasion. In the past the various cysteine proteases have been studied as a group, and the following work examines, one specific protease, TvCP2, in detail through Western blot analysis, immunofluorescent staining, and recombinant expression. The experiments 5 presented here suggest that aT l-CP2 over-expressing transfectant line processes CP2 and sequesters it in cellular compartments. Previous data gives strong evidence of the secretion of cysteine protease CP4 and hints at the possibility of CP2 secretion as well; however, our results show no co-localization between CP2 and CP4 in T l-CP2 over expressing transfectants, suggesting separate trafficking and different roles. To better characterize CP2 function, we attempted to express active, recombinant protein. Although Pichia pastoris serves as a reliable expression vehicle, a processing event following translation ofTvCP2 appears to have cleaved the pro-domain and, along with it, the a-secretion signal, trapping active TvCP2 within the cellular pellet. A thioreoxintagged version ofTvCP2 has been expressed in E. coli, and preliminary experiments show it may auto-activate under certain conditions, but further experimentation is required to confirm the presence of active CP2 within the fraction purified from these cells.

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

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

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.