Introdução: Uma proporção significante de pacientes infectados por HIV-1 (pacientes HIV-1+) tratados com inibidores de protease (IPs) desenvolve mutações de resistência. Estudos recentes têm mostrado que células T CD8+ de pacientes HIV- 1+ reconhecem epitopos de Pol incluindo mutações selecionadas por drogas. Nenhum epitopo CD4+ da protease foi descrito na base de dados de Los Alamos. Objetivo: Considerando que a protease de HIV-1 é alvo de terapia antiretroviral e que essa pressão pode selecionar mutações, nós investigamos se mutações selecionadas por IPs afetariam o reconhecimento de epitopos da protease de HIV-1 por células T CD4+ em pacientes tratados com IPs. Nós investigamos o reconhecimento de três regiões da protease preditas de conter epitopos de células T CD4+ bem como mutações induzidas por IPs por células T CD4+ em pacientes HIV- 1+ tratados com IPs. Materiais e Métodos: Quarenta pacientes HIV-1+ tratados com IPs foram incluídos (30 em uso de Lopinavir/ritonavir, 9 em uso de Atazanavir/Ritonavir e 1 em uso exclusivo de Atazanavir). Para cada paciente determinou-se a seqüência endógena da protease de HIV-1, genotipagem viral e tipagem HLA classe II. Utilizamos o algoritmo TEPITOPE para selecionar peptídeos promíscuos, ligadores de múltiplas moléculas HLA-DR, codificando as três regiões da protease de HIV-1 cepa HXB2 (HXB2 4-23, 45-64, e 76-95) e 32 peptídeos adicionais contidos nas mesmas regiões incorporando as mutações induzidas por IPs mais freqüentes no Brasil. Os 35 peptídeos foram sintetizados. Respostas proliferativas de células T CD4+ e CD8+ aos peptídeos foram determinadas por ensaios de proliferação com diluição do corante CFSE. Ensaios de ligação a alelos HLA classe II foram realizados para confirmar a promiscuidade desses peptídeos e avaliar a habilidade de se ligarem a moléculas HLA presentes em cada paciente. Resultados: Todos os peptídeos foram reconhecidos por pelo menos um paciente e respostas proliferativas de células T CD4+ e CD8+ a pelo menos um peptídeo da protease de HIV-1 foram encontradas em 78% e 75% dos pacientes, respectivamente. A terceira região (Protease 76 95) foi a mais freqüentemente reconhecida. Ao compararmos as respostas de células T às seqüências da protease do HIV-1 endógeno, observamos que a maioria dos pacientes não foi capaz de reconhecer peptídeos idênticos às essas seqüências, porém reconheceram peptídeos variantes diferentes das mesmas regiões. Apenas sete pacientes responderam às seqüências endógenas. Verificamos que diversos peptídeos endógenos que não foram reconhecidos apresentaram ausência de ligação a alelos HLA portados por estes pacientes, sugerindo que mutações selecionadas por pressão imune tenham levado ao escape de apresentação de antígeno e evasão de resposta de linfócitos T CD4+. Alternativamente, isso poderia ser explicado pela presença de um vírus replicante distinto presente no plasma uma vez que somente foram obtidas seqüências provirais. Conclusão: Epitopos selvagens e mutantes da protease do HIV-1 reconhecidos por células T CD4+ foram identificados. Também verificamos que a maior parte dos pacientes não reconheceu as seqüências da protease endógena enquanto que reconheceram seqüências variantes. O reconhecimento de seqüências não-endógenas poderia ser hipoteticamente conseqüência de alvo de populações HIV-1 minoritárias; protease de HERV que contém regiões de similaridade com a protease do HIV-1; ou seqüências de HIV-1 presentes apenas em parceiros virêmicos. A falha de reconhecimento de seqüências endógenas seria mais provável devido ao escape imune, do que ao nível de apresentação ou reconhecimento por células T. Isso implica em uma conseqüência patofisiológica na evasão de respostas de células T contra a protease de HIV-1 e no fato de ser tradicionalmente considerada uma proteína pouco antigênica / Introduction: A significant proportion of protease inhibitor (PI)-treated HIV-1 infected (HIV-1+) patients develop resistance mutations. Recent studies have shown that CD8+ T cells from HIV-1 patients can recognize antiretroviral drug-induced mutant Pol epitopes. No HIV-1 protease CD4 epitopes are described in the Los Alamos database. Aims: Given that the protease of HIV-1 is a target of antiretroviral therapy and this pressure may lead to the selection of mutations, we investigated whether PI-induced mutations affect the recognition of HIV-1 protease epitopes by CD4 + T cells in PI-treated patients. We investigated the recognition of three protease regions predicted to harbor CD4+ T cell epitopes as well as PI-induced mutations by CD4+ T cells of PI-treated HIV-1+ patients. Methods: Forty PI-treated HIV-1+ patients were included (30 undergoing Lopinavir/ritonavir, 9 undergoing Atazanavir/ritonavir and 1 undergoing exclusively Atazanavir treatment). For each patients, the endogenous HIV-1 protease sequence, viral genotype and HLA class II typing were determined. We used the TEPITOPE algorithm to select promiscuous, multiple HLA-DR-binding peptides encoding 3 regions of HIV-1 HXB2 strain protease (HXB2 4-23, 45-64, and 76-95) and 32 additional peptides contained in the same regions, but encompassing the most frequent PI-induced mutations in Brazil. The 35 peptides were thus synthesized. Proliferative responses of CD4+ and CD8+ T cells against peptides were determined by the CFSE dilution assay. HLA class II binding assays were made to confirm the promiscuity of these peptides and evaluate their ability to bind the HLA molecules carried by each patient. Results: All tested peptides were recognized by at least one patient and proliferative responses of CD4+ and CD8+ T cells against at least one HIV-1 protease peptide were found in 78% and 75% patients, respectively. The third region (Protease 76-95) was the most frequently recognized. By comparing T-cell responses to HIV-1 endogenous protease sequences, we found that most patients failed to recognize identical peptides of those sequences, but recognized different variant peptides of the same region. Only seven patients responded to endogenous sequences. We found that several endogenous peptides that failed to be recognized showed no binding to the HLA alleles carried by that given patient, suggesting that mutations selected by immune pressure have led to escape of antigen presentation, as well as direct escape of the CD4+ T cell response. Alternatively, it could have been due to the presence of a different replicating virus in the plasma-since we only obtained proviral sequences. Conclusion: Wild-type and mutant HIV-1 protease epitopes recognized by CD4+ T cells were identified. We also found that most patients failed to recognize their endogenous protease sequences, while they recognized variant sequences. The recognition of non-endogenous sequences could hypothetically be a consequence of targeting a minor HIV-1 population; HERV protease, that contains regions of similarity with HIV-1 protease; or HIV-1 sequences present only in viremic partners. The failure to recognize endogenous sequences is most likely due to immune escape, either at the level of presentation or direct T cell recognition. This may have a pathophysiological consequence on evasion of T cell responses against protease and the fact that it has been considered traditionally a poorly antigenic HIV-1 protein.

L’apprêtement antigénique par les protéases intracellulaires et la présentation des épitopes sont essentiels pour la reconnaissance des cellules infectées par les lymphocytes CD8+. Ici nous avons montré que certains inhibiteurs de la protéase de la VIH (IPs) modulent l’activité de la protéasome et aminopeptidase impliqué dans l’apprêtement antigénique endogène et l’activité cathepsins importante dans l’apprêtement croisée. Deux IPs agissent directement sur les cathepsins et leurs régulateurs en inhibant les activités kinase, NOX2 et en régulant le pH phagolysosomal. Les IPs ont changé la dégradation des protéines viral et la production des épitopes de façon séquence- et cellule-spécifique, ont altéré la présentation direct et croisée des épitopes, et ont partiellement changé l’auto-peptidome des cellules primaires. La modulation par les drogues de l’apprêtement et la présentation des épitopes peut fournir une approche thérapeutique alternative pour moduler la reconnaissance immunitaire. / Antigen processing by intracellular proteases and peptidases and epitope presentation are critical for recognition of pathogen-infected cells by CD8+ T lymphocytes. Here we show that several HIV protease inhibitors (PIs) prescribed to HIV-infected persons variably modulate proteasome and aminopeptidase activities involved in endogenous antigen presentation and cathepsin activities involved in antigen cross-presentation. Two HIV PIs acted directly on cathepsins and on their regulators by inhibiting kinases, NOX2 and the regulation of phagolysosomal pH, subsequently enhancing cathepsin activities. HIV PIs modified HIV protein degradation and epitope production in a sequence- and cell-dependent manner, altered direct- and cross-presentation and T cell-mediated killing, and partly changed the self-peptidome of primary cells. Drug-induced modulation of antigen processing and peptidome may provide an alternate therapeutic approach to modulate immune recognition.

Drug resistance is a global health threat costing society billions of dollars and impacting millions of lives each year. Current drug design strategies are inadequate because they focus on disrupting target activity and not restricting the evolutionary pathways to resistance. Improved strategies would exploit the structural and dynamic changes in the enzyme–inhibitor system integrating data from many inhibitors and variants. Using HIV-1 protease as a model system, I aimed to elucidate the underlying resistance mechanisms, characterize conserved protease-inhibitor interactions, and generate more robust inhibitors by applying these insights. For primary mechanisms of resistance, comparing interactions at the protease–inhibitor interface showed how specific modifications affected potency. For mutations distal to the active site, molecular dynamics simulations were necessary to elucidate how changes propagated to reduce inhibitor binding. These insights informed inhibitor design to improve potency against highly resistant variants by optimizing hydrogen bonding. A series of hybrid inhibitors was also designed that showed excellent potency by combining key moieties of multiple FDA-approved inhibitors. I characterized the structural basis for alterations in binding affinity in HIV-1 protease both from mutations and inhibitors. I applied these strategies to HTLV-1 protease, a potential drug target. I identified the HIV-1 inhibitor darunavir as a viable scaffold and evaluated analogues, leading to a low-nanomolar compound with potential for optimization. Hopefully, insights from this thesis will lead to the development of potent HTLV-1 protease inhibitors. More broadly, these inhibitor design strategies are applicable to other rapidly evolving targets, thereby reducing drug resistance rates in the future.

Failure to process prelamin A, by the enzyme ZMPSTE24, leads to the build up of farnesylated prelamin A, which has been implicated in causing the symptoms experienced in laminopathies and HIV therapy. A common feature to these conditions is the development of an irregular nuclear boundary, often including deep invaginations that form a nucleoplasmic reticulum. Additionally, dysregulated lipid synthesis is frequently associated with improper lamin A processing and I set out to address the molecular mechanisms behind these two features that could explain lipoatrophy experienced in patients. By using siRNA targeted against Zmpste24 I utilised an array of biochemical, molecular and imaging techniques to uncover a mechanism that leads to the production of a nucleoplasmic reticulum that was dependent on both the farnesylated tail of prelamin A and the phosphatidylcholine synthesising enzyme CCTα. The morphology of this structure consisted of an invagination of both the inner and outer nuclear membranes with a cytoplasmic core or just invagination of the inner nuclear membrane. Serial section dual axis electron tomography provided a new insight into the ultrastructural changes at the nuclear periphery that revealed novel structural features. The dysregulation of lipid synthesis was assessed by investigating the effects farnesylated prelamin A has on the distribution and dynamics of the transcription factor SREBP-1 and assessment of the downstream consequences this has on its targets that regulate adipocyte differentiation potential. Finally, the metabolomic profile of an HIV protease inhibitor that leads to prelamin A build up was generated and revealed increases in lipolysis, glycolysis and mediators of inflammation. The research presented offers a new insight into the development of a convoluted nuclear boundary and nucleoplasmic reticulum, in the context of lamin A mutants and how dysregulated lipid synthesis, caused by farnesylated prelamin A, leads to lipoatrophy.

by Lam Tin Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 131-151). / Abstracts in English and Chinese. / Acknowledgment --- p.I / Table of content --- p.II / List of Tables --- p.IX / List of Figures --- p.XI / Abbreviation --- p.XIII / Abstract --- p.XIV / 論文摘要 --- p.XVI / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Acquired immunodeficiency syndrome (AIDS) --- p.1 / Chapter 1.1.1 --- History of AIDS --- p.1 / Chapter 1.1.2 --- Definition of AIDS --- p.2 / Chapter 1.1.3 --- HIV/AIDS Around the World --- p.4 / Chapter 1.1.4 --- HIV/AIDS in Hong Kong --- p.4 / Chapter 1.1.4.1 --- Hong Kong AIDS Update --- p.4 / Chapter 1.1.4.2 --- AIDS Transmission --- p.6 / Chapter 1.1.4.3 --- Main AIDS Complications Occur in Hong Kong --- p.6 / Chapter 1.2 --- Human Immunodeficiency Virus (HIV) --- p.7 / Chapter 1.2.1 --- Classification of HIV --- p.7 / Chapter 1.2.2 --- The Structure of HIV Virion --- p.9 / Chapter 1.2.3 --- The HIV Genome --- p.11 / Chapter 1.2.4 --- The Life Cycle of HIV --- p.12 / Chapter 1.2.4.1 --- Invasion of the Cells --- p.12 / Chapter 1.2.4.2 --- Integration into cell genome --- p.13 / Chapter 1.2.4.3 --- Protease and assembly to the virus --- p.13 / Chapter 1.2.5 --- Three Essential Enzymes for HTV-1 Replication --- p.16 / Chapter 1.2.5.1 --- HIV-1 Reverse Transcriptase (HIV-1 RT) --- p.16 / Chapter 1.2.5.2 --- HIV-1 Integrase (HIV-1 IN) --- p.17 / Chapter 1.2.5.3 --- HIV-1 Protease (HIV-1 PR) --- p.18 / Chapter 1.2.6 --- The Different Stages of HIV Infection --- p.19 / Chapter 1.3 --- AIDS therapy --- p.23 / Chapter 1.3.1 --- Drugs Approved by US Food and Drug Administration (FDA) --- p.23 / Chapter 1.3.2 --- Vaccine --- p.26 / Chapter 1.3.3 --- Chemokine Receptor Inhibitor --- p.27 / Chapter 1.3.4 --- Antisense Oligonucleotides Therpay --- p.28 / Chapter 1.3.5 --- Traditional Chinese Medicine (TCM) --- p.29 / Chapter 1.4 --- Objective of My Project --- p.32 / Chapter CHAPTER 2 --- SCREENING OF TRADITIONAL CHINESE MEDICINAL PLANTS FOR HIV-1 PROTEASE INHIBITION --- p.33 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials and Methods --- p.35 / Chapter 2.2.1 --- Materials --- p.35 / Chapter 2.2.2 --- Extraction Methods --- p.36 / Chapter 2.2.2.1 --- Aqueous Extraction --- p.36 / Chapter 2.2.2.2 --- Methanol Extraction --- p.37 / Chapter 2.2.3 --- Preparation of Recombinant HIV-1 Protease --- p.37 / Chapter 2.2.3.1 --- Selection of Appropriate Clone --- p.37 / Chapter 2.2.3.2 --- Large-scale Expression of Recombinant HIV-1 Protease --- p.38 / Chapter 2.2.2.3 --- Purification of Recombinant HIV-1 Protease by DEAE Sepharose CL-6B Chromatography --- p.38 / Chapter 2.2.3.4 --- Purification of Recombinant HIV-1 Protease by Mono-S Cation Chromatography --- p.39 / Chapter 2.2.3.5 --- Refolding of Purified Recombinant HIV-1 Protease --- p.40 / Chapter 2.2.3.6 --- Protein Concentration Determination --- p.41 / Chapter 2.2.3.7 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.41 / Chapter 2.2.4 --- Characterization of HTV-1 Protease --- p.42 / Chapter 2.2.4.1 --- HIV-1 PR Fluorogenic Assays --- p.42 / Chapter 2.2.4.2 --- HIV-1 PR Assay by Reverse Phase HPLC Separation of Cleavage Products of the Synthetic Peptide Substrate --- p.43 / Chapter 2.3 --- Results --- p.44 / Chapter 2.3.1 --- Functional Analysis of Recombinant HIV-1 PR Activity --- p.44 / Chapter 2.3.2 --- Screening of Crude Extracts for Inhibition of HIV-1 PR Activity --- p.48 / Chapter 2.4 --- Discussion --- p.53 / Chapter CHAPTER 3 --- ISOLATION AND CHARACTERIZATION OF ACTIVE CONSTITUENTS FROM METHANOL EXTRACTS OF WOODWARDIA UNIGEMMATA AGAINST HIV-1 PROTEASE --- p.56 / Chapter 3.1 --- Introduction --- p.56 / Chapter 3.2 --- Materials and Methods --- p.57 / Chapter 3.2.1 --- Materials --- p.57 / Chapter 3.2.2 --- Methods --- p.58 / Chapter 3.2.2.1 --- Methanol Extraction --- p.58 / Chapter 3.2.2.2 --- Removal of Tannins --- p.60 / Chapter 3.2.2.3 --- Glucosidase Digestion --- p.60 / Chapter 3.2.2.4 --- Analytical Thin Layer Chromatographic (TLC) --- p.61 / Chapter 3.2.2.5 --- A cid Hydrolysis --- p.62 / Chapter 3.2.2.6 --- Electrospray Mass Spectrometry --- p.62 / Chapter 3.2.2.7 --- Dose-response Curve --- p.63 / Chapter 3.2.2.8 --- Kinetic Studies --- p.63 / Chapter 3.2.2.9 --- Activity of the HPLC-purified principle (s) on Other Aspartyl Proteases --- p.63 / Chapter 3.3 --- Results --- p.66 / Chapter 3.3.1 --- Purification of Methanol Extracts of Woocdwardia unigemmata --- p.66 / Chapter 3.2.2 --- Removal of Tannins --- p.70 / Chapter 3.2.3 --- Glucosidase Digestion --- p.73 / Chapter 3.2.4 --- Acid Hydrolysis --- p.73 / Chapter 3.2.5 --- Analytical Thin Layer Chromatography --- p.74 / Chapter 3.2.6 --- Electrospray Mass Spectrometry --- p.80 / Chapter 3.2.7 --- Dose-response Inhibition of HIV-1 Protease --- p.80 / Chapter 3.2.8 --- Kinetic Studies --- p.85 / Chapter 3.2.9 --- Effects of HPLC-purified Active Principle on Other Aspartyl Proteases --- p.87 / Chapter 3.3 --- Discussion --- p.89 / Chapter CHATPER 4 --- IDENTIFICATION OF SELECTIVE RNA APTAMERS AGAINST HIV-1 PROTEASE BY SYSTEMATIC EVOLUTION OF LIGANDS BY EXPONENTIAL ENRICHMENT (SELEX) --- p.95 / Chapter 4.1 --- Introduction --- p.95 / Chapter 4.2 --- Materials and Methods --- p.101 / Chapter 4.2.1 --- Materials --- p.101 / Chapter 4.2.2 --- Methods --- p.102 / Chapter 4.2.2.1 --- PCR Amplification for the Generation of a Double-Stranded DNA Library --- p.103 / Chapter 4.2.2.2 --- Preparation of RNA Pools --- p.104 / Chapter 4.2.2.3 --- In vitro Selection of RNA Ligands --- p.104 / Chapter 4.2.2.4 --- Reverse Transcription Reaction of Selected RNA --- p.108 / Chapter 4.2.2.5 --- Cloning of the Amplified cDNA pools --- p.108 / Chapter 4.2.2.6 --- Subcloning of the digested DNA product into pBluescript® IIKS (-) --- p.108 / Chapter 4.2.2.8 --- RNA Labeling with Digoxigenin (DIG) --- p.109 / Chapter 4.2.2.9 --- Binding Affinity of RNA Ligands for HIV-1 PR --- p.109 / Chapter 4.2.2.10 --- Competition Binding Reactions --- p.111 / Chapter 4.2.2.11 --- HIV-1 PR Inhibitory Activities of the Selected RNA Ligands --- p.112 / Chapter 4.3 --- Results --- p.113 / Chapter 4.3.1 --- In Vitro Selection of RNA Ligands --- p.113 / Chapter 4.3.2 --- Sequences of RNA Ligands --- p.114 / Chapter 4.3.3 --- Binding Affinity of RNA Ligands --- p.114 / Chapter 4.3.4 --- Inhibitory Activity of RNA Ligands --- p.119 / Chapter 4.4 --- Discussion --- p.122 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.128 / REFERENCES --- p.132

by Lam Mei Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 127-137). / Abstract also in Chinese. / Acknowledgment --- p.I / Table of content --- p.II / List of figures --- p.VII / List of tables --- p.IX / Abbreviation --- p.X / Abstract --- p.XII / 論文摘要 --- p.XIII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Acquired immunodeficiency syndrome --- p.1 / Chapter 1.1.1 --- Discovery of AIDS --- p.1 / Chapter 1.1.2 --- Definition and symptoms of AIDS --- p.1 / Chapter 1.1.3 --- AIDS transmission --- p.2 / Chapter 1.1.4 --- AIDS epidemic --- p.3 / Chapter 1.2 --- Human immunodeficiency virus --- p.3 / Chapter 1.2.1 --- Discovery of HIV --- p.3 / Chapter 1.2.2 --- The structure of HIV --- p.4 / Chapter 1.2.3 --- Genomic structure of HIV --- p.5 / Chapter 1.2.4 --- Life cycle of HIV --- p.5 / Chapter 1.2.5 --- How HIV is involved in different stages of AIDS --- p.7 / Chapter 1.3 --- Therapeutic targets for treatment of AIDS --- p.8 / Chapter 1.3.1 --- HIV reverse transcriptase (HIV RT) --- p.8 / Chapter 1.3.2 --- HIV integrase (HIV IN) --- p.11 / Chapter 1.3.3 --- HIV protease (HIV PR) --- p.12 / Chapter 1.3.4 --- Chemokine receptors --- p.14 / Chapter 1.3.5 --- Vaccine development --- p.16 / Chapter 1.4 --- AIDS therapy --- p.17 / Chapter 1.4.1 --- Current status of AIDS therapy --- p.17 / Chapter 1.4.1.1 --- Drugs approved by US Food & Drug Administration (FDA) --- p.17 / Chapter 1.4.1.2 --- Combination therapy --- p.19 / Chapter 1.4.1.3 --- Vaccine development --- p.19 / Chapter 1.4.2 --- Alternative treatment --- p.20 / Chapter 1.5 --- Objective of my project --- p.21 / Chapter Chapter 2 --- Screening of traditional Chinese medicinal (TCM) plants for HIV reverse transcriptase inhibition --- p.22 / Chapter 2.1 --- Introduction --- p.22 / Chapter 2.1.1 --- HIV RT structure and function --- p.22 / Chapter 2.1.2 --- Natural product against HIV RT --- p.25 / Chapter 2.1.3 --- Inhibitory activities from plant extracts --- p.27 / Chapter 2.2 --- Materials and Methods --- p.28 / Chapter 2.2.1 --- Materials --- p.28 / Chapter 2.2.2 --- Extraction methods --- p.30 / Chapter 2.2.2.1 --- Methanol extraction --- p.30 / Chapter 2.2.2.2 --- Hot water extraction --- p.30 / Chapter 2.2.2.3 --- Preparation of Prunella vulgaris extract --- p.30 / Chapter 2.2.3 --- Reverse transcriptase assay --- p.31 / Chapter 2.2.4 --- Characterization of active component in extract of Prunella vulgaris --- p.32 / Chapter 2.2.4.1 --- Protease digestion --- p.32 / Chapter 2.2.4.2 --- Glucosidase digestion --- p.32 / Chapter 2.2.4.3 --- Ethanol precipitation --- p.33 / Chapter 2.2.4.4 --- Sodium periodiate oxidization --- p.33 / Chapter 2.2.4.5 --- Polyvinylpyrrolidone (PVP) Precipitation --- p.34 / Chapter 2.2.4.6 --- Polyamide resin binding --- p.34 / Chapter 2.2.5 --- Purification of Prunella vulgaris extract --- p.34 / Chapter 2.2.5.1 --- Polyamide resin column chromatography --- p.34 / Chapter 2.2.5.2 --- Sephadex LH-20 chromatography --- p.35 / Chapter 2.2.5.3 --- Reverse phase HPLC chromatography --- p.36 / Chapter 2.2.6 --- Characterization of purified Prunella vulgaris extract --- p.37 / Chapter 2.2.6.1 --- Paper chromatography --- p.37 / Chapter 2.2.6.2 --- Acid hydrolysis of extract --- p.37 / Chapter 2.2.6.3 --- Thin layer chromatography --- p.38 / Chapter 2.2.6.4 --- Other assays --- p.39 / Chapter 2.2.7 --- Calculation --- p.40 / Chapter 2.3 --- Results --- p.41 / Chapter 2.3.1 --- Screening of Herbs --- p.41 / Chapter 2.3.1.1 --- Screening of methanol extracts --- p.41 / Chapter 2.3.1.2 --- Screening of hot water extracts --- p.41 / Chapter 2.3.2 --- Characterization of active components in Prunella vulgaris crude extracts --- p.44 / Chapter 2.3.2.1 --- Protease digestion --- p.44 / Chapter 2.3.2.2 --- Glucosidase digestion --- p.44 / Chapter 2.3.2.3 --- Ethanol precipitation --- p.44 / Chapter 2.3.2.4 --- Sodium periodate oxidation --- p.48 / Chapter 2.3.2.5 --- Effect of naturally occurring chemicals on inhibition of HIV RT --- p.48 / Chapter 2.3.2.6 --- Effect of removal of polyphenolic components of aqueous extract on inhibition of HTV RT --- p.51 / Chapter 2.3.3 --- Further purification of active components in aqueous extract of Prunella vulgaris --- p.53 / Chapter 2.3.3.1 --- Absorption chromatography by polyamide resin --- p.53 / Chapter 2.3.3.2 --- The Sephadex LH-20 chromatography --- p.53 / Chapter 2.3.3.3 --- Reverse phase high performance liquid chromatography --- p.56 / Chapter 2.3.3.4 --- Recovery of extract --- p.59 / Chapter 2.3.3.5 --- Inhibition from extract of various steps of purification --- p.59 / Chapter 2.3.4 --- Characterization of purified aqueous extract of Prunella vulgaris --- p.62 / Chapter 2.3.4.1 --- Paper chromatography --- p.62 / Chapter 2.3.4.2 --- Dose response curve --- p.62 / Chapter 2.3.4.3 --- Acid hydrolysis of purified extract --- p.68 / Chapter 2.3.4.4 --- Identification of monosaccharide in purified extract by Thin layer chromatography (TLC) --- p.71 / Chapter 2.3.5 --- Specificity of the purified extract on polymerase inhibition --- p.75 / Chapter 2.3.5.1 --- Inhibition of purified Prunella vulgaris extract on Taq polymerase --- p.75 / Chapter 2.3.5.2 --- Inhibition of purified Prunella vulgaris extract on Superscript II --- p.75 / Chapter 2.4 --- Discussion --- p.79 / Chapter Chapter 3 --- Screening of inhibitory activities from traditional Chinese medicinal (TCM) plants extracts to HIV protease --- p.86 / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.1.1 --- HIV Protease structure and function --- p.86 / Chapter 3.1.2 --- Natural products against HIV Protease --- p.87 / Chapter 3.1.3 --- Plant extracts against HIV Protease --- p.89 / Chapter 3.2 --- Materials and Methods --- p.91 / Chapter 3.2.1 --- Materials --- p.91 / Chapter 3.2.2 --- Expression of HIV protease --- p.92 / Chapter 3.2.2.1 --- Expression and purification of HIV protease --- p.92 / Chapter 3.2.2.2. --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.94 / Chapter 3.2.3 --- Characterization of HIV protease --- p.95 / Chapter 3.2.3.1 --- HIV protease assay by fluorometric measurement --- p.95 / Chapter 3.2.3.2 --- HIV protease assay by using reverse phase high performance liquid chromatography --- p.96 / Chapter 3.3 --- Results --- p.98 / Chapter 3.3.1 --- Expression of HIV protease --- p.98 / Chapter 3.3.2 --- HIV protease assay --- p.98 / Chapter 3.3.2.1 --- Protease assay by using reverse phase HPLC --- p.98 / Chapter 3.3.2.2 --- Protease assay by fluorometric measurement --- p.98 / Chapter 3.3.3 --- Screening of crude Chinese medicinal extracts on inhibition of HIV protease --- p.104 / Chapter 3.3.3.1 --- Methanol extracts --- p.104 / Chapter 3.3.3.2 --- Water extracts --- p.105 / Chapter 3.3.4 --- Characterization of herbal extracts on inhibition of HIV protease --- p.110 / Chapter 3.3.4.1 --- Dose response curve of methanol extract of Woodwardia unigemmata --- p.110 / Chapter 3.3.4.2 --- Dose response curve of hot water extract of Prunella vulgaris --- p.110 / Chapter 3.3.4.3 --- Inhibition mode of methanol extract of Woodwardia unigemmata --- p.113 / Chapter 3.3.4.4 --- Inhibition mode of hot water extract of Prunella vulgaris --- p.113 / Chapter 3.3.4.5 --- Effect of partially purified extracts on HIV protease inhibition --- p.116 / Chapter 3.4 --- Discussion --- p.119 / Chapter Chapter 4 --- General discussion --- p.124 / References --- p.127 / Appendix / Appendix 1 Pictures of herbs used in this study --- p.i / Appendix 2 Mass spectrometry of purified Prunella vulgaris extract --- p.vi / Appendix 3 Calibration curve for determination of HIV PR concentration --- p.viii

In the 30 years since the Center for Disease Control's Morbidity and Mortality Weekly Report published the first mention of what later was determined to be AIDS (Acquired immunodeficiency syndrome) and HIV (Human immunodeficiency virus) recognized as the causative pathogen, much has been done to understand this disease’s pathogenesis, development of drugs and emergence of drug resistance under selective drug therapy. Highly Active Antiretroviral Therapy (HAART), a combination of drugs that includes HIV-1 reverse transcriptase, protease, and more recently, integrase and entry inhibitors, have helped stabilize the HIV prevalence at extraordinarily high levels. Despite the recent stabilization of this global epidemic, its dimensions remain staggering with estimated (33-36 million) people living with HIV-AIDS in 2007 alone. This is because the available drugs against AIDS provide treatment for infected individuals, but HIV evolves rapidly under drug pressure and develops resistant strains, rendering the therapy ineffective. Therefore, a better understanding underlying the molecular mechanisms of viral infection and evolution is required to tackle drug resistance and develop improved drugs and treatment regimens. HIV-1 protease is an important target for developing anti-HIV drugs. However, resistant mutations rapidly emerge within the active site of the protease and greatly reduce its affinity for the protease inhibitors. Frequently, these active site drug resistant mutations co-occur with secondary/ non-active site/ associated or compensatory mutations distal to the active site. The role of these accessory mutations is often suggested to be in maintaining viral fitness and stability of protease. Many of the non-active site drug resistant mutations are clustered in the hydrophobic core in each monomer of the protease. Molecular dynamic simulation studies suggest that the hydrophobic core residues facilitate the conformational changes that occur in protease upon ligand binding. There is a complex interdependence and interplay between the inherent adaptability, drug resistant mutations and substrate recognition by the protease. Protease is inherently dynamic and has wide substrate specificity. The PI (protease inhibitor) resistant mutations, perhaps, modulate this dynamics and bring about changes in molecular recognition, such that, in resistant proteases, the substrates are recognized specifically over the PIs for the same binding site. In this thesis research, I have investigated these three complementary phenomena in concert. Chapter II examines the importance of hydrophobic core dynamics in modulating protease function. The hydrophobic core in the WT protease is intrinsically flexible and undergoes conformational changes required for protease to bind its substrates. This study investigated if dynamics is important for protease function by engineering restricted vs. flexible hydrophobic core region in each monomer of the protease, using disulfide chemistry. Under oxidizing conditions, disulfide bond established cross-link at the interface of putative moving domains in each monomer, thereby, restricting motion in this region. Upon reduction of the disulfide bond, the constraining influence was reversed and flexibility returned to near WT. The disulfide cross-linked protease showed significant loss of function when tested in functional cleavage assay. Two protease variants (G16C/L38C) and (R14C/E65C) were engineered and examined for changes in structure and enzymatic activity under oxidizing and reducing conditions. (R14C/E65C) was engineered as an internal control variant, such that cysteines were engineered between putative non-moving domains. Structurally, both the variants were very similar with no structural perturbations under oxidizing or reducing conditions. While significant loss in function was observed for (G16C/L38C) only under oxidizing conditions, (R14C/E65C) did not show any loss of function under oxidizing or reduced conditions, as expected. Successful regain of function for cross-linked (G16C/L38C) was obtained upon reversible reduction of the disulfide bond. Taken together, these data demonstrate that the hydrophobic core dynamics modulates protease function and support the hypothesis that the distal drug resistant mutations, possibly causing drug resistance by modulating hydrophobic core dynamics via long range structural perturbations. Since protease recognizes and cleaves more than 10 substrates at different rates, our further interest is to investigate if there is a differential loss of activity for some specific substrates over the others, and whether the order of polypeptide cleavage is somehow affected by restricted core mobility. In order to better answer these questions it is essential to understand: what determines the substrate binding specificity in protease? A two-pronged approach was applied to address this question as described in chapter III and IV respectively. In chapter III, I investigated the determinants of substrate specificity in HIV-1 protease by using computational positive design and engineered specificity-designed asymmetric protease (Pr3, A28S/D30F/G48R) that would preferentially bind to one of its natural substrates, RT-RH over two other substrates, p2-NC and CA-p2, respectively. The designed protease was expressed, purified and analyzed for changes in structure and function relative to WT. Kinetic studies on Pr3 showed that the specificity of Pr3 for RT-RH was increased significantly compared to the wild-type (WT), as predicted by the positive design. ITC (Isothermal Titration Calorimetry) studies confirmed the kinetic data on RT-RH. Crystal structural of substrate complexes of WT protease and Pr3 variant with RT-RH, CA-p2 and p2-NC were further obtained and analyzed. The structural analysis, however, only partially confirmed to the positive design due to the inherent structural pliability of the protease. Overall, this study supports the positive computational design approach as an invaluable tool in facilitating our understanding of complex proteins such as HIV 1 protease and also proposes the integration of internal protein flexibility in the design algorithms to make the in-silico designs more robust and dependable. Chapter IV probed the substrate specificity determining factors in HIV-1protease system by focusing on the substrate sequences. Previous studies have demonstrated that three N-terminal residues immediate to the scissile bond (P1-P3) are important in determining recognition specificity. This work investigated the structural basis of substrate binding to the protease. Catalytically active WT protease was crystallized with decameric polypeptides corresponding to five of the natural cleavage sites of protease. The structural analyses of these complexes revealed distinct P side product bound in all the structures, demonstrating the higher binding affinity of N terminal substrate for protease. This thesis research successfully establishes that intrinsic hydrophobic core flexibility modulates function in HIV-1 protease and proposes a potential mechanism to explain the role of non-active site mutations in conferring drug resistance in protease. Additionally, the work on specificity designed and N terminal product bound protease complexes advances our understanding of substrate recognition in HIV protease.

Viral proteases have been shown to be effective targets of anti-viral therapies for human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, under the pressure of therapy including protease inhibitors, the virus evolves to select drug resistance mutations both in the protease and substrates. In my thesis study, I aimed to understand the mechanisms of how this protease−substrate co-evolution contributes to drug resistance. Currently, there are no approved drugs against dengue virus (DENV); I investigated substrate recognition by DENV protease and designed cyclic peptides as inhibitors targeting the prime site of dengue protease. First, I used X-ray crystallography and subsequent structural analysis to investigate the molecular basis of HIV-1 protease and p1-p6 substrate coevolution. I found that co-evolved p1-p6 substrates rescue the HIV-1 I50V protease’s binding activity by forming more van der Waals contacts and hydrogen bonds, and that co-evolution restores the dynamics at the active site for all three mutant substrates. Next, I used aprotinin as a platform to investigate DENV protease–substrate recognizing pattern, which revealed that the prime side residues significantly modulate substrate affinity to protease and the optimal interactions at each residue position. Based on these results, I designed cyclic peptide inhibitors that target the prime site pocket of DENV protease. Through optimizing the length and sequence, the best inhibitor achieved a 2.9 micromolar Ki value against DENV3 protease. Since dengue protease does not share substrate sequence with human serine proteases, these cyclic peptides can be used as scaffolds for inhibitor design with higher specificity.

Glycosidases are carbohydrate processing essential enzymes necessary for the growth and development of all organisms such as intestinal digestion, post-translational processing of glycoproteins and the lysosomal catabolism of glycoconjugates. The function of these glycosidases is limited and studies are still in progress to understand their function at cellular level. In recent years, biological role of carbohydrates has resulted in various carbohydrate-based therapeutics2. These carbohydrates serve as a tool to study the function of glycosidases by inhibiting their active site. The concept of inhibition is yet another approach for the discovery of drugs. Glycosidase inhibitors studied are often sugar analogs and a wide range of such inhibitors are reported in the literature.3, 4 Thiosugars, in particular, have gained new perspectives owing to their electronic, geometric, conformational and flexibility differences, as sulfide moiety being less electronegative and more polarizable than the oxygen counter-part.5 These differences make the thiosugars distinct from their oxygen analogs and hence can mimic the active site of the enzyme. Many molecules are reported to be promising glycosidase inhibitors but are not easily accessible due to difficulties in their synthesis. Hence, the chemical synthesis of thio-analogs of carbohydrates, by synthetic routes, remains a major challenge. To address the complexity of synthesis and to make available new strategies, we envisioned the use of benzyltriethylammonium tetrathiomolybdate [BnEt3N]2MoS4, a versatile and efficient sulfur transfer reagent. Objectives of the study: a. Design novel thiosugars as glycosidase inhibitors. b. Devise strategy for the synthesis of novel thiosugars through a simple, practical approach. c. Evaluate the synthesized molecules as glycosidase and HIV-1 protease inhibitors, in silico. d. Study miscellaneous applications of the novel thiosugar-derived thialactones. The thesis is divided into five sections: Section A entitled “Synthesis of deoxythiosugars and thiosugar-based lactones” is divided into two parts, Part A and Part B. Part A – “An introduction and background on thiosugars and sulfur transfer reagents” has been provided. A brief discussion of sulfur transfer reagents in carbohydrate synthesis and earlier work related to the use of benzyltriethylammonium tetrathiomolybdate, [BnEt3N]2MoS4, as an efficient sulfur transfer reagent have been provided. Part B –“Design of inhibitors of glycosidases and HIV-1 protease” deals with the design of inhibitors of glycosidase and HIV-1 protease. The designed thiosugar molecules exhibit the characteristics of sugars and will act as planar molecules to mimic the active site conformation of a good inhibitor. Synthetic methodologies devised and adopted for the synthesis of constrained sugar-derived thialactones include: (a) Double displacement, (b) Displacement-cum-intramolecular thia-Michael addition, (c) Epoxide ring-opening-cum-intramolecular thia-Michael addition, and (d) Displacement-cum-epoxide ring opening in an intramolecular fashion. In all the above mentioned strategies, sulfur transfer step is the crucial step which was achieved by the use of benzyltriethylammonium tetrathiomolybdate [BnEt3N]2MoS46 as the key reagent. (a) Various constrained thialactones synthesized by double displacement strategy using tetrathiomolybdate as the sulfur transfer reagent are shown in Scheme – 1. (b) A number of constrained thialactones were synthesized following nucleophilic displacement-cum-intramolecular thia-Michael addition strategy as shown in Scheme – 2. (c) Synthesis of bicyclic thiolactones was achieved using the strategy of epoxide ring-opening-cum-intramolecular thia-Michael addition. (Scheme – 3) (d) A few bicyclic thialactones were synthesized through displacement-epoxide ring opening-cyclization as shown in Scheme – 4. The methodology was also utilized for the synthesis of thiosugar derivatives and azido-thialactones. (Fig. 1) Figure 1 Synthesis of deoxythiosugars: The bicyclic thialactones (designed as inhibitors) on reduction with borohydride exchange resin (BER) easily furnished the deoxythiosugars (Fig. 2). It is worth mentioning that the synthesis of these thiosugars as reported in the literature involved lengthy procedures whereas the present methodology turns out to be short and concise. Figure 2 Section B entitled “Synthesis of amines, β-amino acids and novel thiosugar-based dehydroamino acids” comprises a brief introduction on the importance of amines, β-amino acids and dehyroamino acids. In this section the effective utilization of benzyltriethylammonium tetrathiomolybdate as a key reagent for reductive transformations and its application in the synthesis of amines, β-amino acids and dehyroamino acids have been presented. A one pot reduction of azides to amines followed by intermolecular aza-Michael addition employing tetrathiomolybdate was achieved to furnish a number of different β-amino esters as shown in Scheme -4: Scheme 4 The study was further extended to the reduction of a few anomeric azides to afford the corresponding anomeric amines and derivatives. (Fig. 3) Figure 3 A one-pot thia-Michael addition-vinyl azide reduction in a tandem fashion employing benzyltriethylammonium tetrathiomolybdate was studied and was shown to be effective for the synthesis of thiosugar derived dehydroamino acid derivatives. (Scheme – 5) Scheme 5 Section C entitled “Molecular docking studies of deoxythiosugar probes” gives an overview of different glycosidases, HIV-1 protease and their inhibitors. This section also deals with a brief introduction on active site conformations of potent inhibitors. In this connection we have studied the crystal conformations of the synthesized molecules whose conformations were the same as that of the existing inhibitors in the active site. (Fig. 4) With this background in silico study of the synthesized deoxythiosugar probes was conducted on human glycosidases: α-mannosidase, α-galactosidase, β-glucosidase and HIV-1 protease, respectively. Figure 4 Molecular docking was carried out using Autodock suite, molecular modeling simulation. Separate docking procedures were employed for the four different receptors. The PDBs representing the four enzyme targets were 2V3D, 3H53, 1X9D and 3I8W for β–glucosidase, α–galactosidase, α–mannosidase and HIV–1 protease respectively. The control compounds used for α–mannosidase were mannostatin and kifunensine. NMB, THK, and BED were the positive controls for HIV–1 protease. Similarly, NBV and cyclophellitol were the controls used for β–glucosidase and NOJ, N–methyl calystegine B2 for α–galactosidase. (Fig. 5) Ligands TGSB68 and TGSB482 had the energy value of –6.49 kcal/mol comparable to that of the average reference value of the positive control, and thus, the potent candidate as identified by molecular docking to HIV-1 protease. (Fig. 6a) The control compounds used for α–mannosidase were mannostatin and kifunensine, which bind with mean binding energy of -9.11 and -5.56. In the case of α–mannosidase, the same compounds TGSB68 and TGSB482 were selected due to comparable energy and a good cluster size with that of positive control. (Fig. 6b) For β– glucosidase, ligands TGSC108 and TGSC236, which had comparable values to that of positive control was identified as the Figure 5 Figure 6 potent candidate. (Fig. 6c) In the case of α–galactosidase, again the ligands TGSB68 and TGSB482 were selected based on binding energies. (Fig. 6d) In conclusion, the concept analogy (deoxy nature, planarity, thiosugar framework, lactone moiety) for the design of inhibitors indeed worked positively. The results are really encouraging. An in vivo study of the synthesized novel thiosugar probes will certainly provide a potent inhibitor. Section D entitled “Research methodology” provides experimental procedures adopted with details of synthesis. Section E entitled “Bibliography” provides the references cited in this work.