Referat: Background: Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease of humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. However, the available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to the probable similarities in cell metabolism among tumor and trypanosoma cells, some of the current registered drugs against HAT were derived from cancer chemotherapeutic research. Here too, for the first time, we have demonstrated that the simple ester, ethyl pyruvate, comprises such properties. On the other hand initial studies have confirmed the efficacy of protease inhibitors in treatment of Trypanosoma cruzi, Plasmodium falciparum and Leishmania major. However, studies on efficacy and specific proteases inhibition using HIV-1 protease inhibitors on T. brucei cells remain untouched. Methodology/Principal findings: The current study covers efficacy and corresponding target evaluation of ethyl pyruvate and HIV-1 protease inhibitors (ritonavir and saquinavir) on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, zymography, phase contrast microscopic video imaging and ex vivo drug toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki=3.0±0.29 mM). The potential of this compound as an anti-trypanosomal drug is also strengthened by its fast acting property, killing cells within three hours post exposure. This was demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, this drug produced minimal side effects in human erythrocytes and is known to easily cross the blood-brain-barrier (BBB) which makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug resistance tests indicate irreversible killing of cells and a low chance of drug resistance development under applied experimental conditions. In addition to ethyl pyruvate our experimental study on HIV-1 protease inhibitors showed that both ritonavir (RTV) (IC50=12.23 µM) and saquinavir (SQV) (IC50=11.49 µM) effectively inhibited T. brucei cells proliferation. The major proteases identified in these cells were the cysteine- (~29kDa Mr) and metallo- (~66kDa Mr) proteases. Their proteolytic activity was, however, not hampered by either of these two protease inhibitors. Conclusion/Significance: Our results present ethyl pyruvate as a safe and fast acting drug. Hence, because of its predefined property to easily cross the BBB, it can probably be a new candidate agent to treat the heamolymphatic as well as neurological stages of sleeping sickness. Similarly, HIV-1 protease inhibitors, SQV and RTV, exhibited their antitrypanosomal potential but require further anlysis to identify their specific targets.

Chapter 1 gives background information on proteases and discusses the concept of protease inhibition as a therapeutic strategy for humans. It introduces the key concept that conformation defines biological activity. It also outlines how proteases almost universally bind their substrate/inhibitors in an extended β-strand conformation. The use of calpain as a prototype protease for the testing of β-strand mimics synthesised later in the thesis is also discussed. Chapter 2 describes how molecular modeling was used to rationalise the structure based activity relationships (SAR) of known calpain inhibitors. Molecular modeling was then used to successfully design a number of acyclic β-strand mimics. The synthesis and testing of eight such inhibitors is described. The most potent β-strand mimic prepared was 2.13. This was determined to have an IC₅₀ of 30 nM against calpain II. Chapter 3 outlines the history and application of ring closing metathesis (RCM) to the synthesis of cyclic compounds. The attempted synthesis of an eight membered cyclic nitrogen to nitrogen conformationally constrained dipeptide is described. The synthesis of a conformationally constrained β-amino acid calpain inhibitor (3.73) is also described. A novel calpain inhibitor motif was designed in Chapter 4. On the basis of this an in-silico combinatorial library of two hundred and eighty eight possible β-strand templates was prepared. Conformational analysis of this library was performed and from this a number of excellent β-strand templates were identified and selected for synthesis. The preparation of ten β-strand templates is described. New microwave irradiation methodology was developed to achieve this. vii The formation of a six-membered catalyst deactivating chelate is also proposed to explain why some dienes fail to undergo RCM. Two methods to circumvent the formation of such a chelate are outlined. The addition of Lewis acid chloro-dicyclohexyl borane to the RCM reaction mixture and chain length alteration are investigated. Chapter 5 describes the design of macrocyclic β-strand mimics using induced fit molecular modelling. The physicochemical properties of these were calculated in-silico. From this analysis a number of Tyr-XX-Gly based and Tyr-XX-Cys based macrocyclic calpain inhibitors were selected for synthesis. The preparation and testing of these are described. In the Tyr-XX-Gly macrocyclic system a number of variables were investigated and numerous SAR implications concluded. Aldehyde 5.14 was identified as the best electrophilic warhead macrocyclic calpain inhibitor with an IC₅₀ against calpain II of 27 nM. The best non-electrophilic warhead macrocycle (5.13) had an IC₅₀ against calpain II of 704 nM. Chapter 6 describes synthetic optimisation for the preparation of calpain inhibitors 2.13, 5.14 and 5.17. Multi-gram quantities of each were prepared. Aldehydes 2.13 and 5.14 were evaluated as anti-cataract agents using in-vivo cataract sheep model. Both of these β-strand mimics were demonstrated to retard cataract development. Macrocycle 5.14 was found to be the most effective, decreasing the rate of cataract development between forty four and forty nine per cent relative to control. Chapter 7 outlines the attempted development of RCM methodology for the chiral synthesis of α-α disubstituted amino acid lactams. In addition, methodology for the stereoselective incorporation of a C-N constrained β-amino acid carbocycle into a peptide or peptidomimetic is described.

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

A enzima Paraoxonase-1 (PON1) possui atividades paraoxonase, arilestearase e lactonase, entre outras. É a mais estuda da família das PONs que é composta pela PON1, PON2 e PON3. Sugere-se, que todas atuam inibindo o processo de peroxidação lipídica de moléculas como a lipoproteína de baixa densidade (LDL) e alta densidade (HDL), caracterizando assim um possível papel anti-aterogênico. O gene da PON1 apresenta dois sítios polimórficos, com a troca de uma Gln192Arg (Q/R) e Met55Leu, que estão associados com diferenças na atividade e concentrações séricas da enzima. Por sua vez, indivíduos soropositivos para o HIV-1 apresentam alterações do metabolismo lipídico, que poderiam estar associados a alterações na atividade da PON1 e a terapia antirretroviral (TARV) com inibidores de protease (IP). O objetivo do estudo foi determinar as atividades séricas da PON1 e da arilestearase (ARE), e as freqüências alélicas dos polimorfismos genéticos da PON1 192QR e 55LM, e ainda, avaliar a correlação destes parâmetros com as alterações lipídicas em indivíduos soropositivos para o HIV-1 tratados com IP. No período de Setembro de 2009 até Junho de 2012, 174 indivíduos soropositivos e 46 soronegativos para o HIV-1 foram estudados. Foi realizada a genotipagem dos polimorfismos da PON1 192QR e 55LM através de PCR-RFLP. A atividade sérica da PON1/ARE foi avaliada por espectrofotometria empregando-se como substratos o paraoxon e o fenilacetato, respectivamente. O RNA-HIV-1 foi quantificado pelo método NASBA, e os linfócitos T-CD4+ e T-CD8+ por citometria de fluxo. Os níveis séricos de colesterol total, HDL, LDL, triglicérides (TG), ApoA1 e ApoB100 foram determinados e os anticorpos IgG anti-oxLDL por ELISA. A atividade sérica da PON1 foi inferior nos grupos de soropositivos, p<0,05, porém, a atividade ARE não apresentou diferenças entre os grupos estudados, p>0,05. Ambas as atividades não apresentaram relação com os genótipos PON1 192QR e 55LM, e estes genótipos apresentaram uma freqüência alélica semelhante ao grupo de soronegativos. Os níveis séricos de TG foram superiores nos grupos de soropositivos com TARV, p<0,05, enquanto o grupo tratado com IP apresentou níveis séricos de HDL e Apo-A1 inferiores aos demais grupos, p<0,05. Níveis séricos de Apo-B100, IgG anti-oxLDL, e o índice de risco aterogênico foram superiores no grupo tratado com IP, p<0,05. Concluí-se, que indivíduos soropositivos para o HIV-1 apresentaram alterações no metabolismo lipídico, principalmente nos tratados com IP, que adicionalmente apresentaram um maior índice de risco aterogênico e maiores níveis de anticorpos IgG anti-oxLDL. Estas alterações não apresentaram relação com os polimorfismos PON1 192QR e 55LM da PON1, e demonstraram que a atividade da enzima PON-1 esta diminuída em indivíduos soropositivos para o HIV-1 / The enzyme Paraoxonase-1 (PON1) has paraoxonase (PON), arylesterase (ARE) and lactonase activities, among others. It is the most studied member of PON family which is composed of PON1, PON2 and PON3. It is suggested that all members acts by inhibiting the peroxidation of lipid molecules as the low-density lipoprotein (LDL) and high-density lipoprotein (HDL), characterizing a potential anti-atherogenic effect. The PON1 gene has two mainly polymorphic sites, with an exchange of Gln192Arg (Q/R) and Met55Leu (L/M), which are associated with differences in activity and serum concentrations of the enzyme. In turn, seropositive individuals for HIV-1 show changes in lipid metabolism, which could be associated with changes in the activity of PON1 and highly active antiretroviral therapy (HAART) with protease inhibitors (PI). The aim of this study was to determinate the serum PON and ARE activities of PON1, the allele frequencies of PON1 192QR and PON1 55LM genetic polymorphisms and evaluate the correlation between these parameters and lipid abnormalities in seropositive patients for HIV-1 treated with IP. In the period from September 2009 until June 2012, 174 seropositive individuals and 46 soronegative individuals for HIV-1 were studied. We performed PON1 192QR and 55LM genotyping by PCR-RFLP. Serum activities PON and ARE of PON1 were evaluated by spectrophotometry using paraoxon and phenylacetate, respectively, as substrates. The HIV-1-RNA was quantified by the NASBA method, and lymphocytes T-CD4+ and T-CD8+, by flow cytometry. Serum levels of total cholesterol, HDL, LDL, triglycerides (TG), apoA1 and ApoB100 were determined. IgG anti-oxLDL antibodies were quantified by ELISA. The serum PON1 activity was lower in the seropositive group, p<0.05, however, ARE activity did not differ between groups, p>0.05. Both activities had no relation with the PON1 192QR and PON1 55LM genotype, and these individuals showed an allele frequency similar to the seronegative group. Serum levels of TG were higher in groups of HIV-positive with HAART, p<0.05, while the IP-treated group showed serum levels of HDL and ApoA1 lower than other groups, p <0.05. Serum levels of ApoB100, IgG anti-oxLDL antibodies, and atherogenic risk indices were higher in the group treated with PI, p<0.05. It was concluded that individuals HIV-1-infected showed changes in lipid metabolism, especially in those treated with IP, which additionally showed a higher rate of atherogenic risk and higher levels of IgG anti-oxLDL antibodies. These changes did not correlated with PON1 192QR and 55LM polymorphisms and demonstrated that the activity of PON1enzyme is decreased in individuals seropositive for HIV-1

INTRODUÇÃO: A hepatite C crônica é uma das principais causas de hepatopatia em todo mundo. A coinfecção pelo vírus C (VHC) e o HIV não é incomum, pois ambos compartilham vias similares de transmissão. Recentemente, a terapêutica da hepatite C crônica foi radicalmente modificada com o advento das drogas antivirais de ação direta (DAAs), elevando as taxas de RVS mesmo na população coinfectada. O VHC é caracterizado pela sua alta taxa replicativa e por grande diversidade populacional. Substituições de ocorrência natural na protease viral associadas a resistência podem comprometer a terapêutica em alguns regimes baseados no uso de inibidores de protease (IPs). OBJETIVOS: Estimar a prevalência de polimorfismos e mutações de ocorrência natural associadas a resistência aos IPs em pacientes monoinfectados e coinfectados pelo VHC e HIV e identificar fatores clínicos e virológicos associados a presença de tais substituições. MATERIAIS E MÉTODOS: Dados epidemiológicos e clínicos foram obtidos de 247 pacientes (135 monoinfectados e 112 coinfectados pelo VHC e HIV). VHC RNA foi extraído do plasma dos indivíduos participantes e um fragmento de 765 pares de base da região NS3 foi amplificado e sequenciado por metodologia populacional (técnica de Sanger). O estadiamento da fibrose hepática foi realizado pelo escore não invasivo FIB- 4. RESULTADOS: 54 indivíduos (21,9%) apresentaram pelo menos uma substituição na região NS3/NS4A do VHC. Somente 14 pacientes (5,7%) apresentaram pelo menos uma mutação de resistência aos IPs (T54S, V55A ou Q80R). A Q80K não foi identificada em nenhuma das amostras. Não houve diferença entre monoinfectados e coinfectados quanto à ocorrência de polimorfismos ou mutações associadas a resistência. As variáveis independentemente associadas com substituições na região da protease foram infecção pelo VHC genótipo 1b, bilirrubinas totais > 1,5 vezes o LSN e níveis de albumina < 3,5 g/dL. Fibrose hepática avançada (FIB-4 > 3.25) não esteve associada a presença de substituições. A análise de diversidade nucleotídica na protease viral revelou maior heterogeneidade do VHC genótipo 1b em relação ao 1a. Contudo, a análise de pressão seletiva não demonstrou maior variabilidade de quasiespécies no grupo de hepatopatia avançada, achado este compatível com uma sequência genômica relativamente conservada. CONCLUSÕES: As substituições na região NS3/NS4 do VHC consistiram majoritariamente por polimorfismos naturais sem impacto clínico num eventual tratamento que envolva o uso de IPs. A prevalência de substituições associadas a resistência foi baixa e compatível com os valores informados pela maioria dos estudos nacionais e internacionais. A coinfecção pelo HIV não parece elevar a frequência de substituições na protease do VHC. A região NS3 do genótipo 1b foi altamente variável em relação ao genótipo 1a, reforçando o conceito de possíveis diferenças geográficas em relação ao perfil genético deste vírus / INTRODUCTION: Chronic hepatitis C is a major cause of liver disease worldwide. Hepatitis C vírus (HCV) and HIV coinfection is not uncommon due to similar transmission routes. Recently developed direct-acting antivirals drugs (DAAs) have increased the rate of SVR even in coinfected patients. HCV has a high replication rate and a lack of proofreading activity, leading to a greatly diverse viral population. Baseline spontaneously occurring resistance substitutions in the protease region may impair the rate of success in some protease inhibitors (PI) based regimens. OBJECTIVE: to determine the prevalence of naturally occurring polymorphisms and resistance associated variants to HCV PIs in mono and coinfected HCV HIV patients and to evaluate potential associations between amino acid substitutions in protease domain and clinical / virological features of those patients. METHODS: Clinical and epidemiological data were retrieved from medical records of 247 subjects in Brazil (135 HCV monoinfected and 112 HIV HCV coinfected patients). HCV-RNA was extracted from plasma and a fragment of 765 base pairs from the NS3 region was amplified and sequenced with Sanger-based technology. Fibrosis staging was assessed by non invasive score (FIB-4). RESULTS: Overall, 54 patients (21.9%) had at least one amino acid substitution in the NS3 region; only 14 patients (5.7%) harboured at least one resistance mutation (T54S, V55A, Q80R). Q80K mutation was not found in any sample. There was no difference between monoinfected and coinfected patients regarding the frequency of natural polymorphisms and resistance mutations. Variables independently associated with amino acid substitution were HCV subtype 1b, total bilirubin level > 1.5 ULN and albumin level < 3.5 g/dL. Advanced liver fibrosis (FIB-4 > 3.25) was not related to NS3 polymorphisms nor resistance associated variants. Examination of HCV protease nucleotide diversity revealed greater heterogeneity in subtype 1b than subtype 1a. Analysis of selective pressure did not reveal a greater quasispecies variability in advanced liver fibrosis group, being such finding consistent with a relatively conserved gene in this setting. CONCLUSION: Baseline HCV NS3 amino acid substitutions depicted herein were considered mostly natural polymorphisms with no clinical impact in a PI based therapy. The prevalence of resistance-associated substitutions was low and compatible with values reported by most national and international studies. HIV coinfection was not associated with a greater frequency of such substitutions in the studied sample. The NS3 region of genotype 1b was highly variable in relation to genotype 1a, highlighting geographic differences concerning HCV genetic profile

Introduction : Le virus de l’hépatite C (VHC), est l’une des premières causes de pathologie hépatique dans le monde. Ce virus à ARN est responsable de l’hépatite C qui aboutit au développement de la cirrhose et du cancer du foie. Selon l’Organisation Mondiale de la Santé, le VHC infecte actuellement plus de 170 millions de personnes dans le monde, soit 3% de la population. L’hépatite C chronique connait toujours en Tunisie un taux de guérison faible pour le génotype 1 car le traitement standard actuellement disponible est la bithérapie interféron pégylé associé à la ribavirine. A l’heure actuelle, le développement de différentes molécules ciblant spécifiquement le VHC, appelées les antiviraux à action directe (AAD), apparait comme une potentielle révolution dans le traitement de l’infection par le VHC.Ces AAD comprennent les inhibiteurs de protéase (IP), les inhibiteurs nucléos(t)idiques (IN) et les inhibiteurs non-nucléosidiques (INN) de la polymérase NS5B ainsi que les inhibiteurs de la protéine NS5A. La quasi-espèce virale est formée d’un mélange complexe de variants viraux parmi lesquels se trouvent des variants associés à des degrés variables à la résistance aux AAD. Ces variants peuvent donc exister naturellement en absence de toute pression médicamenteuse et sont susceptibles d’avoir un impact sur la réponse aux différents traitements par AAD. Notre objectif était de déterminer la prévalence des variants associés à la résistance dans les souches tunisiennes circulantes en préambule à l’introduction deces molécules en Tunisie. Méthodes : L’amplification et le séquençage direct de la protéase NS3, de la polymérase NS5B ainsi que la région NS5A ont été effectuées chez 149 patients tunisiens naïfs de traitement et infectés par le VHC de génotype 1 (génotype 1b = 142 ; génotype 1a = 7). Résultats : Douze séquences NS3 (12/131 ; 9,2%) ont montré des mutations connes pour conférer une résistance aux IP. Une seule séquence (1/95 ; 1,1%) a montré la mutation V321I connue pour conférer une résistance aux IN-NS5B. Trente quatre séquences (34/95 ; 35,8%) ont montré des mutations connues pour diminuer la sensibilité des INN-NS5B. Une seule séquence de génotype 1a (1/7 ; 14,3%) et 17 séquences de génotype 1b (17/112 ; 16,2%) ont montré des mutations connues pour conférer une résistance au inhibiteurs de la protéine NS5A. Conclusions : Notre étude a permis de mettre en évidence la présence de substitutions conférant une diminution de la sensibilité aux AAD chez des patients tunisiens naïfs de tout traitement anti-VHC. Des études in situ seront nécessaires pour évaluer l’impact de ces mutations sur la réponse au traitement. / Introduction: Hepatitis C virus (HCV) is a major cause of liver disease worldwide. This RNA virus is responsible for hepatitis C, which leads to the development of cirrhosis and liver cancer. According to the World Health Organization, HCV infects more than 170 million people worldwide, about 3% of the population. Chronic hepatitis C still know in Tunisia low cure rates for genotype 1, because the currently standard treatment available is combination therapy of pegylated interferon plus ribavirin. At present, the development of different molecules that specifically target HCV, called direct-acting antivirals (DAA) appears as a potential revolution in the treatment of HCV infection. These DAA include protease inhibitors (PI), nucleos(t)ide (NI) and non-nucleoside inhibitors (NNI) for NS5B polymerase and NS5A inhibitors. The viral quasispecies is formed by a complex mixture of viral variants including variants associated with variable degrees of resistance to DAA. These variants may therefore exist naturally in absence of drug pressure and may affect response to different treatments by DAA. Our objective was to determine the prevalence of variants associated with resistance in circulating Tunisian strains preamble to the introduction of these molecules in Tunisia. Methods: Amplification and direct sequencing of NS3 protease, NS5B polymerase and NS5A region were performed in 149 Tunisian naïve patients infected with HCV genotype 1 (genotype 1b = 142; genotype 1a = 7) . Results: Twelve sequences NS3 (12/131; 9.2%) showed mutations known to confer resistance to PI. One sequence (1/95; 1.1%) showed the V321I mutation known to confer resistance to NS5B-IN. Thirty four sequences (34/95; 35.8%) showed mutations known to reduce the sensitivity of NS5B-INN. One genotype 1a sequence (1/7; 14.3%) and 17 genotype 1b sequences (17/112; 16.2%) showed mutations known to confer resistance to NS5A inhibitors.Conclusions: Our study highlighted the presence of substitutions conferring decreased susceptibility to DAA in naïve patients infected with HCV genotype 1. Field studies will be needed to evaluate the impact of these mutations on the treatment response.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Beáta Repeľová Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Study of effects of antiretroviral drugs on transmembrane transport of tenofovir disoproxil fumarate across MDCKII - ABCB1 cell monolayer Tenofovir disoproxil fumarate (TDF) - ester prodrug of tenofovir is considered as one of the most frequently used component of combination antiretroviral therapy. Several ways of application and good patients' tolerability is typical for this compound. TDF is a substrate of dug transporter such as P-glycoprotein (P-gp) therefore its efflux activity may limit the bioavailability after oral administration and distribution of TDF. As many of antiretroviral drugs are also substrates or inhibitors of P-gp, drug - drug interactions with TDF at the level of transmembrane transport could be expected. The aim of the diploma thesis was to describe effects of co-administered antiretroviral drugs on transfer of TDF across MDCKII cell monolayer by using bidirectional transport and concentration equilibrium setups. The results of experiments confirmed that TDF is a substrate of P-gp. High values of efflux ratio describing transmembrane transport of TDF across parental cells have been observed. This...

Referat: Background: Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease of humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. However, the available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to the probable similarities in cell metabolism among tumor and trypanosoma cells, some of the current registered drugs against HAT were derived from cancer chemotherapeutic research. Here too, for the first time, we have demonstrated that the simple ester, ethyl pyruvate, comprises such properties. On the other hand initial studies have confirmed the efficacy of protease inhibitors in treatment of Trypanosoma cruzi, Plasmodium falciparum and Leishmania major. However, studies on efficacy and specific proteases inhibition using HIV-1 protease inhibitors on T. brucei cells remain untouched. Methodology/Principal findings: The current study covers efficacy and corresponding target evaluation of ethyl pyruvate and HIV-1 protease inhibitors (ritonavir and saquinavir) on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, zymography, phase contrast microscopic video imaging and ex vivo drug toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki=3.0±0.29 mM). The potential of this compound as an anti-trypanosomal drug is also strengthened by its fast acting property, killing cells within three hours post exposure. This was demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, this drug produced minimal side effects in human erythrocytes and is known to easily cross the blood-brain-barrier (BBB) which makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug resistance tests indicate irreversible killing of cells and a low chance of drug resistance development under applied experimental conditions. In addition to ethyl pyruvate our experimental study on HIV-1 protease inhibitors showed that both ritonavir (RTV) (IC50=12.23 µM) and saquinavir (SQV) (IC50=11.49 µM) effectively inhibited T. brucei cells proliferation. The major proteases identified in these cells were the cysteine- (~29kDa Mr) and metallo- (~66kDa Mr) proteases. Their proteolytic activity was, however, not hampered by either of these two protease inhibitors. Conclusion/Significance: Our results present ethyl pyruvate as a safe and fast acting drug. Hence, because of its predefined property to easily cross the BBB, it can probably be a new candidate agent to treat the heamolymphatic as well as neurological stages of sleeping sickness. Similarly, HIV-1 protease inhibitors, SQV and RTV, exhibited their antitrypanosomal potential but require further anlysis to identify their specific targets.:Bibliographic description ii Acronyms iii 1. Introduction 1 1.1. Disease background 1 1.2. Epidemiological distribution and disease transmission dynamics 1 1.3. Biology and life cycle of the trypanosomatidea 3 1.4. Public health significance 4 1.5. Clinical stages and disease progression 5 1.6. Current challenges of disease control 6 1.7. Current drugs and their clinical applications 9 1.8. Targets for drug discovery 12 1.8.1. Energy metabolism 12 1.8.2. Proteolysis 17 1.9. Ethyl pyruvate 18 1.10. HIV-1 Protease Inhibitors 21 2. Aim of the study 22 3. Materials and Methods 24 4. Results 31 5. Discussion 45 6. Conclusion 53 7. Supporting information 54 8. Summary 56 9. References 62 Erklärung über die eigenständige Abfassung der Arbeit 77 Curriculum vitae 78 Publications and Presentations 81 Acknowledgement 83

Antiviral drug resistance is a major problem in the treatment of viral infections, including influenza and hepatitis C virus (HCV). Influenza neuraminidase (NA) is a viral sialidase on the surface of the influenza virion and a primary antiviral target in influenza. Two subtypes of NA predominate in humans, N1 and N2, but different patterns of drug resistance have emerged in each subtype. To provide a framework for understanding the structural basis of subtype specific drug resistance mutations in NA, we used molecular dynamics simulations to define dynamic substrate envelopes for NA to determine how different patterns of drug resistance have emerged in N1 and N2 NA. Furthermore, we used the substrate envelope to analyze HCV NS3/4A protease inhibitors in clinical development. In addition, influenza hemagglutinin (HA) is a primary target of neutralizing antibodies against influenza. Novel broadly neutralizing antibodies (BnAbs) against the stem region of HA have been described and inhibit several influenza viral subtypes, but antibody neutralization escape mutations have emerged. We identified potential escape mutations in broadly neutralizing antibody F10 that may impact protein dynamics in HA that are critical for function. We also solved crystal structures of antibody fragments that are important for understanding the structural basis of antibody binding for influenza BnAbs. These studies can inform the design of improved therapeutic strategies against viruses by incorporating an understanding of structural elements that are critical for function, such as substrate processing and protein dynamics, into the development of novel therapeutics that are robust against resistance.

HCV afflicts many millions of people globally, and antiviral therapies are often ineffective and intolerable. The Food and Drug Administration approved the HCV protease inhibitors telaprevir and boceprevir in May 2011, marking an important milestone in anti-HCV research over the past two decades. Nevertheless, severe drug side effects of combination therapy – flu-like symptoms, depression and anemia – limit patient adherence to treatment regimens. The acquisition of resistance challenges the long-term efficacy of antiviral therapies, including protease inhibitors, as suboptimal dosing allows for the selection of drug resistant viral variants. A better understanding of the molecular basis of drug resistance is therefore central to developing future generation protease inhibitors that retain potency against a broader spectrum of HCV strains. To this end, my research characterizes the molecular basis of drug resistance against HCV protease inhibitors. Chapter II defines the mode of substrate recognition by the common volume shared by NS3/4A substrate products – the substrate envelope. Chapter III then correlates patterns of drug resistance to regions where drugs protrude from the substrate envelope. Lastly, Chapter IV elucidates the molecular underpinnings of resistance against four leading protease inhibitors – telaprevir, danoprevir, vaniprevir and MK-5172 – and provides practical approaches to designing novel drugs that are less susceptible to resistance. I ultimately hope my work appeals to the broader biomedical community of virologists, medicinal chemists and clinicians, who struggle to understand HCV and other human pathogens in the face of rapid disease evolution.