Dissertations / Theses on the topic 'Hepacivirus Hepatocytes Viral Proteins'

Drug resistance is a major problem in quickly evolving diseases, including the human immunodeficiency (HIV) and hepatitis C viral (HCV) infections. The viral proteases (HIV protease and HCV NS3/4A protease) are primary drug targets. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition; the drug resistant protease variants are no longer effectively inhibited by the competitive drug molecules but can process the natural substrates with enough efficiency for viral survival. Therefore, the inhibitors that better mimic the natural substrate binding features should result in more robust inhibitors with flat drug resistance profiles. The native substrates adopt a consensus volume when bound to the enzyme, the substrate envelope. The most severe resistance mutations occur at protease residues that are contacted by the inhibitors outside the substrate envelope. To guide the design of robust inhibitors, we investigate the shared and varied properties of substrates with the protein dynamics taken into account to define the dynamic substrate envelope of both viral proteases. The NS3/4A dynamic substrate envelope is compared with inhibitors to detect the structural and dynamic basis of resistance mutation patterns. Comparative analyses of substrates and inhibitors result in a solid list of structural and dynamic features of substrates that are not shared by inhibitors. This study can help guiding the development of novel inhibitors by paying attention to the subtle differences between the binding properties of substrates versus inhibitors.

Drug resistance is a major problem in quickly evolving diseases, including the human immunodeficiency (HIV) and hepatitis C viral (HCV) infections. The viral proteases (HIV protease and HCV NS3/4A protease) are primary drug targets. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition; the drug resistant protease variants are no longer effectively inhibited by the competitive drug molecules but can process the natural substrates with enough efficiency for viral survival. Therefore, the inhibitors that better mimic the natural substrate binding features should result in more robust inhibitors with flat drug resistance profiles. The native substrates adopt a consensus volume when bound to the enzyme, the substrate envelope. The most severe resistance mutations occur at protease residues that are contacted by the inhibitors outside the substrate envelope. To guide the design of robust inhibitors, we investigate the shared and varied properties of substrates with the protein dynamics taken into account to define the dynamic substrate envelope of both viral proteases. The NS3/4A dynamic substrate envelope is compared with inhibitors to detect the structural and dynamic basis of resistance mutation patterns. Comparative analyses of substrates and inhibitors result in a solid list of structural and dynamic features of substrates that are not shared by inhibitors. This study can help guiding the development of novel inhibitors by paying attention to the subtle differences between the binding properties of substrates versus inhibitors.

The Hepatitis C Virus (HCV) is a global health problem as it afflicts an estimated 170 million people worldwide and is the major cause of viral hepatitis, cirrhosis and liver cancer. HCV is a rapidly evolving virus, with 6 major genotypes and multiple subtypes. Over the past 20 years, HCV therapeutic efforts have focused on identifying the best-in-class direct acting antiviral (DAA) targeting crucial components of the viral lifecycle, The NS3/4A protease is responsible for processing the viral polyprotein, a crucial step in viral maturation, and for cleaving host factors involved in activating immunity. Thus targeting the NS3/4A constitutes a dual strategy of restoring the immune response and halting viral maturation. This high priority target has 4 FDA approved inhibitors as well as several others in clinical development. Unfortunately, the heterogeneity of the virus causes seriously therapeutic challenges, particularly the NS3/4A protease inhibitors (PIs), which suffer from both the rapid emergence of drug resistant mutants as well as a lack of pan-genotypic activity. My thesis research focused on filling two critical gaps in our structural understanding of inhibitor binding modes. The first gap in knowledge is the molecular basis by which macrocyclization of PIs improves antiviral activity. Macrocycles are hydrophobic chains used to link neighboring chemical moieties within an inhibitor and create a structurally pre-organized ligand. In HCV PIs, macrocycle come in two forms: a P1 - P3 and P2 - P4 strategy. I investigated the structural and thermodynamic basis of the role of macrocyclization in reducing resistance susceptibility. For a rigorous comparison, we designed and synthesized both a P1 - P3 and a linear analog of grazoprevir, a P2 - P4 inhibitor. I found that, while the P2 - P4 strategy is more favorable for achieving potency, it does not allow the inhibitor sufficient flexibility to accommodate resistance mutations. On the other hand, the P1 - P3 strategy strikes a better balance between potency and resistance barrier. The second gap my thesis addresses is elucidating the structural basis by which highly potent protease inhibitors function in genotype 1 but not in genotype 3, despite having an 87% sequence similarity. After mapping the amino acids responsible for this differential efficacy in genotypes 1 and 3, I engineered a 1a3a chimeric protease for crystallographic studies. My structural characterization of three PIs in complex with both the 1a3a and genotype 1 protease revealed that the loss of inhibitor efficacy in the 1a3a and GT-3 proteases is a consequence of disrupted electrostatic interactions between amino acids 168 and 155, which is critical for potent binding of quinoline and isoindoline based PIs. Here, I have revealed details of molecular and structural basis for the lack of PI efficacy against GT-3, which are needed for design of pan-genotypic inhibitors.

The Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs) have become a mainstay of newer all-oral combination therapies. Despite improvements in potency of this inhibitor class, drug resistance remains a problem with the rapid emergence of resistance-associated substitutions (RASs). In this thesis I elucidate the molecular mechanisms of drug resistance for PIs against a resistant variant and apply insights toward the design of inhibitors with improved resistance profiles using structural, biochemical and computational techniques. Newer generation PIs retain high potency against most single substitutions in the protease active site by stacking on the catalytic triad. I investigated the molecular mechanisms of resistance against the Y56H/D168A variant. My analysis revealed that the Y56H substitution disrupts these inhibitors’ favorable stacking interactions with the catalytic residue His57. To further address the impact of drug resistance, I designed new inhibitors that minimize contact with known drug resistance residues that are unessential in substrate recognition. The initially designed inhibitors exhibited flatter resistance profiles than the newer generation PIs but lost potency against the D168A variant. Finally, I designed inhibitors to extend into the substrate envelope (SE) and successfully regained potency against RAS variants maintaining a flat profile. These inhibitors both pack well in the enzyme and fit within the SE. Together these studies elucidate the molecular mechanisms of PI resistance and highlight the importance of substrate recognition in inhibitor design. The insights from this thesis provide strategies toward the development of diverse NS3/4A PIs that may one day lead to the eradication of HCV.

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.

Aujourd’hui, le Virus de l'Hépatite C (VHC) infecte plus 70 millions de personnes dans le monde. L’Organisation mondiale de la santé prévoit l’élimination du virus VHC d’ici 2030, grâce aux récentes découvertes dans le milieu du développement médical. Ces derniers ont conduit à la production des antiviraux pangenotypiques à action directe (ADD). Le VHC est un virus enveloppé de l’ARN, avec une polarité positive. Il est constitué de nucléocapside entouré d’une membrane lipidique. La nucléocapside contient l’acide ribonucléique (ARN) et la protéine core. La membrane lipidique quant à elle contient à la surface les glycoprotéines E1 et E2. Ainsi ces protéines, sont les premières à rencontrer les hépatocytes, c’est donc grâce à elles que le virus parvient à entrer dans les cellules. Parmi les deux protéines, l’E2 a été la mieux caractérisée pour ses fonctions de liaisons aux récepteurs spécifiques. De plus les anticorps neutralisants ciblent majoritairement cette protéine. En se basant sur le fait que ce virus est membre de la famille des Flaviviridae, il a été suggéré par analogie, que le VHC contient des protéines de fusion de classe II et que la protéine E2 est la protéine de fusion. Cependant, les structures cristallines récentes d’E2 ont révélé qu'il lui manquait les caractéristiques structurelles des protéines de fusion de classe II. Ainsi, tous les regards se sont tournés sur la glycoprotéine E1, suggérant qu’elle est responsable de l’étape de fusion, seule ou à l’aide d’E2. En effet, la partie N-terminale de l'ectodomaine E1 a été récemment cristallisée. La caractérisation des résidus conservés dans cette région a démontré son importance pour l'infectivité du virus, pour l'interaction entre E1 et E2, ainsi que pour son implication dans l'interaction avec les récepteurs du VHC. En soutenant le rôle potentiel d'E1 dans le processus de fusion, différents segments de l'extrémité C-terminale de l'ectodomaine seraient impliqués dans les interactions avec les membranes modèles. Nous avons étudié en particulier deux régions d’intérêt. La première située dans la zone du peptide de fusion putatif (PFP) entre les acides aminés 270 et 291. Cette région se compose des séquences hydrophobes, soutenant son implication dans l'étape de fusion. La deuxième région englobant les acides aminés 314-342, d’une activité membranotrope située à proximité de la zone transmembranaire d’E1, a été démontrée par la cristallographie aux rayons X et les études de RMN comme comprenant deux hélices α (α2 et α3).Nous avons introduit 22 mutations dans la partie C-terminale de l'ectodomaine E1 dans le contexte d'un clone infectieux JFH1. Nous avons remplacé les résidus les plus conservés par de l'alanine, puis analysé l'effet des mutations sur le cycle de vie du virus. Vingt des vingt-deux mutants ont été atténué ou ont perdu leur pouvoir infectieux, ce qui indique leur importance dans le cycle viral. Nous avons observé différents phénotypes; certaines mutations ont modulé la dépendance du virus vis-à-vis des récepteurs CLDN1 et SRBI pour l’entrée cellulaire. Plusieurs mutations dans la région PFP, ont affecté la sécrétion et l'assemblage du virus, ainsi que l'hétérodimérisation E1E2. D’autres mutations, telles que les mutations de l'hélice α2 ont entraîné une atténuation grave ou une perte complète d'infectivité, sans affecter le repliement d’E1 et E2, ni la morphogenèse virale. Une caractérisation plus poussée de certains mutants au sein de la région hélice α2 a suggéré l'implication de cette région dans une étape tardive de l'entrée du VHC. Enfin, nos résultats montrent le rôle important joué par la glycoprotéine E1 dans l'hétérodimérisation de E1E2, la morphogenèse du virus, ainsi que son interaction avec les récepteurs du VHC et son implication potentielle dans l'étape de fusion
Hepatitis C virus is currently estimated to infect around 71 million people around the world. However, recent advances in drug development led to the generation of pangenotypic direct acting antivirals (DAA), which may make it possible to eliminate HCV by 2030 as planned by the World health organization (WHO). HCV is a small RNA enveloped virus of positive sense. The RNA is encapsidated and surrounded by a lipid bilayer in which the E1 and E2 envelope glycoproteins are anchored on the surface. Thus, E1 and E2 are the first viral proteins to encounter the hepatocytes and mediate the entry step. HCV entry into hepatocytes is a sophisticated process that includes several steps ranging from interaction of glycoproteins with cellular host attachment factors and HCV specific-receptors, which is followed by internalization via clathrin-mediated endocytosis. Finally, viral and endosomal membranes merge at acidic pH leading to the release of viral RNA into the cytoplasm. Among the two glycoproteins, E2 has been the better characterized, as it is responsible for binding to cellular receptors and targeted by neutralizing antibodies. As a member of the Flaviviridae family, it has been suggested by analogy that HCV encodes class II fusion proteins and that E2 is the fusion protein. Nevertheless, the recent crystal structures of E2 revealed that it lacks structural features of class II fusion proteins. Thus, E1 glycoprotein became under the spotlight with the assumption that it is responsible for the fusion step whether alone or with the help of E2. Indeed, the N-terminal part of E1 ectodomain was recently crystallized, and the characterization of conserved residues within this region demonstrated its importance for virus infectivity, E1E2 interaction as well as its involvement in the interplay with HCV receptors. Supporting the potential role of E1 in the fusion process, different segments in the C-terminal of the ectodomain have been reported to be involved in interactions with model membranes. In particular, we investigated two regions of interest. The first one located in the putative fusion peptide (PFP) region between amino acid 270 and 291, containing hydrophobic sequences, supporting its involvement in the fusion step. The second region spanning amino acids 314-342, a membranotropic region located proximal to the transmembrane region of E1 and has been shown by X-ray crystallography and NMR-studies to comprise two α-helices (α2 and α3). We introduced 22 mutations in the C-terminal part of E1 ectodomain in the context of a JFH1 infectious clone. We replaced the most conserved residues with alanine and analyzed the effect of the mutations on the viral life cycle. Twenty out of the 22 mutants were either attenuated or lost their infectivity, indicating their importance for the viral life cycle. We observed different phenotypes; some mutations modulated the dependence of the virus on CLDN1 and SRBI receptors for cellular entry. Most mutations in the PFP region affected virus secretion and assembly as well as E1E2 heterodimerization. Nevertheless, the majority of mutations in the α2-helix (aa 315-324) led to severe attenuation or complete loss of infectivity without affecting E1E2 folding or viral morphogenesis. Further characterization of some mutants within this region suggested the involvement of the α2-helix in a late step of HCV entry. Finally, our results show the important role of E1 played in E1E2 heterodimerization, virus morphogenesis, interaction with HCV receptors and its potential involvement in the fusion step

Two decades after the discovery of the Hepatitis C Virus (HCV), Hepatitis C infection still persists to be a global health problem. With the recent approval of the first set of directly acting antivirals (DAAs), the rate of sustained viral response for HCV-infected patients increased significantly. However, a complete cure has not been found yet. Drug development efforts primarily target NS3/4A protease, bifunctional serine protease-RNA helicase of HCV. HCV NS3/4A is critical in viral function; protease domain processes the viral polyprotein and helicase domain aids replication of HCV genome by unwinding double stranded RNA transcripts produced by NS5B, RNA-dependent RNA polymerase of HCV. Protease and helicase domains can be isolated, expressed and purified separately while retaining function. Isolated domains of HCV NS3/4A have been extensively used in biochemical and biophysical studies for scientific and therapeutic purposes to evaluate functional capability and mechanism. However, these domains are highly interdependent and modulate the activities of each other bidirectionally. Interdomain dependence was demonstrated in comparative studies where activities of isolated domains versus the full length protein were evaluated. Nevertheless, specific factors affecting interdependence have not been thoroughly studied. Chapter II investigates the domain-domain interface formed between protease and helicase domains as a determinant in interdependence. Molecular dynamics simulations performed on single chain NS3/4A constructs demonstrated the importance of interface in the coupled dynamics of the two domains. The role of the interface in interdomain communication was experimentally probed by disrupting the domain-domain interface through Ala-scanning mutations in selected residues in the interface with significant buried surface areas. These interface mutants were assayed for both helicase and protease related activities. Instead of downregulating the activities of either domain, interface mutants caused enhancement of protease and helicase activities. In addition, the interface had minimal effect in RNA unwinding activity of the helicase domain, the mere presence of the protease domain was the main protagonist in elevated RNA unwinding activity. In conclusion, I suspect that the interface formed between the domains is transient in nature and plays a regulatory role more than a functional role. In addition, I found results supporting the suggestion that an alternate domain-domain arrangement other than what is observed in crystal structures is the active, biologically relevant conformation for both the helicase and the protease. Chapter III investigates structural features of HCV NS3/4A protease inhibitors in relation to effects on inhibitor potency, susceptibility to drug resistance and modulation of potency by the helicase domain. Nearly all NS3/4A protease inhibitors share common features, with major differences only in bulky P2 extension groups and macrocyclization statuses. Enzymatic inhibition profiles of different drugs were analyzed for wildtype isolated protease domain and single chain NS3/4A helicase-protease construct, their multi drug resistant variants, and additional helicase mutants. Inhibitor potency was mainly influenced by macrocyclization, where macrocyclic drugs were significantly more potent compared to acyclic variants. Potency loss with respect to resistance mutations primarily depended on the P2 extension, while macrocyclization had minimal effect except for P2-P4 macrocyclic compounds which were up to an order of magnitude more susceptible to mutations A156T and, in lesser extent, D168A. Modulation by helicase domain was also dependent on P2 extension, although opposite trends were observed for danoprevir analogs versus others. In conclusion, this study provides a basis for future inhibitor development in both avoiding drug resistance and exploitation of the helicase domain for additional efficacy. In this thesis, I have provided evidence further supporting and revealing the details of domain-domain dependency in HCV NS3/4A. Lessons learned here will aid future research for dissecting the interdependency to gain a better understanding of HCV NS3/4A function, which can possibly be extended to all Flaviviridae NS3 protease-helicase complexes. In addition, interdomain dependence can be exploited in future drug development efforts to create better drugs that will pave the way to an effective cure.