Hepatitis C Virus: Structural Insights into Protease Inhibitor Efficacy and Drug Resistance: A Dissertation

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