Avoiding Drug Resistance by Substrate Envelope-Guided Design: Toward Potent and Robust HCV NS3/4A Protease Inhibitors

ABSTRACT Hepatitis C virus (HCV) infects millions of people worldwide, causing chronic liver disease that can lead to cirrhosis, hepatocellular carcinoma, and liver transplant. In the last several years, the advent of direct-acting antivirals, including NS3/4A protease inhibitors (PIs), has remarkably improved treatment outcomes of HCV-infected patients. However, selection of resistance-associated substitutions and polymorphisms among genotypes can lead to drug resistance and in some cases treatment failure. A proactive strategy to combat resistance is to constrain PIs within evolutionarily conserved regions in the protease active site. Designing PIs using the substrate envelope is a rational strategy to decrease the susceptibility to resistance by using the constraints of substrate recognition. We successfully designed two series of HCV NS3/4A PIs to leverage unexploited areas in the substrate envelope to improve potency, specifically against resistance-associated substitutions at D168. Our design strategy achieved better resistance profiles over both the FDA-approved NS3/4A PI grazoprevir and the parent compound against the clinically relevant D168A substitution. Crystallographic structural analysis and inhibition assays confirmed that optimally filling the substrate envelope is critical to improve inhibitor potency while avoiding resistance. Specifically, inhibitors that enhanced hydrophobic packing in the S4 pocket and avoided an energetically frustrated pocket performed the best. Thus, the HCV substrate envelope proved to be a powerful tool to design robust PIs, offering a strategy that can be translated to other targets for rational design of inhibitors with improved potency and resistance profiles. IMPORTANCE Despite significant progress, hepatitis C virus (HCV) continues to be a major health problem with millions of people infected worldwide and thousands dying annually due to resulting complications. Recent antiviral combinations can achieve >95% cure, but late diagnosis, low access to treatment, and treatment failure due to drug resistance continue to be roadblocks against eradication of the virus. We report the rational design of two series of HCV NS3/4A protease inhibitors with improved resistance profiles by exploiting evolutionarily constrained regions of the active site using the substrate envelope model. Optimally filling the S4 pocket is critical to avoid resistance and improve potency. Our results provide drug design strategies to avoid resistance that are applicable to other quickly evolving viral drug targets.

Combatting Drug Resistance: Lessons from the viral proteases of HIV and HCV

Drug resistance negatively impacts the lives of millions of patients and costs our society billions of dollars by limiting the longevity of many of our most potent drugs. Drug resistance can be caused by a change in the balance of molecular recognition events that selectively weakens inhibitor binding but maintains the biological function of the target. To reduce the likelihood of drug resistance, a detailed understanding of the target's function is necessary. Both structure at atomic resolution and evolutionarily constraints on its variation is required. "Resilient" targets are less susceptible to drug resistance due to their key location in a particular pathway. This rationale was derived through crystallographic studies elucidating substrate recognition and drug resistance in HIV-1 protease and Hepatitis C (HCV) NS3/4A protease. Both are key therapeutic targets and are potentially "resilient" targets where resistant mutations occur outside of the substrate binding site. To reduce the probability of drug resistance inhibitors should be designed to fit within what we define as the "substrate envelope". These principals are likely more generally applicable to other quickly evolving diseases where drug resistance is quickly evolving. http://www.umassmed.edu/schifferlab/index.aspx