Abstract
Drug resistance is a principal concern in the treatment of quickly evolving diseases. The viral protease NS3/4A is a primary drug target for the hepatitis C virus (HCV) and is known to evolve resistance mutations in response to drug therapy. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition by NS3/4A; the drug resistant protease variants are no longer effectively inhibited by the competitive active site inhibitors but can still process the natural substrates with enough efficiency for viral survival. In previous works we have developed the "substrate envelope" hypothesis, which posits that inhibitors should be less susceptible to drug resistance if they better mimic the natural substrate molecular recognition features. In this work, we perform molecular dynamics simulations on four native substrates bound to NS3/4A and discover a clearly conserved dynamic substrate envelope. We show that the most severe drug resistance mutations in NS3/4A occur at residues that are outside the substrate envelope. Comparative analysis of three NS3/4A inhibitors reveals structural and dynamic characteristics of inhibitors that could lead to resistance. We also suggest inhibitor modifications to improve resistance profiles based on the dynamic substrate envelope. This study provides a general framework for guiding the development of novel inhibitors that will be more robust against resistance by mimicking the static and dynamic binding characteristics of natural substrates.