Improving the Resistance Profile of Hepatitis C NS3/4A Inhibitors: Dynamic Substrate Envelope Guided Design

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.

Hepatitis C Virus NS3/4A Protease Inhibitors Incorporating Flexible P2 Quinoxalines Target Drug Resistant Viral Variants

“…39,40 Compound 2 is an attractive scaffold for exploring this strategy due to the unique structural features: (1) the P2 quinoxaline moiety that predominantly interacts with the highly conserved catalytic residues Asp81 and His57 and (2) the conformational flexibility that allows the inhibitor to efficiently accommodate structural changes in the S2 subsite due to resistance mutations. Despite these promising features, optimization of substituents at the P2 quinoxaline and the N-terminal capping may be key to discovering analogues with improved potency and resistance profiles.…”

Elucidating the Interdependence of Drug Resistance from Combinations of Mutations

“…The rigorous pre-equilibration model was employed as described elsewhere 47 . Briefly, a series of restrained minimization steps was performed to gradually relax the system.…”

Improving Viral Protease Inhibitors to Counter Drug Resistance

“…For example, the P1’ moiety in DRV can be extended without violating the substrate envelope, a strategy that resulted in inhibitors even more potent and more robust than DRV [51–53]. Similarly, a comparison of HCV NS3/4A inhibitors with the substrates reveals a large portion of the substrate envelope in the P4-P5 region not utilized by any current inhibitor [54] (Figure 3). Extending inhibitors to this untapped region of the protease active site can enhance inhibitor potency.…”