Structural and Thermodynamic Effects of Macrocyclization in HCV NS3/4A Inhibitor MK-5172

Abstract

Recent advances in direct-acting antivirals against Hepatitis C Virus (HCV) have led to the development of potent inhibitors, including MK-5172, that target the viral NS3/4A protease with relatively low susceptibility to resistance. MK-5172 has a P2–P4 macrocycle and a unique binding mode among current protease inhibitors where the P2 quinoxaline packs against the catalytic residues H57 and D81. However, the effect of macrocyclization on this binding mode is not clear, as is the relation between macrocyclization, thermodynamic stabilization, and susceptibility to the resistance mutation A156T. We have determined high-resolution crystal structures of linear and P1–P3 macrocyclic analogs of MK-5172 bound to WT and A156T protease and compared these structures, their molecular dynamics, and experimental binding thermodynamics to the parent compound. We find that the “unique” binding mode of MK-5172 is conserved even when the P2–P4 macrocycle is removed or replaced with a P1–P3 macrocycle. While beneficial to decreasing the entropic penalty associated with binding, the constraint exerted by the P2–P4 macrocycle prevents efficient rearrangement to accommodate the A156T mutation, a deficit alleviated in the linear and P1–P3 analogs. Design of macrocyclic inhibitors against NS3/4A needs to achieve the best balance between exerting optimal conformational constraint for enhancing potency, fitting within the substrate envelope and allowing adaptability to be robust against resistance mutations.

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

“…Comparison of the WT- 2 and A156T- 2 (PDB code: 5EPY) structures shows subtle changes in inhibitor interactions with the mutant protease. 38 In the A156T- 2 structure the P2 quinoxaline largely maintains interactions with the catalytic residues, but the ethyl group is shifted away from Arg155 side chain toward A156T. Moreover, to accommodate a larger Thr side-chain, the Asp168 side chain adopts another conformation, moving away from Arg155.…”

“…51 Structures were solved by molecular replacement using PHASER. 52 The WT- 2 complex structure (PDB code: 5EPN) 38 was used as the starting structure for all structure solutions. Model building and refinement were performed using Coot 53 and PHENIX, 54 respectively.…”

Molecular and Dynamic Mechanism Underlying Drug Resistance in Genotype 3 Hepatitis C NS3/4A Protease

“…36 We have previously described the molecular basis for resistance to many of these inhibitors in terms of the substrate envelope and macrocyclization. 37–41 Disturbingly, these inhibitors lack cross-genotypic activity, especially second- and third-generation PIs such as simeprevir, danoprevir, GS-9451, and grazoprevir, which are considerably less potent against GT-3 compared to GT-1 with 140- to 1500-fold loss in activity in viral subgenomic replicon models. 15,16 Naturally occurring polymorphisms in GT-3 protease relative to GT-1 include drug resistance sites in GT-1 (residues 36, 123, 132, 168 and 170), several of which are outside the active site.…”

Improving Viral Protease Inhibitors to Counter Drug Resistance

“…The macrocycle is thought to stabilize the inhibitor in a conformation competent for protease binding, thus increasing the entropic contribution to the free energy of binding. However, macrocycle design may need to be optimized between the rigidity required for enhancing entropy versus the flexibility required for adapting to a mutated active site [55, 58]. …”