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. 2020 Mar 31;11(2):e00172-20.
doi: 10.1128/mBio.00172-20.

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

Ashley N Matthew #  1 , Jacqueto Zephyr #  1 , Desaboini Nageswara Rao  1 , Mina Henes  1 , Wasih Kamran  1 , Klajdi Kosovrasti  1 , Adam K Hedger  1 , Gordon J Lockbaum  1 , Jennifer Timm  1 , Akbar Ali  1 , Nese Kurt Yilmaz  2 , Celia A Schiffer  2
Affiliations

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

Ashley N Matthew et al. mBio. .

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.

Keywords: X-ray crystallography; drug design; drug resistance mechanisms; hepatitis C virus; structural biology.

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Figures

FIG 1
FIG 1
Chemical structures of designed HCV NS3/4A protease inhibitors. (a) Grazoprevir (MK-5172) is an FDA-approved PI. Change of the macrocycle location (5172-mcP1P3) and optimization of the P2 quinoxaline moiety led to the parent compound (35) modified in this study. The canonical nomenclature for drug moiety positioning and the P4 moiety altered are indicated. (b) The inhibitors designed based on the parent compound (i) to optimally fill the S4 pocket by modifying the P4 capping group (P4-1 to P4-7) and (ii) to extend into the substrate envelope by incorporating a P4 moiety with a P5 capping group (P4P5-1A to P4P5-6).
FIG 2
FIG 2
Resistance profile of HCV NS3/4A protease inhibitors against GT1a WT (WT1a) and D168A variant. (a) Enzyme inhibition constants of the P4-cap inhibitors against wild-type and D168A proteases, as indicated (a), and fold change of enzyme inhibitory activity against the D168A variant with respect to that of the wild-type NS3/4A protease (b). (c) Enzyme inhibition constant. (d) Fold change of the P4-P5-cap inhibitors. PC, parent compound; GZR, grazoprevir.
FIG 3
FIG 3
Binding of grazoprevir and designed PIs to WT and D168A protease active sites. Crystal structures of grazoprevir (GZR) (a), the parent compound (PC) (b), P4-4 (c), and P4P5-2A (d) bound to the wild-type and D168A proteases, as indicated. The protease active site is in surface representation, with the side chains of catalytic triad and S4 subsite residues shown as sticks. Water molecules are shown as nonbonded spheres (red), and hydrogen bonds (gray dashed lines) that stabilize S4 pocket side chains are displayed. Black dashed lines outline the surface of the S4 pocket where the D168A mutation is located.
FIG 4
FIG 4
Fit of NS3/4A protease inhibitors within the substrate envelope. Inhibitors grazoprevir (a), parent compound (b), P4 series (P4-1, P4-2, P4-3, P4-4, P4-5, P4-6, and P4-7) (c), and P4P5 series (P4P5-2A, P4P5-2B, P4P5-4, P4P5-5, and P4P5-6) (d) are shown as sticks (orange) in the substrate envelope (blue). The side chains of the catalytic triad and residues surrounding the S4 pocket are shown in the substrate-bound conformations as yellow and green sticks, respectively.
FIG 5
FIG 5
Filling the S4 subsite of the HCV NS3/4A protease active site. (a) Crystal structures of P4-1, P4-2, P4-4, and P4-7, as indicated, bound to D168A HCV protease variant. The protease active site is in surface representation, with the residues that make up the S4 pocket (white) and the catalytic triad (yellow) side chains displayed as sticks. (b) Intermolecular van der Waals (vdW) contact energies for inhibitors with residues forming the S4 pocket in the D168A crystal structures. (c) Change in vdW contacts (ΔvdW) relative to those of the parent compound (PC). Negative values indicate enhanced contacts compared to those of the parent compound.
FIG 6
FIG 6
Fit of P4 capping groups and the conformation of R123 reshaping the S4 pocket of HCV NS3/4A protease. P4-4 (a), P4-7 (b), and P4-6 (c) cocrystal structures are shown with the D168A variant. The protease is in surface representation, with residues in and around the S4 pocket (white) and the catalytic triad (yellow) side chains in stick representation. The contour of the S4 pocket is outlined in dotted lines. (d) Superposition of P4-4, P4-7, and P4-6, as indicated, showing the alternate conformations of Arg123 (in respective color of the inhibitors) in the cocrystal structures.
FIG 7
FIG 7
Binding mode of P4P5 inhibitors relative to that of the parent compound. (a) Superposition of cocrystal structures of the parent compound (PC), P4P5-2A, P4P5-4, P4P5-5, and P4P5-6, as indicated. The protease is in surface representation, and side chains of residues in and around the S4 pocket (white) and the catalytic triad (yellow) are displayed as sticks. The R123 can adopt two conformations (R123a and R123b). In all of the structures, R123 is in the commonly observed conformation (white; R123a) except for the P4P5-5 complex (green), where both conformations are observed (green). The contour of the S4 pocket is outlined in black dotted lines. The cyan-to-pink arrow indicates the displacement of tert-butyl group in the parent compound relative to that in P4P5-2A, and the green arrow shows a shift of the P5 extension away from the protease surface in P4P5-5 relative to that of the other compounds. (b) Superposition of P4P5-2A and P4P5-6 bound to D168A protease. The inhibitors are displayed as sticks with a mesh surface representation of the van der Waals surface. (c) Change in vdW contacts (ΔvdW) relative to those of the parent compound (PC) for P4P5-2A, P4P5-4, P4P5-5, and P4P5-6 with S4 residues.
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