Third generation Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs), glecaprevir and voxilaprevir, are highly effective across genotypes and against many resistant variants. Unlike earlier PIs, these compounds have fluorine substitutions on the P2-P4 macrocycle and P1 moieties. Fluorination has long been used in medicinal chemistry as a strategy to improve physicochemical properties and potency. However, the molecular basis by which fluorination improves potency and resistance profile of HCV NS3/4A PIs is not well understood. To systematically analyze the contribution of fluorine substitutions to inhibitor potency and resistance profile, we used a multi-disciplinary approach involving inhibitor design and synthesis, enzyme inhibition assays, co-crystallography, and structural analysis. A panel of inhibitors in matched pairs were designed with and without P4 cap fluorination, tested against WT protease and the D168A resistant variant, and a total of 22 high-resolution co-crystal structures were determined. While fluorination did not significantly improve potency against the WT protease, PIs with fluorinated P4 caps retained much better potency against the D168A protease variant. Detailed analysis of the co-crystal structures revealed that PIs with fluorinated P4 caps can sample alternate binding conformations that enable adapting to structural changes induced by the D168A substitution. Our results elucidate molecular mechanisms of fluorine-specific inhibitor interactions that can be leveraged in avoiding drug resistance.
Hepatitis C virus (HCV) infects about 71 million people worldwide and is responsible for 400,000 deaths per year. HCV infection eventually leads to chronic liver disease and is the leading cause of hepatocellular carcinoma. Current treatment involves a combination of direct-acting antivirals (DAAs) targeting viral proteins NS5A, NS5B and NS3/4A protease. The earlier generation HCV NS3/4A protease inhibitors (PIs) were readily susceptible to drug resistance and effective against only certain genotypes. These PIs were linear covalent peptidomimetics (telaprevir, boceprevir) or noncovalent P1-P3 macrocycles (simeprevir, paritaprevir). Currently, only three (grazoprevir, glecaprevir, and voxilaprevir) of the seven FDA-approved PIs are used in clinic. All three of these PIs are P2-P4 macrocycles sharing a very similar chemical scaffold, with fluorine atoms incorporated at the P2+ and P1 moieties of glecaprevir (GLE) and voxilaprevir (VOX). These first pan-genotypic inhibitors are able to target the challenging genotype 3 (GT-3) NS3/4A protease, a milestone in HCV treatment.
Figure 1.Inhibitor design strategy (a) Grazoprevir, (b) P1-P3 macrocyclic inhibitor scaffold, and (c) non-fluorinated and fluorinated P4 capping groups used in this study. The moieties of grazoprevir and the inhibitor scaffold with macrocyclization between P2 and P4 (mcP2P4), and P1 and P3 (mcP1P3) side chains, respectively, are labeled.
Figure 2.Structural analysis of Glecaprevir (a) Overall binding mode of Glecaprevir to WT (PDB ID: 6P6L), and (b) fluorine specific interactions. Residues of the S4 pocket and around the fluorine atoms are labeled, gray dotted lines represent an orthogonal multipolar interaction (with distance and angle provided). The orange dotted lines (2.2 and 2.6 Å) represent fluorine induced hydrogen bonds of the P1 moiety. The electrostatic potential (blue for positive and red for negative) are highlighted.
Although GZR, GLE and VOX are pan-genotypic inhibitors, they are still susceptible to common resistance-associated substitutions (RASs), including A156T and D168A in GT1 protease. The P2-P4 macrocycles of GZR, GLE and VOX protrude beyond the HCV substrate envelope and clash with the larger Thr side chain in the A156T resistant variant. Loss of inhibitor potency is also caused by the D168Q polymorphism in GT-3 protease. GLE is robust against the D168Q polymorphism, but similar to GZR is highly susceptible to the D168A RAS which decreases the potency of all PIs by increasing protein dynamics and weakening inhibitor interactions in the S4 pocket. The fluorine atoms in GLE and VOX are at least partially responsible for the improved potency across genotypes compared with GZR.
Incorporation of fluorine atoms in the scaffold of lead compounds can serve several purposes, such as improving potency, metabolic stability, physiochemical properties, and conformational selectivity. Furthermore, fluorination in drug discovery has been used as an approach to combat drug resistance in a variety of targets. The benefits of fluorination are associated with the strong electronegativity and relatively small atomic radius of fluorine. Hydrogen to fluorine substitution yields a carbon-fluorine bond that is highly polarized causing the fluorine atom to carry a partial negative charge. Recent studies have explored fluorination in HCV NS3/4A PIs GZR, asunaprevir, and simeprevir at the P1, P2+ and P4 moieties. Overall, these studies show that fluorine incorporation improved potency and antiviral activity against resistant variants and across genotypes.
The HCV NS3/4A protease substrate envelope, which is defined as the consensus volume occupied by natural substrates, serves as a tool to understand the molecular basis of drug resistance and potency. Relocating the macrocycle from P2-P4 to P1-P3 and staying within the substrate envelope we demonstrated that we could design inhibitors that avoid susceptibility to RASs at A156. Further modifications to the P1-P3 macrocyclic scaffold also ameliorated susceptibility to the D168A RAS by staying within the constraints of the protease substrate envelope and achieving shape complementarity with the contours of the S4 pocket.
Moreover, we recently published an extensive structure-activity relationship study (SAR) focusing on two series of compounds with different P2+ quinoxalines moiety in combination with diverse non-fluorinated and fluorinated P4 capping groups of varying size and shape. Our SAR results indicated that P4 capping groups that optimally fill the S4 pocket led to PIs with both excellent potencies and resistance profiles. Furthermore, incorporating fluorine motifs at the P4 capping groups was successful at improving potency against common resistant variants (D168A and A156T) and GT-3. Our strategy of using fluorinated P4 caps to target the variable S4 pocket proved effective, and emphasized the need for structural data to understand the contribution of fluorination to potency, resistance profile, and inhibitor binding mode.
In the current study, we investigate the impact of fluorination on molecular interactions underlying potency and resistance profile using a panel of NS3/4A PIs with and without fluorine substitutions through structural analysis of co-crystal structures. Seven sets of fluorinated and non-fluorinated analogues that differ by 1-3 hydrogen-to-fluorine substitution at the P4 capping groups were compared. The PIs share an identical P1-P3 macrocyclic scaffold containing a flexible quinoxaline moiety at the P2+ position and diverse acyclic and cyclic P4 capping groups. While all PIs lost potency due to the D168A RAS, PIs with fluorinated P4 cap groups retained better potency compared to the non-fluorinated analogues. A total of 22 high-resolution co-crystal structures, 10 with the WT NS3/4A protease and 12 with the D168A variant, were determined and compared with our previously reported co-crystal structures of nonfluorinated analogues. Detailed structural analysis revealed that increased van der Waals (vdW) contacts, as well as electrostatic and fluorine-induced intramolecular interactions contribute to the improved potency of fluorinated PIs. When in complex with the D168A protease variant, PIs with fluorinated P4 caps sampled alternate binding conformations that enabled PIs to adapt to structural changes in the S4 pocket. The results provide insights into the molecular mechanism by which inhibitor fluorination can lead to improved robustness against drug resistance.
Figure 3.Inhibitor potencies and fold change analyses Inhibition constants of PIs with non-fluorinated and fluorinated P4 caps, GRZ and GLE against (a) WT1a and (b) D168A variant. (c) Fold change in potency against the D168A resistant variant as a result of fluorination.
Seven sets of inhibitors were analyzed to determine the impact of fluorination on potency and efficacy against drug resistant variant D168A of HCV NS3/4A. The selected PIs contained non-fluorinated acyclic P4 capping groups tert-butyl (1), isopropyl (2), and cyclopropylethyl (3); and cyclic P4 capping groups 1-methylcyclocbutyl (4), cyclobutyl (5), 1-methylcyclopentyl (6), and cyclopentyl (7). The corresponding PIs with fluorinated acyclic P4 groups contain methyl to trifluoromethyl substitution, trifluoro t
