RESUMO
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.
Assuntos
Ácidos Aminoisobutíricos , Ciclopropanos , Desenho de Fármacos , Farmacorresistência Viral , Inibidores da HCV NS3-4A Protease , Lactamas Macrocíclicas , Leucina/análogos & derivados , Prolina/análogos & derivados , Quinoxalinas , Sulfonamidas , Proteases Virais , Ácidos Aminoisobutíricos/química , Ácidos Aminoisobutíricos/farmacologia , Ciclopropanos/química , Ciclopropanos/farmacologia , Farmacorresistência Viral/genética , Flúor/química , Inibidores da HCV NS3-4A Protease/química , Inibidores da HCV NS3-4A Protease/farmacologia , Halogenação , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Hepacivirus/genética , Humanos , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/farmacologia , Leucina/química , Leucina/genética , Leucina/farmacologia , Prolina/química , Prolina/genética , Prolina/farmacologia , Quinoxalinas/química , Quinoxalinas/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Proteases Virais/química , Proteases Virais/genéticaRESUMO
The three pan-genotypic HCV NS3/4A protease inhibitors (PIs) currently in clinical use-grazoprevir, glecaprevir, and voxilaprevir-are quinoxaline-based P2-P4 macrocycles and thus exhibit similar resistance profiles. Using our quinoxaline-based P1-P3 macrocyclic lead compounds as an alternative chemical scaffold, we explored structure-activity relationships (SARs) at the P2 and P4 positions to develop pan-genotypic PIs that avoid drug resistance. A structure-guided strategy was used to design and synthesize two series of compounds with different P2 quinoxalines in combination with diverse P4 groups of varying sizes and shapes, with and without fluorine substitutions. Our SAR data and cocrystal structures revealed the interplay between the P2 and P4 groups, which influenced inhibitor binding and the overall resistance profile. Optimizing inhibitor interactions in the S4 pocket led to PIs with excellent antiviral activity against clinically relevant PI-resistant HCV variants and genotype 3, providing potential pan-genotypic inhibitors with improved resistance profiles.