Discovery of Quinoxaline-Based P1–P3 Macrocyclic NS3/4A Protease Inhibitors with Potent Activity against Drug-Resistant Hepatitis C Virus Variants

Abstract

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.

Defining the substrate envelope of SARS-CoV-2 main protease to predict and avoid drug resistance

“…Over the years, we and others have shown that regions where inhibitors protrude from the substrate envelope are susceptible to resistance mutations 16 , 26 , 28 , 37 – 40 . Thus, our newly determined substrate envelope of M pro provides a predictive tool to assess likelihood that resistance to a specific inhibitor will emerge.…”

Hepatitis C: The Story of a Long Journey through First, Second, and Third Generation NS3/4A Peptidomimetic Inhibitors. What Did We Learn?

“…162 Next, P1−P3 macrocyclization was explored as P1−P3 cycles better accommodate the A156T mutation and allow flexibility of the P2 moiety. 58 Inhibitors containing the 3-methyl-quinoxaline group at the P2 Pro, in combination with several P4capping groups, were prepared. Almost all the compounds retained low antiviral potency against the A156T variant (nanomolar range).…”

“…The first generation of HCV PIs includes agents with potent activity against gt1; unfortunately, they were not effective against all genotypes. The precursors of first generation PIs were N-terminal peptide products derived from the cleavage of sequences of HCV NS3 protease substrates, corresponding to the P6–P1 residues from the NS4A/4B and NS4B/5A, also-called breakthrough structures. , These inhibitors were transition state mimics and were found to act as competitive inhibitors of the enzyme (feedback inhibition). − Later, less peptidelike product-based ones were designed, by peptide truncation and extension of the side chain of the P2 proline (Pro) site and by P1 optimization.…”

Anno 2021: Which antivirals for the coming decade?

“…The macrocyclic scaffold structure shared by approved HCV NS3/4A protease inhibitors has been modified to modulate both potency and drug-like properties of the inhibitors as well as prevent resistance due to pre-existing resistance-associated substitutions (RASs) and/or baseline polymorphisms among diverse genotypes. 200 , 201 …”