Design and Evaluation of Next-Gen Inhibitors Targeting SARS-CoV-2 3CL Protease Clinical Variants

Abstract The 3C-like protease (3CLpro), also known as main protease (Mpro), is currently the target of the commercially available drugs nirmatrelvir and simnotrelvir which are used to treat SARS-CoV-2 infections. Global sequencing efforts of clinical isolates of SARS-CoV-2 from infected patients treated with nirmatrelvir have identified several 3CLpro clinical variants that are potentially drug resistant. Two variants, E166 and S144, have been widely studied and confirmed to have drug resistance in vitro. Using the Coronavirus 3D database, we identified and characterized ten additional SARS-CoV-2 3CLpro clinical variants deemed likely to render drug resistance. Of these, we identified a new variant, V186G, as being highly drug resistant, i.e., 116-times, 44-times, and 25-times more resistant to nirmatrelvir, simnotrelvir, and the inhibitor GRL-051-22, respectively, than the wild type. The V186G variant also displayed a 23-fold decrease in dimerization affinity. Surprisingly, no changes in drug resistance or dimerization affinity were observed for the V186F or V186I variants. X-ray structures of WT, V186G, V186F, M165I, M49I, and T190I in complex with the inhibitor GRL-051-22 were determined at resolutions between 1.3 Å and 1.9 Å revealing only small structural changes associated with any of the variants, e.g., <1.5 Å in the S2 loop of the V186G variant. Structure-based design was then used to overcome SARS-CoV-2 V186G 3CLpro drug resistance, producing the compound GRL-157-23 which is 6.5-times more potent against the V186G variant compared with the wild type. Structure–activity data for six designed compounds and their interactions with the ten variants revealed an important crosstalk between the SARS-CoV-2 3CLpro S2 and S4 subsites that plays a key role in the kinetic activity and inhibition of these variants. These results demonstrate that it is possible to design and optimize drug scaffolds that target 3CLpro variants to overcome potential drug resistance.