TG-5

The SARS-CoV-2 M^pro protease from COVID-19 cleaves the pp1a and pp2b polyproteins at 11 sites during viral maturation and is the target of Nirmatrelvir, one of the two components of the frontline treatment sold as Paxlovid. We used the YESS 2.0 platform, combining protease and substrate expression in the yeast endoplasmic reticulum with fluorescence-activated cell sorting and next-generation sequencing, to carry out the high-resolution substrate specificity profiling of SARS-CoV-2 M^pro as well as the related SARS-CoV M^pro from SARS 2003. Even at such a high level of resolution, the substrate specificity profiles of both enzymes are essentially identical. The population of cleaved substrates isolated in our sorts is so deep, the relative catalytic efficiencies of the different cleavage sites on the SARS-CoV-2 polyproteins pp1a and pp2b are qualitatively predicted. These results not only demonstrated the precise and reproducible nature of the YESS 2.0/NGS approach to protease substrate specificity profiling but also should be useful in the design of next generation SARS-CoV-2 M^pro inhibitors, and by analogy, SARS-CoV M^pro inhibitors as well.

As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor. While most covalent inhibitors of SARS-CoV-2 3CLPro reported to date contain an amide as a Gln mimic at P1, MK-7845 bears a difluorobutyl substituent at this position. SAR analysis and X-ray crystallographic studies indicate that this group interacts with His163, the same residue that forms a hydrogen bond with the amide substituents typically found at P1. In addition to promising in vivo efficacy and an acceptable projected human dose with unboosted pharmacokinetics, MK-7845 exhibits favorable properties for both solubility and absorption that may be attributable to the unusual difluorobutyl substituent.