Studies on antiviral resistant SARS-CoV-2 Mpro mutants

Abstract

SARS-CoV-2 Mpro is a cysteine protease responsible for 11/14 cleavage events that liberate and activate the viral proteins from the SARS-CoV-2 viral polyprotein. Mpro is essential for viral replication and is well conserved throughout coronaviruses. Due to the high degree of conservation of Mpro throughout coronaviruses, Mpro is viewed as a target for broad spectrum antivirals to treat coronavirus infections. Proof of concept antivirals exist such as GC-376 which was developed to treat Feline Infectious Peritonitis but also exhibits activity against SARS-CoV-2 Mpro. In the context of the ongoing SARS-CoV-2 pandemic, Mpro inhibitors such as Nirmatrelvir/Ritonavir (aka Paxlovid) have been developed and have demonstrated success in reducing the death rate from SARS-CoV-2 infections. The expected emergence of resistance mutations to existing antivirals demands the development of novel antiviral compounds. We are performing an assay based on the cleavage of a synthetic peptide bearing a fluorophore on one terminus and a black hole quencher on the opposite terminus. Cleavage of the peptide by Mpro allows the excitation of the fluorophore. This biochemical assay provides quantitative kinetic data. Recent research by Moghadasi et al led to the development of a gain of function live cell based assay based on GFP fluorescence upon the inhibition of Mpro. Together, these two assays can distinguish between effective Mpro inhibitors, ineffective Mpro inhibitors, and toxic Mpro inhibitors. We are currently studying novel resistance mutants to Nirmatrelvir and Ensitrelvir as well as other small molecule inhibitors of Mpro. We are also using crystallographic methods to further characterize the structural basis of resistance to antiviral compounds.