High throughput bioluminescent assay to characterize and monitor the activity of SARS-CoV-2 methyltransferases.

ABSTRACT

The fast rate of viral mutations of SARS CoV-2 result in decrease in the efficacy of the vaccines that have been developed before the emergence of these mutations. Thus, it is believed that using additional measures to combat the virus is not only advisable but also beneficial. Two antiviral drugs were authorized for emergency use by the FDA, namely Pfizer's two-drug regimen sold under the brand name Paxlovid, and Merck's drug Lagevrio. Pfizer's two-drug combination consists of nirmatrelvir, a protease inhibitor that blocks coronavirus ability to multiply and another antiviral, ritonavir, that lowers the rate of drug clearance to boost the longevity and activity of the protease inhibitor. Merck's drug Lagevrio (molnupiravir) is a nucleoside analogue with a mechanism of action that aims to introduce errors into the genetic code of the virus. We believe the armament against the virus can be augmented by the addition of another class of enzyme inhibitors that are required for viral survival and its ability to replicate. Enzymes like nsp14 and nsp10/16 methyltransferases (MTases) represent another class of drug targets since they are required for viral RNA translation and evading the host immune system. In this communication, we have successfully verified that the MTase-Glo, which is universal and homogeneous MTase assay can be used to screen for inhibitors of the two pivotal enzymes nsp14 and nsp16 of SARS CoV-2. Furthermore, we have carried out extensive studies on those enzymes using different RNA substrates and tested their activity using various inhibitors and verified the utility of this assay for use in drug screening programs. We anticipate our work will be pursued further to screen for large libraries to discover new and selective inhibitors for the viral enzymes particularly that these enzymes are structurally different from their mammalian counterparts.