posted on 2024-03-13, 03:29authored byJulia
M. Flynn, Sarah N. Zvornicanin, Tenzin Tsepal, Ala M. Shaqra, Nese Kurt Yilmaz, Weiping Jia, Stephanie Moquin, Dustin Dovala, Celia A. Schiffer, Daniel N. A. Bolon
The appearance and spread of mutations that cause drug
resistance
in rapidly evolving diseases, including infections by the SARS-CoV-2
virus, are major concerns for human health. Many drugs target enzymes,
and resistance-conferring mutations impact inhibitor binding or enzyme
activity. Nirmatrelvir, the most widely used inhibitor currently used
to treat SARS-CoV-2 infections, targets the main protease (Mpro) preventing it from processing the viral polyprotein into active
subunits. Our previous work systematically analyzed resistance mutations
in Mpro that reduce binding to inhibitors; here, we investigate
mutations that affect enzyme function. Hyperactive mutations that
increase Mpro activity can contribute to drug resistance
but have not been thoroughly studied. To explore how hyperactive mutations
contribute to resistance, we comprehensively assessed how all possible
individual mutations in Mpro affect enzyme function using
a mutational scanning approach with a fluorescence resonance energy
transfer (FRET)-based yeast readout. We identified hundreds of mutations
that significantly increased the Mpro activity. Hyperactive
mutations occurred both proximal and distal to the active site, consistent
with protein stability and/or dynamics impacting activity. Hyperactive
mutations were observed 3 times more than mutations which reduced
apparent binding to nirmatrelvir in recent studies of laboratory-grown
viruses selected for drug resistance. Hyperactive mutations were also
about three times more prevalent than nirmatrelvir binding mutations
in sequenced isolates from circulating SARS-CoV-2. Our findings indicate
that hyperactive mutations are likely to contribute to the natural
evolution of drug resistance in Mpro and provide a comprehensive
list for future surveillance efforts.