Nirmatrelvir Resistance in SARS-CoV-2 Omicron_BA.1 and WA1 Replicons and Escape Strategies

Abstract

The antiviral component of Paxlovid, nirmatrelvir (NIR), forms a covalent bond with Cys145 of SARS-CoV-2 nsp5. To explore NIR resistance we designed mutations to impair binding of NIR over substrate. Using 12 Omicron (BA.1) and WA.1 SARS-CoV-2 replicons, cell-based complementation and enzymatic assays, we showed that in both strains, E166V imparted high NIR resistance (~55-fold), with major decrease in WA1 replicon fitness (~20-fold), but not BA.1 (~2-fold). WA1 replicon fitness was restored by L50F. These differences may contribute to a potentially lower barrier to resistance in Omicron than WA1. E166V is rare in untreated patients, albeit more prevalent in paxlovid-treated EPIC-HR clinical trial patients. Importantly, NIR-resistant replicons with E166V or E166V/L50F remained susceptible to a) the flexible GC376, and b) PF-00835231, which forms additional interactions. Molecular dynamics simulations show steric clashes between the rigid and bulky NIR t-butyl and β-branched V166 distancing the NIR warhead from its Cys145 target. In contrast, GC376, through-wiggling and jiggling- accommodates V166 and still covalently binds Cys145. PF-00835231 uses its strategically positioned methoxy-indole to form a beta-sheet and overcome E166V. Drug design based on strategic flexibility and main chain-targeting may help develop second-generation nsp5-targeting antivirals efficient against NIR-resistant viruses.

Rapid resistance profiling of SARS-CoV-2 protease inhibitors

“…Other mutants such as A173V show modest 1.5 to 3-fold decreases in relative activity, and a few such as E166V are severely compromised. L50F partly restores the activity of E166A and E166V mutants consistent with prior reports [2][3][4][5][6][7][8][9] .…”

Discovery of Nirmatrelvir Resistance Mutations in SARS-CoV-2 3CLpro: A Computational-Experimental Approach

“…Mutations L50F and T21I have previously been reported to restore replicative fitness. 14,18 Therefore, the addition of these mutations may help to restore fitness of the Y54A/S144A and S144A/E166A 3CLpro mutants.…”

“…Similar to our discussion on the reduced enzymatic activity of our mutants (see above), the addition of mutations L50F and T21I in 3CLpro has previously shown to restore the replicative fitness of SARS-CoV-2. 14,18 As the potential of drug resistance mutations also depends on the fitness of the virus, additional compensatory mutations that increase virus fitness would likely need to accompany Y54A/ S144A and S144A/E166A mutations in SARS-CoV-2 3CLpro.…”

Rapid resistance profiling of SARS-CoV-2 protease inhibitors

“…However, as for drugs developed to treat other viruses 1 and for first-generation SARS-CoV-2 vaccines, there is a high probability that variants will emerge that resist nirmatrelvir. Indeed, a flurry of recent studies has described a variety of candidate nirmatrelvir-resistance mutations 2–9 . Thus, considerable urgency exists to develop next-generation M pro inhibitors with different resistance mechanisms and, in parallel, robust systems to rapidly assess the potential impact of candidate resistance mutations.…”

“…We recently developed a gain-of-signal system for facile quantification of M pro inhibition 12 , and subsequently used it together with an evolution- and structure-guided approach to characterize candidate nirmatrelvir- and ensitrelvir-resistance mutations 2 . Here, an expanded panel of M pro single and double mutants based on recent studies by our group and others 2–9 is leveraged to determine resistance profiles of these two drugs, as well as FB2001, a potential next-generation therapy (heatmap of results in Figure 1B ; quantification summary in Table 1 ; representative dose responses in Figure S1 ).…”