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

Lan, S.; Neilsen, G.; Slack, R. L.; Cantara, W. A.; Emanuelli Castaner, A.; Lorson, Z. C.; Lulkin, N. D.; Zhang, H.; Lee, J.; Cilento, M. E.; Tedbury, P. R.; Sarafianos, S. G.

doi:10.1101/2022.12.31.522389

bioRxiv · 10.1101/2022.12.31.522389

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

Lan, S.·Neilsen, G.·Slack, R. L.·Cantara, W. A.·Emanuelli Castaner, A.·Lorson, Z. C.·Lulkin, N. D.·Zhang, H.·Lee, J.·Cilento, M. E.·Tedbury, P. R.·Sarafianos, S. G.

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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 {beta}-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.

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Lan, S., Neilsen, G., Slack, R. L., Cantara, W. A., Emanuelli Castaner, A., Lorson, Z. C., Lulkin, N. D., Zhang, H., Lee, J., Cilento, M. E., Tedbury, P. R., Sarafianos, S. G.. 2023-01-03. Nirmatrelvir Resistance in SARS-CoV-2 Omicron_BA.1 and WA1 Replicons and Escape Strategies. https://doi.org/10.1101/2022.12.31.522389

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