Uridine cytidine kinases govern molnupiravir bioactivation and anti-SARS-CoV-2 activity

Abstract

Molnupiravir is a nucleoside analogue antiviral drug against RNA viruses, including its clinical indication SARSCoV2. Whilst its mechanismofaction is well defined, host factors that regulate its therapeutic responses have not been thoroughly deci phered and characterized. Here we show that uridine cytidine kinases (UCKs), key enzymes in pyrimidine salvage, effectively phosphorylate and thereby bioactivate N4hydroxycytidine (NHC) – the active compound of molnupiravir, thus dictating its antiSARSCoV2 efficacy and furthermore selectivity. In vitro, both isoforms of UCKs (UCK1 and UCK2) effectively phosphorylated NHC, where the structural basis of the catalysis was further deciphered via the first complete substrate bound cocrystal structure of UCK, i.e., UCK1NHCAMPPNP. In SARSCoV2infected cells, UCK2 knockdown via siRNA hampered the intracellular accumulation of the triphosphorylated antiviral metabolite of NHC, resulting in a 10fold reduction of the antiviral efficacy, and surprisingly, 2fold reduction of its selectivity, which were critically recapitulated in a dosedependent manner using a panUCK inhibitor. Altogether, this work underscores UCKs as pivotal players in upholding molnupiravir efficacy and therapeutic window of molnupiravir, and furthermore as pharmacologi cally tractable targets for tailoring the drug response.