Molnupiravir is not a selective CES2 probe substrate: in vitro evidence for CES1 involvement

Molnupiravir is an orally administered prodrug that requires enzymatic hydrolysis to generate N-hydroxycytidine, a pharmacologically active ribonucleoside analog. Previous reports suggested that molnupiravir hydrolysis was selectively catalyzed by the serine hydrolase carboxylesterase (CES) 2 and recommended its use as a CES2-selective marker reaction in vitro. However, upon reexamination of the data, it appeared that molnupiravir hydrolysis was not completely inhibited by remdesivir, a CES2 selective time-dependent inhibitor, thereby raising the possibility of additional hepatic serine hydrolases contributing to molnupiravir bioactivation. Consequently, the overarching aim of the present study was to reexamine the in vitro enzymatic basis of molnupiravir hydrolysis. Using pooled human liver and intestinal microsomes, as well as recombinant CES1, CES2, and arylacetamide deacetylase, CL_int,app values indicated substantial contributions from both CES isoforms, with minimal activity attributable to arylacetamide deacetylase. Activity-abundance correlations in human liver microsomes from 48 donors yielded moderate but statistically significant (P < .001) relationships for CES1 (Spearman r = 0.54) and CES2 (r = 0.46). Multiple linear regression analysis incorporating CES1 and CES2 abundances explained ∼42% of the interdonor variability in hydrolysis, with both enzymes contributing independently to the model. Consistent with these findings, chemical inhibition experiments showed only partial attenuation of hydrolysis by CES1 and CES2 inhibitors, whereas complete inhibition was observed with the pan serine hydrolase inactivator 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride. Collectively, these results provide compelling evidence that molnupiravir hydrolysis is catalyzed by both CES isoforms, rather than being selective for CES2, thereby indicating that molnupiravir may not be a suitable in vitro probe substrate for CES2. SIGNIFICANCE STATEMENT: This study challenges the prevailing view that molnupiravir hydrolysis is selectively catalyzed by CES2, demonstrating instead that both hepatic CES isoforms and potentially other hepatic serine hydrolases contribute to its bioactivation in vitro. These findings refine the characterization of molnupiravir metabolism, highlighting the involvement of CES1 in its hydrolysis and questioning its suitability as a selective CES2 probe substrate, with direct implication for hydrolase inhibition assays and drug development strategies.