Mechanistic insights into human carboxylesterase 2 (CES2) inhibition by the CES1 prodrug substrate remdesivir.

COVID-19 remains a significant health threat, particularly to people over the age of 65 with existing comorbidities like diabetes, hypertension, cancer, and viral infections. Despite expedited drug approvals, drug-drug interaction profiles for COVID-19 antiviral drugs have not yet been completely defined. The antiviral drugs remdesivir and molnupiravir are ester prodrugs with carboxylesterases (CES) playing a critical role in their bioactivation. In this study, we investigated the effect of COVID-19 antiviral drugs on CES hydrolysis activity. Of the 3 drugs tested, remdesivir inhibited 50% of CES2 activity in a nanomolar concentration range. Furthermore, time-dependent inhibition of CES2 activity by remdesivir was identified with an IC50 shift of nearly 3-fold from 0.188 μM after 5-minute preincubation to 0.068 μM following 40-minute preincubation. Remdesivir inactivation of CES2 was characterized by a kinact/KI value of 1.6 × 103 M-1.s-1. Through equilibrium dialysis and substrate protection experiments, we were able to further substantiate previous findings of the irreversible CES2 inhibition by remdesivir. Finally, in silico docking analysis of remdesivir to CES2 supported the proposed mechanism for covalent modification of the CES2 active site involving the catalytic triad serine via the phosphate group of remdesivir. SIGNIFICANCE STATEMENT: This study provides insights into human carboxylesterase inhibition by COVID-19 drugs and these findings demonstrate the underlying inhibition mechanism by remdesivir of recombinant human carboxylesterase 2.