Abstract: doi: 10.1111/fcp.12643 SHORT COMMUNICATION Remdesivir potently inhibits carboxylesterase-2 through covalent modifications: signifying strong drug-drug interactions Yue Shen, William Eades, Bingfang Yan Division of Pharmaceutical Sciences, James L. Winkle College of Pharmacy, University of Cincinnati, Cincinnati, OH, USA Keywords COVID-19, remdesivir, carboxylesterases, drug safety Received 15 November 2020; revised 23 December 2020; accepted 23 December 2020 ABSTRACT Remdesivir was recently approved to treat COVID-19. While this antiviral agent delivers clinical benefits, several safety concerns in many cases have been raised. This study reports that remdesivir at nanomolar concentrations inhibits carboxylesterase-2 (CES2) through covalent modifications. CES2 is a major drug-metabolizing enzyme. The combination of high potency with irreversible inhibition concludes that cautions must be exercised when remdesivir is used along with drugs hydrolyzed by CES2. Correspondence Bingfang Yan, Division of Pharmaceutical Sciences, James L. Winkle College of Pharmacy, University of Cincinnati, Cincinnati, OH 45229, USA. Email: yanbg@uc.edu FUNDING This work was supported by National Institutes of Health Grants R01 EB018748 and R21AI153031-01 (Yan B). The pandemic of coronavirus disease 2019 (COVID-19) has become a health crisis with the global death toll passing one million. So far, there are limited options to treat COVID-19. Remdesivir was granted emergency use authorization based on promising clinical benefits.1-3 Even with remdesivir, the rate of serious adverse events and mortality was high.1-3 The precise mechanisms remain unclear. Nevertheless, COVID-19 patients frequently receive multiple drugs and remdesivir requires metabolism for its therapeutic activity1-3; therefore, metabolism-based interactions are likely contributing factors. 432 Remdesivir is an ester prodrug and undergoes hydrolysis, most likely by carboxylesterase-1 (CES1). In addition, remdesivir has a core-structure alanine (boxed in Figure 1a) as seen with orlistat and sofosbuvir, two covalent inhibitors of CES2.4,5 We therefore hypothesized that remdesivir irreversibly inhibits CES2. To test this hypothesis, human liver microsomes pooled from 23 donors, incubated with remdesivir (0, 0.2 and 1 µmol/L), electrophoretically separated and tested for the remaining hydrolytic activity in the gel. Electrophoresis removed unbound but not covalently bound inhibitor, establishing an involvement of te  Francßaise de Pharmacologie et de The rapeutique ª 2020 Socie Fundamental & Clinical Pharmacology 35 (2021) 432–434 433 Potent inhibition of CES2 by remdesivir Figure 1 Irreversible inhibition of CES2 by remdesivir, and its effect on the hydrolysis of tenofovir disoproxil fumarate (TDF). (a) Structure of remdesivir (alanine boxed). (b) Native gel electrophoresis stained for hydrolytic activity. Microsomes pooled from 23 individual donors (5 µg) were incubated with remdesivir at 0, 0.2 or 1.0 µmol/L and subjected to native gel electrophoresis stained for esterase activity with 4-methylumbelliferylacetate, and the staining intensity was captured by Bio-Rad ChemoDoc Imager. (c) Inhibition of liver microsomal hydrolysis of TDF by remdesivir. Pre-incubation format: pooled liver microsomes (0.06 µg/µL) were pre-incubated at 37°C for 120 min with remdesivir at 0, 0.2 or 1.0 µmol/L followed by TDF at 10 µmol/L. Simultaneous format: pooled liver microsomes (0.06 µg/µL) were..