Substrate Report for: Molnupiravir

Members of the carboxylesterase 2 (Ces2/CES2) family have been studied intensively with respect to their hydrolytic function on (pro)drugs, whereas their physiological role in lipid and energy metabolism has been realized only within the last few years. Humans have one CES2 gene which is highly expressed in liver, intestine, and kidney. Interestingly, eight homologous Ces2 (Ces2a to Ces2h) genes exist in mice and the individual roles of the corresponding proteins are incompletely understood. Mouse Ces2c (mCes2c) is suggested as potential ortholog of human CES2. Therefore, we aimed at its structural and biophysical characterization. Here, we present the first crystal structure of mCes2c to 2.12 resolution. The overall structure of mCes2c resembles that of the human CES1 (hCES1). The core domain adopts an alpha/beta hydrolase-fold with S230, E347, and H459 forming a catalytic triad. Access to the active site is restricted by the cap, the flexible lid, and the regulatory domain. The conserved gate (M417) and switch (F418) residues might have a function in product release similar as suggested for hCES1. Biophysical characterization confirms that mCes2c is a monomer in solution. Thus, this study broadens our understanding of the mammalian carboxylesterase family and assists in delineating the similarities and differences of the different family members.

Molnupiravir is one of the two COVID-19 oral drugs that were recently granted the emergency use authorization by the Food and Drug Administration (FDA). Molnupiravir is an ester and requires hydrolysis to exert antiviral activity. Carboxylesterases constitute a class of hydrolases with high catalytic efficiency. Humans express two major carboxylesterases (CES1 and CES2) that differ in substrate specificity. Based on the structural characteristics of molnupiravir, this study was performed to test the hypothesis that molnupiravir is preferably hydrolyzed by CES2. Several complementary approaches were used to test this hypothesis. As many as 24 individual human liver samples were tested and the hydrolysis of molnupiravir was significantly correlated with the level of CES2 but not CES1. Microsomes from the intestine, kidney and liver but not lung all rapidly hydrolyzed molnupiravir and the magnitude of hydrolysis was related closely to the level of CES2 expression among these organs. Importantly, recombinant CES2 but not CES1 hydrolyzed molnupiravir, collectively establishing that molnupiravir is a CES2-selective substrate. In addition, several CES2 polymorphic variants (e.g., R180H) differed from the wild-type CES2 in the hydrolysis of molnupiravir. Molecular docking revealed that wild-type CES2 and its variant R180H used different sets of amino acids to interact with molnupiravir. Furthermore, molnupiravir hydrolysis was significantly inhibited by remdesivir, the first COVID-19 drug granted the full approval by the FDA. The results presented raise the possibility that CES2 expression and genetic variation may impact therapeutic efficacy in clinical situations and warrants further investigation. Significance Statement COVID-19 remains a global health crisis, and molnupiravir is one of the two recently approved oral COVID-19 therapeutics. In this study, we have shown that molnupiravir is hydrolytically activated by CES2, a major hydrolase whose activity is impacted by genetic polymorphic variants, disease mediators, and many potentially co-administered medicines. these results presented raise the possibility that CES2 expression and genetic variation may impact therapeutic efficacy in clinical situations and warrants further investigation.