Discovery of natural pentacyclic triterpenoids as potent and selective inhibitors against human carboxylesterase 1.

Human carboxylesterase 1 (CES1), primarily expressed in the liver and adipocytes, is responsible for the hydrolysis of endogenous esters (such as cholesteryl esters and triacylglycerols) and the metabolism of xenobiotic esters (such as clopidogrel and oseltamivir), thus participates in physiological and pathological processes. In this study, a series of natural pentacyclic triterpenoids were collected and their inhibitory effects against CES1 and CES2 were assayed using D-luciferin methyl ester (DME) and N-(2-butyl-1,3-dioxo-2,3-dihydro-1H-benzo[de] isoquinolin- 6-yl)- 2-chloroacetamide (NCEN) as specific optical substrate for CES1, and CES2, respectively. To this end, betulinic acid (BA) was found with strong inhibitory effect on CES1 (IC50, 15 nM) and relative high selectivity over CES2 (>2400-fold). Primary structure-activity relationships (SAR) analysis and docking simulations revealed that the carboxyl group at the C-28 site of BA is very essential for CES1 inhibition. The inhibition kinetic analyses demonstrated that BA was a potent competitive inhibitor against CES1-mediated DME hydrolysis. Further investigation on the inhibitory effect of BA in living cells (HepG2) based assays demonstrated that BA displayed potent inhibitory effects on intracellular CES1 activities, with the low IC50 value of 1.30 μM. These results demonstrated that BA is potent and highly selective CES1 inhibitor, which might be used as the promising tool for exploring the biological functions of CES1 in complex biological systems.