Pancreatic lipase (PL) is a well-known key target for the prevention and treatment of obesity. Human carboxylesterase 1A (hCES1A) has become an important target for the treatment of hyperlipidaemia. Thus, the discovery of potent dual-target inhibitors based on PL and hCES1A hold great potential for the development of remedies for treating related metabolic diseases. In this study, a series of natural triterpenoids were collected and the inhibitory effects of these triterpenoids on PL and hCES1A were determined using fluorescence-based biochemical assays. It was found that oleanolic acid (OA) and ursolic acid (UA) have the excellent inhibitory effects against PL and hCES1A, and highly selectivity over hCES2A. Subsequently, a number of compounds based on the OA and UA skeletons were synthesised and evaluated. Structure-activity relationship (SAR) analysis of these compounds revealed that the acetyl group at the C-3 site of UA (compound 41) was very essential for both PL and hCES1A inhibition, with IC(50) of 0.75 microM and 0.014 microM, respectively. In addition, compound 39 with 2-enol and 3-ketal moiety of OA also has strong inhibitory effects against both PL and hCES1A, with IC(50) of 2.13 microM and 0.055 microM, respectively. Furthermore, compound 39 and 41 exhibited good selectivity over other human serine hydrolases including hCES2A, butyrylcholinesterase (BChE) and dipeptidyl peptidase IV (DPP-IV). Inhibitory kinetics and molecular docking studies demonstrated that both compounds 39 and 41 were effective mixed inhibitors of PL, while competitive inhibitors of hCES1A. Further investigations demonstrated that both compounds 39 and 41 could inhibit adipocyte adipogenesis induced by mouse preadipocytes. Collectively, we found two triterpenoid derivatives with strong inhibitory ability on both PL and hCES1A, which can be served as promising lead compounds for the development of more potent dual-target inhibitors targeting on PL and hCES1A.
Dipeptidyl peptidase-IV (DPP-IV) plays a critical role in glucose metabolism and has become an important target for type 2 diabetes mellitus. We previously reported a two-photon fluorescent probe glycyl-prolyl-N-butyl-4-amino-1,8-naphthalimide (GP-BAN) for DPP-IV detection with high specificity and sensitivity. In this study, a high-throughput screening (HTS) method for DPP-IV inhibitors using human plasma as the enzyme source was established and optimized. Further investigations demonstrate that the IC50 value of sitagliptin (listed as the DPP-IV inhibitor) determined with human recombinant DPP-IV (36.22 nM) is very similar to that in human plasma (39.18 nM), and sitagliptin acts as a competitive inhibitor against human plasma DPP-IV-mediated GP-BAN hydrolysis. These results indicate that expensive human recombinant DPP-IV can be replaced by human plasma in this GP-BAN-based assay. On this basis, GP-AMC (commercial probe) was used as a comparison to verify this method, and the catalytic efficacy (Vmax/Km) for GP-AMC (0.09 min-1) hydrolysis in human plasma is lower than that for GP-BAN (0.21 min-1). Further analysis of inhibition kinetics (sitagliptin) and molecular docking (GP-BAN and GP-AMC) showed that GP-BAN has better specificity and affinity for enzymes than GP-AMC. Finally, the optimized method was used for the HTS of DPP-IV inhibitors in 69 natural alkaloids.
In this study, a highly specific ratiometric two-photon fluorescent probe GP-BAN was developed and well-characterized to monitor dipeptidyl peptidase IV in plasma and living systems. GP-BAN was designed on the basis of the catalytic properties and substrate preference of DPP-IV, and it could be readily hydrolyzed upon addition of DPP-IV under physiological conditions. Both reaction phenotyping and inhibition assays demonstrated that GP-BAN displayed good reactivity and high selectivity towards DPP-IV over other human serine hydrolases including FAP, DPP-VIII, and DPP-IX. The probe was successfully used to monitor the real activities of DPP-IV in complex biological systems including diluted plasma, while it could be used for high throughput screening of DPP-IV inhibitors by using human plasma or tissue preparations as enzyme sources. As a two-photon fluorescent probe, GP-BAN was also successfully used for two-photon imaging of endogenous DPP-IV in living cells and tissues, and showed high ratiometric imaging resolution and deep-tissue penetration ability. Taken together, a ratiometric two-photon fluorescent probe GP-BAN was developed and well-characterized for highly selective and sensitive detection of DPP-IV in complex biological systems, which could serve as a promising imaging tool to explore the biological functions and physiological roles of this key enzyme in living systems.
