1. A high-throughput screening assay for dipeptidyl peptidase-IV inhibitors using human plasma
- Author
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Xing-Kai Qian, Gui-Min Zhang, Li-Wei Zou, Pei-Fang Song, Jing Zhang, Hong Huo, An-Qi Wang, Jing-Chun Yao, and Xiao-Dong Li
- Subjects
General Chemical Engineering ,010402 general chemistry ,01 natural sciences ,Analytical Chemistry ,law.invention ,Hydrolysis ,law ,medicine ,Humans ,IC50 ,Dipeptidyl-Peptidase IV Inhibitors ,chemistry.chemical_classification ,010405 organic chemistry ,Sitagliptin Phosphate ,General Engineering ,Fluorescence ,High-Throughput Screening Assays ,0104 chemical sciences ,Molecular Docking Simulation ,Enzyme ,Diabetes Mellitus, Type 2 ,Biochemistry ,chemistry ,Human plasma ,Sitagliptin ,Recombinant DNA ,medicine.drug - Abstract
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.
- Published
- 2021