A high-throughput screening assay for dipeptidyl peptidase-IV inhibitors using human plasma. Issue 24 (25th May 2021)

• Record Type: Journal Article
• Authors: Zhang, Jing; Qian, Xing-Kai; Song, Pei-Fang; Li, Xiao-Dong; Wang, An-Qi; Huo, Hong; Yao, Jing-Chun; Zhang, Gui-Min; Zou, Li-Wei
• Is Part Of: Analytical methods. Volume 13:Issue 24(2021)
• Page Start: 2671
• Page End: 2678
• Publication Date: 2021-05-25
• Subjects: Chemistry, Analytic -- Periodicals; Analytical biochemistry -- Periodicals; Chemical laboratories -- Standards -- Periodicals; 543.1905
• DOI: 10.1039/d1ay00415h
• Languages: English
• ISSNs: 1759-9660

Abstract

Abstract : A high-throughput screening assay for dipeptidyl peptidase-IV inhibitors. 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 ( V max / K m ) 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.