Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.
Dalian Institute of Chemical Physics, China.
Anal Methods. 2021 Jun 24;13(24):2671-2678. doi: 10.1039/d1ay00415h.
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.
二肽基肽酶-IV（DPP-IV）在葡萄糖代谢中发挥着关键作用，已成为 2 型糖尿病的重要靶点。我们之前报道了一种用于 DPP-IV 检测的双光子荧光探针甘氨酰脯氨酰-N-丁基-4-氨基-1,8-萘酰亚胺（GP-BAN），其具有高特异性和灵敏度。在这项研究中，建立并优化了一种使用人血浆作为酶源的高通量筛选（HTS）方法来检测 DPP-IV 抑制剂。进一步的研究表明，西他列汀（列为 DPP-IV 抑制剂）在人重组 DPP-IV 中的 IC50 值（36.22 nM）与人血浆中的非常相似（39.18 nM），西他列汀作为一种竞争性抑制剂作用于人血浆 DPP-IV 介导的 GP-BAN 水解。这些结果表明，在这种基于 GP-BAN 的测定中，昂贵的人重组 DPP-IV 可以用人血浆代替。在此基础上，使用 GP-AMC（商业探针）进行比较验证，人血浆中 GP-AMC（0.09 min-1）水解的催化效率（Vmax/Km）低于 GP-BAN（0.21 min-1）。对抑制动力学（西他列汀）和分子对接（GP-BAN 和 GP-AMC）的进一步分析表明，GP-BAN 对酶具有比 GP-AMC 更好的特异性和亲和力。最后，优化后的方法用于对 69 种天然生物碱中的 DPP-IV 抑制剂进行 HTS。
