High-Throughput Screening of DPPIV Inhibitors Antagonizing GLP-1 Degradation Using an Enzymatic Activated Fluorescent Probe

Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) is an exopeptidase widely expressed on various cell surfaces that selectively cleaves N-terminal dipeptides from diverse substrates. In recent years, DPPIV inhibitors have been extensively utilized in the treatment of hepatitis mellitus (DM). In this study, we designed a far-red fluorescent probe, DBX-AP, through molecular docking simulations and by leveraging the functional characteristics of DPPIV. This probe enables rapid, highly selective, and real-time monitoring of DPPIV activity both in vitro and in vivo. Using DBX-AP, we developed a visual high-throughput screening technique for the detection of DPPIV inhibitors. From a library of 4828 compounds, three inhibitors (K784-2660, 6484-0066, and E699-0153) were identified for their strong inhibitory effects on DPPIV. These inhibitors not only suppressed DPPIV activity in the ileum of mice, thereby reducing GLP-1 degradation, but also effectively inhibited DPPIV activity in gut microbiota. The successful application of DBX-AP in visual detection technology highlights its potential for evaluating DPPIV activity and identifying novel DPPIV inhibitors for diabetes mellitus treatment.