LC-MS based assay method for DPP-IV inhibitor screening and substrate discovery

The traditional methods for dipeptidyl peptidase IV (DPP-IV) inhibitor screening using fluorescence and chromogenic detections have a number of limitations. Interference with assay readout may occur if compounds have strong absorption at the wavelength region used for the detection. Some commonly used fluorescent and chromogenic DPP-IV substrates have poor aqueous solubility and require organic solvents such as DMSO for solubilization. The use of organic co-solvents in enzymatic assays for DPP-IV may lead to unreliable results. Furthermore, some fluorescent and chromogenic DPP-IV substrates are unstable in aqueous solution and undergo hydrolysis in the absence of DPP-IV. We have developed an LC-MS based method for DPP-IV activity assay which allowed the use of wider selections of substrates than the fluorescent and chromogenic methods. The LC-MS method was validated with several known DPP-IV inhibitors in comparison with a chromogenic assay method and was used to test library compounds to discover new inhibitors of DPP-IV. In addition, being a more universal detection technology, LC-MS method facilitates the discovery of new substrates. A mini-library of tripeptides was synthesized and screened for the discovery of new substrates with improved properties for DPP-IV assay.