Dissertation > Medicine, health > Pharmacy > Pharmacology

Application of LC-MS Based Assay in DPP-IV Inhibitor Screening and Respective Substrate Discovery

Author LiuJingJing
Tutor FuLei
School Shanghai Jiaotong University
Course Medicinal Chemistry
Keywords LC-MS activity screening DPP-IV inhibitor substrate tripeptide
Type Master's thesis
Year 2012
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Dipeptidyl peptidaseⅣ(DPP-Ⅳ) is an enzyme of considerable biomedical interest because it is upregulated in certain diseases. Study DPP-Ⅳas a target is meaningful and with commercial value. For example, DPP-Ⅳinhibitors discovery was the most hot topic in recent decennium.The traditional methods for DPP-Ⅳinhibitor 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-Ⅳsubstrates have poor aqueous solubility and require organic solvents such as DMSO for solubilization. The use of organic co-solvents in enzymatic assays for DPP-Ⅳmay lead to unreliable results. Furthermore, some fluorescent and chromogenic DPP-Ⅳsubstrates are unstable in aqueous solution and undergo hydrolysis in the absence of DPP-Ⅳ.We have developed an LC-MS based method for DPP-Ⅳactivity assay which allowed the use of wider selections of substrates than the fluorescent and chromogenic methods. We use both chromogenic method and LC-MS method in measuring the DPP-Ⅳactivity Unit, substrate Km and positive IC50 value detecting. The LC-MS method was validated with several known DPP-Ⅳinhibitors 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-Ⅳassay. The tripeptide Gly-Pro-AspOMe and Gly-Pro-LeuOMe showed some interesting properties. In comparison with the commonly used chromogenic DPP-Ⅳsubstrate Gly-Pro-pNA, they both have better water-solubility. Gly-Pro-AspOMe reacted quickly and Gly-Pro-LeuOMe have lower Km value.

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