In-Capillary Screening of Matrix Metalloproteinase Inhibitors by Electrophoretically Mediated Microanalysis with Fluorescence Detection

摘要

A capillary electrophoresis-based method with enzymatinreaction inside the capillary for the screening of matrinmetalloproteinase (MMP) inhibitors has been developednMMP-2 and MMP-9, which have been considered anpromising targets for cancer therapy, were selected as thnmodel enzymes. The hydrolysis of a fluorogenic substratncatalyzed by MMPs was determined by measuring thnincrease in fluorescence. For high-throughput screeningnthe short-end injection was employed. The enzyme, subnstrate containing inhibitors, and enzyme solutions werninjected from the outlet of the capillary via the sandwichnmode. They were mixed by alternating the potential anpositive and negative polarities. Online hydrolysis, sepanration, and detection were achieved in 70 s with apnproximately 0.87 fmol of MMP required for each assaynTheapplicabilityofelectrophoreticallymediatedmicroanalynsis (EMMA) with fluorescence detection to estimate thninhibitory mechanism and to determine the IC50 valuenwas evaluated for two natural inhibitors, epigallocatnechin gallate and oleic acid. A few other naturancompounds such as resveratrol, quercetin, caffeic acidnglucosamine, and doxycycline were also screened tontest their inhibitory potency. The results obtained werncompared with those obtained by offline enzyme assanand confirm the effectiveness of the present methodnA rapid, cost-effective, and fully automated method fonMMP inhibitor screening is proposed.