title: In-capillary screening of matrix metalloproteinase inhibitors by electrophoretically mediated microanalysis with fluorescence detection.
abstract: A capillary electrophoresis-based method with enzymatic reaction inside the capillary for the screening of matrix metalloproteinase (MMP) inhibitors has been developed. MMP-2 and MMP-9, which have been considered as promising targets for cancer therapy, were selected as the model enzymes. The hydrolysis of a fluorogenic substrate catalyzed by MMPs was determined by measuring the increase in fluorescence. For high-throughput screening, the short-end injection was employed. The enzyme, substrate containing inhibitors, and enzyme solutions were injected from the outlet of the capillary via the sandwich mode. They were mixed by alternating the potential at positive and negative polarities. Online hydrolysis, separation, and detection were achieved in 70 s with approximately 0.87 fmol of MMP required for each assay. The applicability of electrophoretically mediated microanalysis (EMMA) with fluorescence detection to estimate the inhibitory mechanism and to determine the IC(50) values was evaluated for two natural inhibitors, epigallocatechin gallate and oleic acid. A few other natural compounds such as resveratrol, quercetin, caffeic acid, glucosamine, and doxycycline were also screened to test their inhibitory potency. The results obtained were compared with those obtained by offline enzyme assay and confirm the effectiveness of the present method. A rapid, cost-effective, and fully automated method for MMP inhibitor screening is proposed.
authors.affiliation: Laboratory for Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: Xin
authors.initials: X
authors.lastname: Hai
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.forename: Mohamed
authors.initials: M
authors.lastname: El-Attug
authors.forename: Erwin
authors.initials: E
authors.lastname: Adams
authors.forename: Jos
authors.initials: J
authors.lastname: Hoogmartens
authors.forename: Ann
authors.initials: A
authors.lastname: Van Schepdael
mesh_terms: D001688:Biological Products / Q000032:analysis / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D046209:Electrophoresis, Microchip / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000378:metabolism; D009862:Online Systems; D010455:Peptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D001688:Biological Products; D005456:Fluorescent Dyes; D061965:Matrix Metalloproteinase Inhibitors; D010455:Peptides; D011480:Protease Inhibitors; D020782:Matrix Metalloproteinases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9