title: Rapid screening of aminopeptidase N inhibitors by capillary electrophoresis with electrophoretically mediated microanalysis.
abstract: In this work, an electrophoretically mediated microanalysis (EMMA) method with a partial-filling technique was setup to evaluate the inhibitory potency of novel compounds toward aminopeptidase N (APN). It was necessary to optimize the electrophoretic conditions with respect to the kinetic constraints and for attaining high sensitivity. In our setup, a part of the capillary was filled with the incubation buffer for the enzyme reaction, whereas the rest was filled with a suitable BGE for the separation of substrates and products. To monitor the performance of the newly developed method, the kinetic constants (Km and Vmax) for the catalyzed dissociation of L-Leucine-p-nitroanilide in the presence of APN as well as the inhibition constant (IC50 ) of a known competitive inhibitor, that is bestatin, were determined and these results were compared with those obtained by a classical spectrophotometric assay. The developed EMMA method was subsequently applied to the screening of 30 APN inhibitors. Whereas the inhibition potency of these inhibitors (expressed in IC50 values) were significantly underestimated by the EMMA method, the order of the inhibitory potential of these various compounds was found in agreement with the literature.
authors.affiliation: School of Pharmaceutical Sciences, Shandong University, Jinan, P. R. China.
authors.forename: Hairong
authors.initials: H
authors.lastname: Wang
authors.forename: Wenfang
authors.initials: W
authors.lastname: Xu
authors.forename: Jiangying
authors.initials: J
authors.lastname: Cao
authors.forename: Weihong
authors.initials: W
authors.lastname: Wang
mesh_terms: D000813:Anilides / Q000378:metabolism; D018826:CD13 Antigens / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D015203:Reproducibility of Results; D013054:Spectrophotometry, Atomic
chemical_list: D000813:Anilides; D004791:Enzyme Inhibitors; C001536:1-leucine-4-nitroanilide; D018826:CD13 Antigens; D007930:Leucine; C012211:ubenimex
keywords: Aminopeptidase N; Electrophoretically mediated microanalysis; Inhibitor screening; Kinetic constants