Rapid screening of aminopeptidase N inhibitors by capillary electrophoresis with electrophoretically mediated microanalysis

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 (K_m and V_max) 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.