Online Screening Of Aminopeptidase N Inhibitors By Capillary Electrophoresis

Aminopeptidase N (APN), an ectoenzyme located on cell surface, is a zinc-dependent metalloprotease containing zinc at its active centre. APN is widely present in several brain, kidney, liver, and angiogenic vessels in adults. Over-expression of APN is reported to be associated with extracellular matrix degradation, tumor proliferation, invasion, angiogenesis, and metastasis. APN has thus become an attractive therapeutic target for the inhibition of tumor vascularization and growth. Therefore, it is important to develop APN-targeting new drugs for tumor therapy. The availability of appropriate screening methods allowing the selection of new synthesized compounds is an essential prerequisite in such a drug discovery process. Therefore, it is absolutely necessary to develop effective and rapid methods to assess the inhibitory potency of synthetic test compounds against APN targets.Capillary Electrophoresis (CE) is one of the most powerful analytical tools due to its low sample consumption, high resolution, fast analysis, degree of automation and multiplexing capabilities. Besides being an excellent separation technique, CE represents an attractive alternative to several other enzymatic assays aimed for inhibitor screening and characterization. CE-based enzyme assays are economical and efficient, and they require only small volumes of biochemical reagents. An in-capillary enzyme assay denominated as the electrophoretically mediated microanalysis (EMMA), has been proven to be very significant for studying enzyme reactions and for enzyme inhibitor screening as it combines the enzyme-catalyzed reaction, separation, and detection all in a single capillary. The purpose of the present work is the developing of an effective and reliable two online CE method for enzymatic activity studies of APN and inhibitor screening.1. An EMMA method has been developed to evaluate the inhibitory potency of novel compounds toward APN. It was necessary to optimize the electrophoretic conditions with respect to the kinetic constraints and for attaining high sensitivity. A partial filling technique was applied; incubation buffer (50 mM phosphate buffer, pH 7.2) was injected into a fused-silica capillary (50 cm,75μm i.d.), followed by enzyme and subsequent injection of substrate solution and incubation buffer. Upon the application of ±2.0 kV each for 6 s, the enzyme and substrate solutions were mixed. The reaction was initiated by the application of 1 kV voltage for 2 min. Then a constant voltage of 25 kV was applied to separate the product, unreacted substrate and internal standard within 8 min using running buffer (HEPES buffer with DOC, pH 7.7). To monitor the performance of the newly developed method, the kinetic constants (Km and Vmax) for the catalyzed dissociation of L-Leucine-p-nitroanilide (Leu-pNA) in the presence of APN as well as the inhibition constant (IC50) of a known competitive inhibitor, i.e. bestatin, were determined and these results were compared with those obtained by a classical spectrophotometric assay. The developed EMMA method was applied to quantitatively determine the inhibition of the enzymatic activity of 29 synthesized compounds (including 2 unpurified compounds present as crude products). 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. This method permitted the evaluation of the enzyme activity and the screening of the effect of APN inhibitors within a few minutes with high accuracy and reproducibility.2. CE-based cell membrane screening method is a good technology for studying the interactions between drug and cell membrane, membrane receptor and enzyme. In this way, it retains best the integrity and enzymatic activity of cell membrane, the stereo-structure and surroundings of membrane enzyme. In this study, a CE-based cell membrane screening method was setup to evaluate the inhibitory potency of novel compounds toward APN expressed on the surface of cancer cell. The important content of this work is the preparation of high expression APN cell membrane. Gaining good active and purity of cell membranes by differential centrifugal, we inspected the cell membrane access of broken way on the cell membrane and influence of the APN activity. In our set-up inlet of the capillary was filled with cell membrane in incubation buffer for the enzyme reaction, whereas the rest was filled with a suitable background electrolyte for the separation of substrates, products and internal standard. To monitor the performance of the newly developed method, the inhibition constant (IC50) of a known positive inhibitor, i.e. bestatin, were determined and these results were compared with that obtained by a classical spectrophotometric assay. Cisplatin, as a negative inhibitor, and the cell membrane of K562 which express low level of APN and were injected to verify the reliability of the method as well as the kinetic constants (Km) for the catalyzed dissociation of Leu-pNA in the presence of APN in cell membrane was determined. The developed CE-based cell membrane screening method was also applied to quantitatively determine the inhibition of the enzymatic activity of 29 synthesized compounds (including 2 unpurified compounds present as crude products). The inhibition potency of these inhibitors determined by the CE-based cell membrane screening method was found in agreement with the literature.