Screening for alcohol dehydrogenase inhibitors in natural compounds by electrophoretically mediated microanalysis.

Alcohol dehydrogenase (ADH), a pivotal enzyme in ethanol metabolism, has gained prominence as a promising therapeutic target. This study developed an electrophoretically mediated microanalysis (EMMA) platform integrated with rapid polarity switching to enable efficient mixing of reactants and rapid screening of ADH inhibitors from natural compounds. Systematic optimization of capillary electrophoresis separation and enzymatic reaction parameters yielded precise kinetic characterization, with a Michaelis constant (Km) of 2.55 mM and a half-maximal inhibitory concentration (IC50) of 0.27 mM for the positive inhibitor 4-methylpyrazole. The optimized EMMA method identified three natural compounds (myricetin, emodin, and kaempferol), exhibiting significant ADH inhibitory activities. Parallel ultravioletvisible spectrophotometric assays confirmed these effects and elucidated distinct inhibition mechanisms: myricetin acted via competitive inhibition, emodin through non-competitive inhibition, and kaempferol via mixed-type inhibition. Molecular docking simulations corroborated these findings by resolving molecular level ligand-binding interactions at the ADH active site. This work demonstrates a sensitive and reliable screening strategy for ADH inhibitors and provides critical insights into the mechanistic diversity of natural enzyme inhibitors.