Aim: To develop high‐throughput screening (HTS) assays for monoamine oxidase (MAO)‐A and MAO‐B inhibitors. Methods: A fluorescence probe based method measuring MAO‐A and MAO‐B activity was established and optimized, with its sensitivity, stability and specificity evaluated. Reaction conditions including enzyme sources, substrate concentrations, incubation volume and reaction time in 384‐well format were optimized to achieve sensitive and low consumptive goal. Results: In optimized conditions, dynamic parameters of MAO‐A and MAO‐B were obtained. The Km value of serotonin to MAO‐A was 1.66 μmol/L, while that of benzylamine to MAO‐B was 0.80 μmol/L. The IC50 value of clorgyline to MAO‐A was 2.99 nmol/L, and that of deprenyl to MAO‐B was 7.04 nmol/L, matching those obtained from traditional spectrometric assays. Among tested samples, one compound exerted an inhibitory effect on MAO‐A activity with IC50 as 0.36 umol/L, and three compounds had an inhibitory effect on MAO‐B activity with IC50 as 0.13, 0.19, and 0.13 μmol/L. The Z′ factor was 0.71±0.03 and 0.75±0.03 in MAO‐A‐inhibitor and MAO‐B‐inhibitor HTS system, respectively. Conclusion: The established assays can be well applied to MAO‐A and MAO‐B inhibitor screening with high quality, precision and reproducibility.
Acetylcholinesterase (AChE) inhibitors are an important class of medicinal agents used for the treatment of Alzheimer's disease. A screening model of AChE inhibitor was used to evaluate the inhibition of a series of phenyl pentenone derivatives. The assay result showed that some compounds displayed higher inhibitory effects. In order to study the relationship between the bioactivities and the structures, 26 compounds with phenyl pentenone scaffold were analyzed. A 3D-QSAR model was constructed using the method of comparative molecular field analysis (CoMFA). The results of cross-validated R(2)cv=0.629, non-cross-validated R(2)=0.972, SE=0.331, and F=72.41 indicate that the 3D-model possesses an ability to predict the activities of new inhibitors, and the CoMFA model would be useful for the future design of new AChE inhibitors.
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