High-throughput screening for monoamine oxidase-A and monoamine oxidase-B inhibitors using one-step fluorescence assay
AbstractAim: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 μmol/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.