Acta Pharmacologica Sinica 2006 June; 27 (6): 760-766; doi: 10.1111/j.1745-7254.2006.00336.x

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 K_m 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.

Keywords: fluorescence assay; monoamine oxidase; preclinical drug evaluation