High-throughput screening for monoamine oxidase-A and monoamine oxidase-B inhibitors using one-step fluorescence assay.
OBJECTIVE
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 micromol/L, while that of benzylamine to MAO-B was 0.80 micromol/L. The IC(50) 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 IC(50) as 0.36 micromol/L, and three compounds had an inhibitory effect on MAO-B activity with IC(50) as 0.13, 0.19, and 0.13 micromol/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.


CONCLUSIONS
The established assays can be well applied to MAO-A and MAO-B inhibitor screening with high quality, precision and reproducibility.
Guang
Hong-Mei
HM
Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, China.
Du
Guan-Hua
GH
D000818:Animals; D001921:Brain / Q000201:enzymology*; D003010:Clorgyline / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D020128:Inhibitory Concentration 50; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D008995:Monoamine Oxidase / Q000378:metabolism*; D008996:Monoamine Oxidase Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012642:Selegiline / Q000494:pharmacology; D012680:Sensitivity and Specificity
D008996:Monoamine Oxidase Inhibitors; D012642:Selegiline; D008995:Monoamine Oxidase; D003010:Clorgyline