A high-throughput screen utilizing the fluorescence of riboflavin for identification of lumazine synthase inhibitors.
A high-throughput screening method based on the competitive binding of a lumazine synthase inhibitor and riboflavin to the active site of Schizosaccharomyces pombe lumazine synthase was developed. This assay is sensitive, simple, and robust. During assay development, all of the known active inhibitors tested were positively identified. Preliminary high-throughput screening in 384-well format resulted in a Z factor of 0.7. The approach utilizes a thermodynamic assay to bypass the problems associated with the instabilities of both lumazine synthase substrates that complicate the use of a kinetic assay in a high-throughput format, and it removes the time element from the assay, thus simplifying the procedure.
Chen
Jinhua
J
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Purdue University, West Lafayette, IN 47907, USA.
Illarionov
Boris
B
Bacher
Adelbert
A
Fischer
Markus
M
Haase
Ilka
I
Georg
Gunda
G
Ye
Qi-Zhuang
QZ
Ma
Zeqiang
Z
Cushman
Mark
M
D001665:Binding Sites; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000302:isolation & purification*; D008024:Ligands; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D012256:Riboflavin / Q000737:chemistry*; D012568:Schizosaccharomyces / Q000201:enzymology; D013050:Spectrometry, Fluorescence
D004791:Enzyme Inhibitors; D008024:Ligands; D009097:Multienzyme Complexes; C044822:6,7-dimethyl-8-ribityllumazine synthase; D012256:Riboflavin