Identification of inhibitors of bacterial transcription/translation machinery utilizing a miniaturized 1536-well format screen

This report presents the miniaturization of a HTS screen to identify inhibi tors of prokaryotic transcription-translation in a 1536-well format. The in vitro assay design utilized the bacterial expression machinery to drive ex pression of a firefly luciferase reporter gene, which was read as an endpoi nt luminesence measurement. This multicomponent system permits identificati on of inhibitors at different steps in this pathway. Successful miniaturiza tion required integration of homogeneous assay formats, robust liquid-handl ing workstations, and second-generation imaging systems. Comparison of data from a triplicate 1536-well screen of a subset of a target library that ha d been previously validated and followed up for hit confirmation in a 384-w ell plate format confirmed that triplicate screening yields data of higher confidence and quality, eliminates the time-consuming and potentially error -prone step of cherry-picking, and reduces the number of false positives an d negatives. The substantial savings of reagents and reduction of the numbe rs of plates to process obtained in a 1536-well format as compared to a 384 -well format allowed a full triplicate evaluation of the entire library of 183,000 compounds at lower cost and in less time. The triplicate-screen sta tistics are consistent with a highly reliable data set with a coefficient o f variation of 14.8% and Z' and Z values of 0.57 and 0.25, respectively. Th is screen resulted in the identification of 1,149 hits (0.63% hit rate), re presenting a compound population at 2.5 standard deviations from the mean c utoff. Furthermore, the data demonstrate good agreement between IC50 values derived for this assay in a 1536-well format and 384-well format.