Halley, .F, Reinshagen, J., Ellinger, B., Wolf, M., Niles, A.L., Evans, N.J., Kirkland, T.A., Wagner, J.M., Jung, M., Gribbon, P. and Gul, S.
Notes: This paper describes the evaluation of the HDAC-Glo™ I/II and SIRT-Glo™ Assays. For all enzymes assayed (HDAC-I, HDAC-3, HDAC-6 and SIRT-1), the Km values obtained using the luminogenic assays were comparable to those reported in the literature. Furthermore, the assays tolerated well the concentrations of DMSO used to deliver known HDAC inhibitors. The authors used the assays to generate dose-response curves and IC50 values for standard inhibitors of several HDAC isoforms. They indicate that all data sets were of high quality and that the IC50 values obtained were comparable to those reported in the literature. The authors screened four HDAC isoforms (HDAC-1, HDAC-3, HDAC-6 and SIRT-1) in duplicate against 640 FDA-approved drugs, and they screened HDAC-6 and SIRT-1 against the Hypha Discovery MycoDiverse natural products library. For both screens, “hits” were classified as compounds that showed >50% inhibition at their screening concentration. For the 640-drug screen, the confirmation of hits was >80%, and the false-positive hit rate was <20%. The Z´-factor values were 0.65 for the HDAC-6 screen and >0.85 for the SIRT-1 in the Hypha Discovery MycoDiverse natural products library screen. A Z´ factor of 0.5 or greater is indicative of an assay with low data variability but good dynamic range. The authors conclude that the HDAC-Glo™ I/II and SIRT-Glo™ Assays met the criteria of sensitivity, signal stability, low background, DMSO tolerance, data variability and dynamic range, and scalability required for a suitable drug-screening assay. (4137)