Given the nature of this HTS method, we speculated a significant portion of the primary active compounds were assay artifacts. Therefore, active compounds were subjected to a series of filters, counter-screens and orthogonal assays to identify compounds that truly inhibited histone acetylation. First, primary actives were computationally filtered for PAINS and other undesirable structural components such as maleimides, thiols and certain functional groups with terminal sulfur atoms. This step effectively triaged one-half of the primary screen actives. Compounds were then subjected to a counter-screen that mimicked the HTS assay conditions, except that proteins were omitted and the acetyl-CoA substrate was replaced with the CoA reaction ASP1517 product. Given the possibility that compounds could both interfere and still inhibit enzyme-catalyzed histone acetylation, we chose to triage most compounds with greater than 50% interference. Compounds with intermediate interference levels were triaged and passed depending on several factors, including chemical diversity, the potential for compound-thiol reactivity and the interference patterns observed for each compound. Next, library compounds were assessed for their ability to generate a dose-response curve. Compounds were scored according to curve shape. The overall paucity of bona fide HAT inhibitors reported in the literature coupled with the observation that a recent, well-designed screen versus a human HAT discovered only one true hit suggested the number of true-positives in our HTS may also be relatively low. Therefore, only compounds with no observable dose-response were triaged, which eliminated more than one-half of the remaining compounds. We obtained solid powder samples for all the remaining compounds and tested them for IC50 confirmation. Approximately one-half of the purchased solid samples had measureable IC50 values, typically in the 5�C 25 mM range. IC50 EX 527 HDAC inhibitor values were determined in two separate experiments with similar values and curve shapes, further demonstrating the assay output is reproducible. Nearly all of these samples did not show interference in a repeat of the CoA-based counter-screen. Orthogonal assays were performed next to verify if the test compounds inhibited the actual enzyme-catalyzed acetylation of histones. Based on the shape of the dose-response curves, the chemotype clusters in the confirmed actives, and other medicinal chemistry considerations, 54 compounds were tested using the orthogonal slot blot assay to probe for decreases in the actual histone acetylation.