Table 1.
Rtt109-Vps75 HTS protocol steps.
Table 2.
Rtt109-Vps75 HTS protocol notes.
Figure 1.
Fluorometric Rtt109-Vps75 HTS schematic.
The Rtt109-Vps75 KAT complex catalyzes the transfer of an acetyl group from acetyl-CoA to specific histone lysine residues within the Asf1-dH3-H4 substrate complex. The resulting CoA contains a free thiol group (-SH), which can react with the sulfhydryl-sensitive probe CPM to form a fluorescent adduct, thereby permitting the quantification of free CoA as a measure of KAT activity.
Figure 2.
Assay design and optimization.
(A) Titration matrix of CoA and CPM in buffer-only conditions to determine the optimal assay levels of acetyl-CoA and CPM. (B) Titration matrix of CPM and acetyl-CoA in buffer-only conditions to verify acetyl-CoA and CPM do not form fluorescent adducts under HTS conditions. (C) Time-course study of CoA titrations with 20 µM CPM in buffer-only conditions to determine the optimal time for the reaction involving CoA and CPM. (D) HTS plate template. Arrows denote chosen HTS conditions.
Figure 3.
Assay validation using the LOPAC ± detergent.
(A) Z' factors for replicate LOPAC experiments ± detergent. (B–C) Comparison of duplicate runs of the LOPAC ± detergent. Each point represents the activity of a discrete compound from the LOPAC. (D) Comparison of the LOPAC results ± detergent. Percent inhibitions represent the means of the replicate LOPAC experiments. Trend line (solid, red), ideal correlation line (dashed, blue). (E) Percent inhibition distribution of the averaged LOPAC results ± detergent, binned in 5% intervals.
Figure 4.
(A) Z' factors for each HTS plate, arranged in order screened. (B) Percent inhibition distribution pattern of compounds screened, binned in 1% intervals (outliers not shown). Cumulative results are pooled from HTS1 and HTS2. (C) Percent inhibition of each compound arranged in order screened. (D) Heat map of each assayed compound, arranged by well position and assay plate in order screened. HTS1 spans plates 1–259 while HTS2 spans plates 260–715. Active compounds were calculated separately for HTS1 and HTS2. HTS1 = no detergent; HTS2 = detergent.
Table 3.
Rtt109-Vps75 HTS summary.
Table 4.
Activity breakdown of Rtt109-Vps75 HTS.
Figure 5.
Example dose-response and orthogonal assays using garcinol, a previously reported KAT inhibitor.
(A) CPM-based assay dose-response experiments with garcinol. Fluconazole = negative control. (B) Orthogonal slot blot assay to detect the presence of H3K56ac, H3K27ac and H3K9ac by Western blot. Equal protein content was verified for each membrane by Ponceau S staining. (C) Dose-response experiments with garcinol using a secondary [3H]-acetyl-CoA-based orthogonal assay.
Figure 6.
Noteworthy PAINS substructures in the primary Rtt109-Vps75 HTS.
Italics denote the original names of published PAINS substructures [51]. For individual substructures, the ratios denote the number of primary active compounds divided by the number of compounds tested for each HTS production run.
Figure 7.
(A) Post-HTS triage schematic. Italics = HTS library samples. (B) Summary of confirmatory dose-responses, as classified by shape. Full response: >95% percent inhibition span and two horizontal asymptotes; partial response: >30% span; marginal response: >15% span. (C) IC50 profile of purchased solid samples. (D) Chemical structures of representative artifact compounds identified by the orthogonal slot blot assay.
Figure 8.
Example active compound discovered with the Rtt109 HTS and post-HTS triage.
(A) CPM-based assay dose-response with compound 1. Fluconazole = negative control compound. (B) Orthogonal slot blot assay to detect the presence of H3K56ac by Western blot. Equal protein content was verified for each membrane by Ponceau S staining. (C) Dose-response of compound 1 using a [3H]-acetyl-CoA orthogonal assay.