Identification of HDAC Inhibitors Using a Cell-Based HDAC I/II Assay

Abstract

Histone deacetylases (HDACs) are a class of epigenetic enzymes that regulate gene expression by histone deacetylation. Altered HDAC function has been linked to cancer and neurodegenerative diseases, making HDACs popular therapeutic targets. In this study, we describe a screening approach for identification of compounds that inhibit endogenous class I and II HDACs. A homogeneous, luminogenic HDAC I/II assay was optimized in a 1536-well plate format in several human cancer cell lines, including HCT116 and human neural stem cells. The assay confirmed 37 known HDAC inhibitors from two libraries of known epigenetics-active compounds. Using the assay, we identified a group of potential HDAC inhibitors by screening the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection of 2527 small-molecule drugs. The selected compounds showed similar HDAC I/II inhibitory potency and efficacy values in both HCT116 and neural stem cells. Several previously unidentified HDAC inhibitors were further evaluated and profiled for their selectivity against a panel of 10 HDAC I/II isoforms using fluorogenic HDAC biochemical assays. In summary, our results show that several novel HDAC inhibitors, including nafamostat and piceatannol, have been identified using the HDAC I/II cell-based assay, and multiple cell types have been validated for high-throughput screening of large chemical libraries.

Keywords: cancer; epigenetics; histone deacetylase; neurodegenerative disease; qHTS.

Figure 1. Optimization and validation of the histone deacetylase (HDAC)-Glo I/II assay in 1536-well format

A. Optimal concentration titration of HDAC-Glo I/II activity in HCT116 (average IC₅₀ of 0.25 μM), HEK293 (average IC₅₀ of 0.09 μM), HepG2 (average IC₅₀ of 0.05 μM), human neural stem cells (hNSC) (average IC₅₀ of 0.44 μM), and SU-DHL-6 (average IC₅₀ of 0.10 μM) treated with trichostatin A. B. Time course of HDAC-Glo I/II activity in HCT116 cells after treatment of trichostatin A, with average IC₅₀s of 0.02 μM for 1 hour, 0.11 μM for 3 hours, 0.22 μM for 6 hours, and 0.29 μM for 18 hours, respectively. C. Assay selectivity as shown as concentration-response curves of HCT116 cells treated with two known HDAC inhibitors (average IC₅₀s of 0.77 μM for vorinostat/suberoylanilide hydroxamic acid (SAHA) and 0.07 μM for trichostatin A) and a sirtuin (SIRT) inhibitor (selistat, inactive). Each data point was presented as mean ± SEM from three replicates.

Figure 2. Histone deacetylase (HDAC) inhibitor hits identified from National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection

A. Concentration-response curves and chemical structures of nafamostat (average IC₅₀ of 0.36 μM) and camostat (average IC₅₀ of 0.95 μM). B. Concentration-response curves and chemical structures of piceatannol (average IC₅₀ of 4.18 μM) and resveratrol (average IC₅₀ of 7.43 μM). Each data point in the concentration-response curves was measured in human neural stem cells (hNSCs) and presented as mean ± SD from three replicates.

Figure 3. Biochemical profiling of isoform selectivity of histone deacetylase (HDAC) inhibitor hits

In total, 20 μM of the 15 selected potential HDAC inhibitors were tested with individual recombinant HDAC class I and II enzymes and their corresponding fluorogenic substrates. Efficacy values were averaged from three replicates and shown in heat map.

Figure 4. Confirmation of nafamostat and piceatannol identified by both cell-based and biochemical-based Histone deacetylase (HDAC) assays

A. Concentration-response curves of nafamostat in HDAC class I assays (average IC₅₀s of 4.67 μM for HDAC1, 4.69 μM for HDAC2, 4.04 μM for HDAC3, and 1.52 μM for HDAC8, respectively). B. Concentration-response curves of nafamostat in HDAC class II assays (average IC₅₀s of 1.49 μM for HDAC4, 1.33 μM for HDAC5, 4.39 μM for HDAC6, and 1.55 μM for HDAC9, respectively). C. Concentration-response curves of piceatannol in HDAC class I assays (average IC₅₀s of 4.28 μM for HDAC1, 5.04 μM for HDAC2, 23.29 μM for HDAC3, and 3.42 μM for HDAC8, respectively). D. Concentration-response curves of piceatannol in HDAC class II assays. Each data point was presented as mean ± SD from three replicates (IC₅₀s of 0.89 μM for HDAC5, 8.22 μM for HDAC6, and 9.95 μM for HDAC9, respectively).