2.2. Histone deacetylase inhibition assays

The free thiol form (CT-101S) of the parent compound CT-101 (Largazole peptide isostere) was provided by Dr. Robert Williams (Colorado State University, Fort Collins, CO). The other HDAC inhibitors, entinostat (#S1053), vorinostat (#S1047), panobinostat (#13280), belinostat (#S1085) and romidepsin (#R425060), were purchased from Cedarlane Laboratories, Ontario, Canada. The HDACi reference compounds Trichostatin A and TMP269 were added as internal controls to validate Class I and Class IIa inhibition profiles [29]. For in vitro HDAC inhibition assays (Reaction Biology Corp, Malvern, PA) recombinant HDAC isoforms 1–9 and HDAC -11 were combined with base reaction buffer (50 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl₂) and bovine serum albumin (1 mg/ml). Test articles were dissolved in dimethyl sulfoxide (DMSO) and added to the base reaction mixture to 1% of the total volume. Fluorogenic HDAC substrates were added to each sample to initiate the reaction. After incubation at 30 °C, developer was added to stop reactions and kinetic measurements were made for 1.5 h; endpoint readings were recorded once development reached a plateau. For all test articles, the IC₅₀ values were calculated from the 10-point inhibition curves for individual HDAC isoforms and results plotted as a heat plot (Fig. 1A). Trichostatin A and TMP269 inhibitory profiles were as established.

CT-101 shows selective histone deacetylase inhibition. A) In vitro HDAC inhibition assay was performed using the active free thiol form of CT-101 (CT-101S) and compared to currently marketed competitive compounds.Shown below is the heat plot index. B) The impact of select compounds on γ-globin RNA including to CT-101 and CT-108 (Largazole depsipeptide oxazole-thiazoline) compared to sodium 2,2-dimethylbutyrate (ST-20), valproic acid (VA) and sodium phenyl butyrate (SPB). Expression of γ-globin mRNA by RT-qPCR is represented as fold change compared to control samples.