Chemical phylogenetics of histone deacetylases

Abstract

The broad study of histone deacetylases in chemistry, biology and medicine relies on tool compounds to derive mechanistic insights. A phylogenetic analysis of class I and II histone deacetylases (HDACs) as targets of a comprehensive, structurally diverse panel of inhibitors revealed unexpected isoform selectivity even among compounds widely perceived as nonselective. The synthesis and study of a focused library of cinnamic hydroxamates allowed the identification of, to our knowledge, the first nonselective HDAC inhibitor. These data will guide a more informed use of HDAC inhibitors as chemical probes and therapeutic agents.

Figure 1

Development of a platform for biochemical profiling of human deacetylases. (a) Chemical structure of substrates 3 - 6. (b) Comparative enzymatic activity of HDAC1-9 with tripeptide substrate 3 and trifluoro acetyl-lysine tripeptide substrate 6, studied at equivalent substrate concentration (10 μM). Data represent mean values of three measurements ± s.d. Substrate 6 allows miniaturized, kinetic study of HDAC4, 5, 7, 8 and 9.

Figure 2

Chemical phylogenetic analysis of HDACs identifies unexpected selectivity of HDAC inhibitors. (a) Hierarchical clustering of HDACs and a representative panel of structurally-diverse HDAC inhibitor tool and investigational compounds 1, 2, 7-20 weighted by inhibitory potency (K_i). Complete quantitative data are shown in Supplementary Table 1. Data based on mean values of triplicate measurements. (b,c) Chemical structure and enzymatic selectivity profile of (b) MS-275 19 and (c) SAHA 1, overlaying molecular phylogeny. HDAC dendrograms are adapted from Supplementary Figure 4. Circles are proportionate in size to K_i on a logarithmic scale, as shown.

Figure 3

Inhibition of Class IIa HDACs by acetylated lysine based substrates. Inhibition of trifluoroacetyl-lysine substrate 6 processing by (a) acetyl-lysine substrate 3 (b) acetyl-lysine substrate 21 is presented in dose-response format for Class IIa HDACs. Data represent mean values of triplicate measurements ± s.d. IC₅₀ curves were fit by logistic regression.

Figure 4

Synthesis and testing of an HDAC-biased chemical library and identification of a non-selective HDAC inhibitor. (a) Library design of meta- and para-substituted hydroxamic acid HDAC inhibitors, utilizing parallel condensation of aldehydes, efficiently samples chemical diversity at the capping feature. (b) Biochemical profiling data for the para-substituted sub-library (n=160 compounds), presented in dose-response format for inhibition of HDAC5. Structural variation in the capping feature was observed to confer a broad range of potency, as illustrated with the most (IC₅₀= 18 nM) and least (IC₅₀ = 55 μM) potent small molecules tested. (c) Comparative biochemical profiling of meta- (red) and para-substituted (blue) sub-libraries for relative inhibition of HDAC2 and HDAC3. The complete library was studied and is displayed at a range of concentrations (0.03, 0.3, 3.0 and 30.0 μM). Compounds of this structural class do not discriminate between HDAC2 and HDAC3. (d) Comparative biochemical profiling of meta- (red) and para-substituted (blue) sub-libraries for relative inhibition of HDAC5 and HDAC7. The complete library was studied and is displayed at a range of concentrations (0.03, 0.3, 3.0 and 30.0 μM). Para-substituted cinnamic hydroxamic acids exhibit increased potency for HDAC5, relative to meta-substituted regioisomers. (e) Specificity profile of pandacostat 22 overlaying molecular phylogeny. HDAC dendrograms are adapted from Supplementary Figure 4. Circles are proportionate in size to K_i on a logarithmic scale, as shown. (f) Immunoblot of Jurkat cells treated with pandacostat for 24 hours and stained for acetylated histones (AcH3K18), acetylated alpha-tubulin (AcTub) or GAPDH. (g) Chemical structure of pandacostat 22.