Targeting the epigenome: Screening bioactive compounds that regulate histone deacetylase activity

Abstract

Scope: Nutrigenomics is a rapidly expanding field that elucidates the link between diet-genome interactions. Recent evidence demonstrates that regulation of the epigenome, and in particular inhibition of histone deacetylases (HDACs), impact pathogenetic mechanisms involved in chronic disease. Few studies, to date, have screened libraries of bioactive compounds that act as epigenetic modifiers. This study screened a library of 131 natural compounds to determine bioactive compounds that inhibit Zn-dependent HDAC activity.

Methods and results: Using class-specific HDAC substrates, we screened 131 natural compounds for HDAC activity in bovine cardiac tissue. From this screen, we identified 18 bioactive compound HDAC inhibitors. Using our class-specific HDAC substrates, we next screened these 18 bioactive compounds against recombinant HDAC proteins. Consistent with inhibition of HDAC activity, these compounds were capable of inhibiting activity of individual HDAC isoforms. Lastly, we report that treatment of H9c2 cardiac myoblasts with bioactive HDAC inhibitors was sufficient to increase lysine acetylation as assessed via immunoblot.

Conclusion: This study provided the first step in identifying multiple bioactive compound HDAC inhibitors. Taken together, this report sets the stage for future exploration of these bioactive compounds as epigenetic regulators to potentially ameliorate chronic disease.

Keywords: Acetylation; Bioactive compounds; Dietary HDAC inhibitors; HDACs; Histone deacetylases.

Figure 1. Schematic representation of histone deacetylases (HDACs)

Zinc-dependent HDACs fall into three categories (Class I, II, IV; represented in blue), where class II are subdivided into IIa and IIb. Class III HDACs (Sirtuins; represented in green) are NAD⁺-dependent.

Figure 2. HDAC fluorogenic substrate specificity in bovine cardiac tissue

Bovine cardiac tissue was treated for 2 hours with pan- or class-specific HDAC inhibitors prior to incubation with cell permeable fluorogenic HDAC substrates for 2 hours and developer solution for 20 minutes. A) Trichostatin A (TSA) acted as a pan-HDAC inhibitor, while B) MGCD0103 (MGCD; Class I), C) DPAH (Class IIa) and D) Tubastatin A (TubA; Class IIb) worked as class-specific HDAC inhibitors.

Figure 3. Identification of bioactive HDAC inhibitors in bovine cardiac tissue

Bovine cardiac tissue were treated with increasing concentrations of indicated non-flavonoids (A&B) and flavonoids (C&D) for 2 hours. Bovine cardiac tissue was subsequently incubated with fluorogenic HDAC substrates for 2 hours prior to addition of stop solution for 20 minutes. Fluorescence was assessed via BioTek Synergy plate reader and demonstrated pan-HDAC inhibition of all compounds.

Figure 4. Bioactive compounds inhibit recombinant HDAC activity

Recombinant HDACs were incubated with non-flavonoids (A&B) and flavonoids (C&D) for 2 hours prior to the addition of cell permeable HDAC substrates. Non-flavonoids A) emodin and B) gossypol as well as flavonoids C) luteolin and D) quercetin dihydrate inhibited activity of most class I, IIa, and IIb HDAC isoforms.

Figure 5. Bioactive compound HDAC inhibitors increased lysine acetylation in H9c2 cells

H9c2 cells were incubated with indicated bioactive compounds for 2 hours prior to protein harvest. Cells were lysed and acetyl-lysine (Ac-Lysine), acetyl-histone 3 (Ac-H3 K9/14) and total histone 3 (Total-H3) were assessed via immunoblot. Acetylation of lysine increased for all compounds (A, C, E & G), whereas Ac-H3 (K9/14) only increased for A) emodin, B) gossypol and G) quercetin dihydrate. Acetyl proteins were normalized to total H3 and quantitation performed (B, D, F & H) via Image J software.