New Insights into the Epigenetic Activities of Natural Compounds

Background: With their varied pharmacophores, natural products are interesting tools to open the drug discovery pipeline. Several plant secondary metabolites belong to our diet and have conflictual documented epigenetic activities which need clarification. Methods: Seventy-one different natural products plus 17 controls were assembled for screening. First localized DNA methylation (DNAm) was studied on a stretch of the Retinoic Acid Receptor geneRAR followed then by a wider measure of all genomic 5 methylated Cytosine (5mC) by High Performance Liquid Chromatography tandem Mass Spectrometry (HPLC-MS/MS). DNA Methyl Transferase 1 (DNMT1) enzymatic activity was measured for selected compounds. Levels of Histone H3 trimethylation at lysine 9 and 27 (me3H3K9 and me3H3K27) were measured by Western blot. Global histone deacetylase inhibition (HDAC) was assayed first via a Bioluminescent Resonance Energy Transfer-based (BRET) assay and then with enzymatic fluorescence based-assays for HDAC class 1: 1, 2 and 3. As for HDAC6 inhibition, it was measured by Western blot screening. Sirtuine 2 (Sirt) inhibition was assessed first by a thermal shift assay and then by enzymatic assays for Sirt 1 and 2 using the SIRTainty™ Class III HDAC assay. Results: Several compounds decreased RAR methylation levels; but only Curcumin and (-) -Epigallocatechin gallate (EGCG) inhibited the DNMT1 catalytic activity; most other compounds worked probably, as S-Adenosyl-L-homocysteine (SAH) producers, with different potency under the assay conditions used here. Localized DNA demethylation was not mirrored by the global measure of 5mC levels. D-Glucuronic acid and anthraquinones decreased the level of me3H3K9 and 27 by un-deciphered mechanisms. When screening for HDAC class 1 inhibition, most flavonoids were emissive for enzymatic assays (excitation (Exec) = 340-360 nm and emission (Em) =440-465 nm). It was also the case when screening for Sirt activity by thermal shift (Exec= 365 nm and Em= 460 nm). This was overrun by the SIRTainty™ Class III HDAC (Exec= 420 nm and Em= 450 nm) assays by opposition to the FLUOR DE LYS® HDAC assay (Exec=360 nm and Em=460 nm). Within such limitations, we found that alkaloids like Brucine and Papaverin and anthraquinones are new epigenetic modifiers; this opens the epigenetic chemical space. Conclusions: To study diversity compound libraries, high through-put alpha screens are used at first. To study DNAm, they have large pitfalls. Indeed, finding unmethylated RARalleles does not indicate what a given compound is doing at the level of the entire genome for a given lapse of time and a given dose of compound. The measure of the DNMT1 enzymatic activity is not useful since most natural compounds are not direct enzymatic inhibitors. When studying histone methylation, Western blot is laborious but remains a cheap functional assay when several histone methylases (KDMs) or demethylases could be at the basis of histones methylation level decreases. Genome reversible epigenetic modifications remain desirable targets for nutrition oriented preventive strategies. However, reliable HDAC inhibition assays remain necessary before claiming that flavanols have true HDAC modulating activities.