Novel Selective Activators Of Human Histone Deacetylase-8(HDAC-8)

Post-translational modification of lysine side chains of nucleosomes via acetylation play a critical role in regulating multitude diverse cellular functions,including energy and nutrients metabolism,protein folding,transcription and translation.The dynamic equilibrium of acetylation and deacetylation lysine side chains of histone catalyzed respectively by histone acetyl transferase?HATs?and histone deacetylases?HDAC_s?is associated with epigenetic control.HDACs are broadly classified as NAD⁺-dependents and Zn⁺²-dependent HDACs with sub-classes,?,?,?,and ?.Classes ?,?,and ? are Zn⁺²-dependent whereas class ??i.e the sirtuins?are NAD+-dependent HDACs.Histone deacetylase 8?HDAC8?is a class ? zinc-dependent HDAC.It is a unique member of this family due to its highly structural and kinetic characterization.Since its discovery in 2000,little is known about HDAC8 activators.Meanwhile,circumstantial evidence suggests the potential role of activation of specific isoforms of HDAC_s in promoting physiologically desirable metabolic processes.Activators hold great potential in the future toward a fuller exploration of HDAC8/HDAC_s biology and pharmacology as well as toward developing novel therapeutics for metabolic and age-related diseases as well as cancer.In this study,?i?we designed,synthesized,purified,characterized and tested the activities of seven?7?novel compounds side-by-side with a control compound?TM-2-51?on HDAC8 enzyme in an HPLC-based enzymatic assay system with 7-amino-4-methylcoumarine?AMC?containing?i.e FLUOR DE LYS?-HDAC8?substrate as the enzymatic substrate and measured quantitatively the fold of activation of each test compound as a means of assessing the potencies of the compounds compared with the control,TM-2-51;?ii?the seven compounds were tested alongside the control with an HDAC8 AMC-less substrate(CH₃-CO-FG-K_ac-FSW-NH₂).Again the activation folds were quantitatively determined in contrast to TM-2-51 to confer whether these novel activators are substrate-specific or not;?iii?subsequently,isoform selectivity assay was performed with HeLa nuclear extracts?containing nuclear HDAC_s such as HDAC-1 and-2?and AMC substrate in the same assay system to see whether these novel activators are HDAC8 selective or not;?iv?to further confirm the selectivity of the compounds,we substituted AMC substrate with an AMC-less HeLa nuclear extract substrate(CH₃-CO-RHK_Ac K_Ac-CO-NH₂)for another round of enzymatic assay;?v?the best activator?i.e compound-7?out of the seven novel compounds was selected and then used to measure both HDAC-8 substrates' Km in the absence and presence of the activator.In a nutshell,our novel compounds exhibited activation in both cases of utilizing AMC substrate and AMC-Less substrate(CH₃-CO-FG-K_ac-FSW-NH₂)of HDAC8 as the enzymatic substrates in our HPLC-based enzymatic assay at the expense of the control?which activated HDAC8 only in the presence of the former substrate?and were selective activators of HDAC8 according to results obtained from the HeLa nuclear extract assays.We can however,to some extent say that our compounds are not substrate specific compared with the N-acetyl thiourea series of activators by Singh et al with compound 7 as the best activator on average.This study and that of the pioneers could serve as a fertile ground to explore more selective activators of the HDAC8 enzyme in the future and hence may aid in the detail understanding of the molecular,and kinetic mechanisms with which these small molecules activate the enzyme.