Continuous Activity Assay for HDAC11 Enabling Reevaluation of HDAC Inhibitors

Abstract

Histone deacetylase 11 (HDAC11) preferentially removes fatty acid residues from lysine side chains in a peptide or protein environment. Here, we report the development and validation of a continuous fluorescence-based activity assay using an internally quenched TNFα-derived peptide derivative as a substrate. The threonine residue in the +1 position was replaced by the quencher amino acid 3′-nitro-l-tyrosine and the fatty acyl moiety substituted by 2-aminobenzoylated 11-aminoundecanoic acid. The resulting peptide substrate enables fluorescence-based direct and continuous readout of HDAC11-mediated amide bond cleavage fully compatible with high-throughput screening formats. The Z′-factor is higher than 0.85 for the 15 μM substrate concentration, and the signal-to-noise ratio exceeds 150 for 384-well plates. In the absence of NAD+, this substrate is specific for HDAC11. Reevaluation of inhibitory data using our novel assay revealed limited potency and selectivity of known HDAC inhibitors, including Elevenostat, a putative HDAC11-specific inhibitor.

The Roles of Histone Deacetylases and Their Inhibitors in Cancer Therapy

“…This effect leads to type I interferon receptor chain 1 (IFNαR1) polyubiquitylation and degradation, as well as downregulation of IFN signaling ( Cao et al, 2019 ). HDAC11-specific inhibitors, such as elevenostat, FT895, and SIS17, might represent promising future treatments that target lipid metabolic dysregulation in cancers ( Martin et al, 2018 ; Kutil et al, 2019 ; Son et al, 2019 ). SIRT3 has a role in mitochondrial fatty-acid β-oxidation by regulating long-chain acyl-CoA dehydrogenase (LCAD) ( Hirschey et al, 2010 ).…”

The Emerging Role of HDACs: Pathology and Therapeutic Targets in Diabetes Mellitus

“…Class IV HDAC, HDAC11, is a relatively new member of the HDAC family, which can preferentially remove fatty acid residues from lysine side chains of a protein or peptide [66]. HDAC11 has been regarded to regulate metabolism and obesity [67].…”

“…Inhibition of HDAC11 is regarded to promote energy expenditure by triggering UCP1 activation in brown adipose tissue [68]. Several small molecules, such as elevenostat, FT895, 2-carboxamidothiophene-based zinc ion chelating carbohydrazides, etc., can selectively inhibit HDAC11 [66]. Additionally, some pan-HDACs, such as romidepsin and TSA have been shown to inhibit HDAC11 in nM concentration [66].…”

“…Several small molecules, such as elevenostat, FT895, 2-carboxamidothiophene-based zinc ion chelating carbohydrazides, etc., can selectively inhibit HDAC11 [66]. Additionally, some pan-HDACs, such as romidepsin and TSA have been shown to inhibit HDAC11 in nM concentration [66]. Thus, these compounds could probably be potential agents to correct T2DM by HDAC11 inhibition.…”

Selective Targeting of Epigenetic Readers and Histone Deacetylases in Autoimmune and Inflammatory Diseases: Recent Advances and Future Perspectives

“…Genetic depletion of individual HDACs has demonstrated that these proteins mediate specific and unique functions [ 57 ], suggesting therapeutic relevance for selective isoform targeting. While achieving this is a challenge due to conserved structural similarity between HDAC isoforms [ 12 ], HDACi has been reported with selectivity for HDAC1, HDAC2, HDAC3 [ 33 , 58 , 59 , 60 , 61 ], HDAC8 [ 62 , 63 ], HDAC6 [ 64 ], HDAC11 [ 65 ], SIRT1, and SIRT2 [ 66 , 67 ]. However, caution should be taken in interpreting the specificity of the effects of these published inhibitors given the variability of available HDAC assays and the residual dose-dependent effects on other isoforms (Table 1).…”