Continuous Activity Assay for HDAC11 Enabling Reevaluation of HDAC Inhibitors

0579099 - ÚMG 2024 RIV US eng J - Journal Article
Kutil, Z. - Mikešová, J. - Zessin, M. - Meleshin, M. - Nováková, Z. - Alquicer, Glenda - Kozikowski, A. - Sippl, W. - Bařinka, C. - Schutkowski, M.
Continuous Activity Assay for HDAC11 Enabling Reevaluation of HDAC Inhibitors.
ACS Omega. Roč. 4, č. 22 (2019), s. 19895-19904. ISSN 2470-1343. E-ISSN 2470-1343
R&D Projects: GA MŠMT(CZ) ED1.1.00/02.0109
Institutional support: RVO:68378050
Keywords : histone deacetylase inhibitors * sir2 family * posttranslational modifications * fluorescent-probe * peptide arrays * in-vitro * substrate-specificity * metabolic-regulation * fluorogenic probe * activity profiles
OECD category: Biochemistry and molecular biology
Impact factor: 2.870, year: 2019
Method of publishing: Open access
https://pubs.acs.org/doi/10.1021/acsomega.9b02808

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 TNF alpha-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 mu M substrate concentration, and the signal-to-noise ratio exceeds 150 for 384-well plates. In the absence of NAD(+), this substrate is specific for HDAC 11. Reevaluation of inhibitory data using our novel assay revealed limited potency and selectivity of known HDAC inhibitors, including Elevenostat, a putative HDAC11-specific inhibitor.
Permanent Link: https://hdl.handle.net/11104/0347965