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Continuous Activity Assay for HDAC11 Enabling Reevaluation of HDAC Inhibitors
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SYSNO ASEP 0579099 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Continuous Activity Assay for HDAC11 Enabling Reevaluation of HDAC Inhibitors Author(s) Kutil, Z. (CZ)
Mikešová, J. (CZ)
Zessin, M. (DE)
Meleshin, M. (DE)
Nováková, Z. (CZ)
Alquicer, Glenda (UMG-J)
Kozikowski, A. (US)
Sippl, W. (DE)
Bařinka, C. (CZ)
Schutkowski, M. (DE)Number of authors 10 Source Title ACS Omega. - : American Chemical Society - ISSN 2470-1343
Roč. 4, č. 22 (2019), s. 19895-19904Number of pages 10 s. Language eng - English Country US - United States 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 R&D Projects ED1.1.00/02.0109 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) Method of publishing Open access Institutional support UMG-J - RVO:68378050 UT WOS 000499133200042 EID SCOPUS 85075614812 DOI 10.1021/acsomega.9b02808 Annotation 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. Workplace Institute of Molecular Genetics Contact Nikol Škňouřilová, nikol.sknourilova@img.cas.cz, Tel.: 241 063 217 Year of Publishing 2024 Electronic address https://pubs.acs.org/doi/10.1021/acsomega.9b02808
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