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.

Department of Enzymology Institute of Biochemistry and Biotechnology Charles Tanford Protein Centre Martin Luther University Halle Wittenberg Kurt Mothes Straße 3a 06120 Halle Germany

Department of Medicinal Chemistry Institute of Pharmacy Martin Luther University Halle Wittenberg 06120 Halle Germany

Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Prumyslova 595 252 50 Vestec Czech Republic

StarWise Therapeutics LLC 505 S Rosa Road Suite 27 Madison Wisconsin 53719 1235 United States

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Chen Y.; Sprung R.; Tang Y.; Ball H.; Sangras B.; Kim S. C.; Falck J. R.; Peng J.; Gu W.; Zhao Y. Lysine propionylation and butyrylation are novel post-translational modifications in histones. Mol. Cell. Proteomics 2007, 6, 812–819. 10.1074/mcp.M700021-MCP200. PubMed DOI PMC

Nishida Y.; Rardin M. J.; Carrico C.; He W.; Sahu A. K.; Gut P.; Najjar R.; Fitch M.; Hellerstein M.; Gibson B. W.; Verdin E. SIRT5 Regulates both Cytosolic and Mitochondrial Protein Malonylation with Glycolysis as a Major Target. Mol. Cell 2015, 59, 321–332. 10.1016/j.molcel.2015.05.022. PubMed DOI PMC

Peng C.; Lu Z.; Xie Z.; Cheng Z.; Chen Y.; Tan M.; Luo H.; Zhang Y.; He W.; Yang K.; Zwaans B. M. M.; Tishkoff D.; Ho L.; Lombard D.; He T.-C.; Dai J.; Verdin E.; Ye Y.; Zhao Y. The first identification of lysine malonylation substrates and its regulatory enzyme. Mol. Cell. Proteomics 2011, 10, M111.01265810.1074/mcp.M111.012658. PubMed DOI PMC

Zhang Z.; Tan M.; Xie Z.; Dai L.; Chen Y.; Zhao Y. Identification of lysine succinylation as a new post-translational modification. Nat. Chem. Biol. 2011, 7, 58–63. 10.1038/nchembio.495. PubMed DOI PMC

Tan M.; Peng C.; Anderson K. A.; Chhoy P.; Xie Z.; Dai L.; Park J.; Chen Y.; Huang H.; Zhang Y.; Ro J.; Wagner G. R.; Green M. F.; Madsen A. S.; Schmiesing J.; Peterson B. S.; Xu G.; Ilkayeva O. R.; Muehlbauer M. J.; Braulke T.; Mühlhausen C.; Backos D. S.; Olsen C. A.; McGuire P. J.; Pletcher S. D.; Lombard D. B.; Hirschey M. D.; Zhao Y. Lysine glutarylation is a protein posttranslational modification regulated by SIRT5. Cell Metabol. 2014, 19, 605–617. 10.1016/j.cmet.2014.03.014. PubMed DOI PMC

Tan M.; Luo H.; Lee S.; Jin F.; Yang J. S.; Montellier E.; Buchou T.; Cheng Z.; Rousseaux S.; Rajagopal N.; Lu Z.; Ye Z.; Zhu Q.; Wysocka J.; Ye Y.; Khochbin S.; Ren B.; Zhao Y. Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification. Cell 2011, 146, 1016–1028. 10.1016/j.cell.2011.08.008. PubMed DOI PMC

Xie Z.; Zhang Di.; Chung D.; Tang Z.; Huang H.; Dai L.; Qi S.; Li J.; Colak G.; Chen Y.; Xia C.; Peng C.; Ruan H.; Kirkey M.; Wang D.; Jensen L. M.; Kwon O. K.; Lee S.; Pletcher S. D.; Tan M.; Lombard D. B.; White K. P.; Zhao H.; Li J.; Roeder R. G.; Yang X.; Zhao Y. Metabolic Regulation of Gene Expression by Histone Lysine β-Hydroxybutyrylation. Mol. Cell 2016, 62, 194–206. 10.1016/j.molcel.2016.03.036. PubMed DOI PMC

Cui Y.; Li X.; Lin J.; Hao Q.; Li X. D. Histone Ketoamide Adduction by 4-Oxo-2-nonenal Is a Reversible Posttranslational Modification Regulated by Sirt2. ACS Chem. Biol. 2017, 12, 47–51. 10.1021/acschembio.6b00713. PubMed DOI

Galligan J. J.; Rose K. L.; Beavers W. N.; Hill S.; Tallman K. A.; Tansey W. P.; Marnett L. J. Stable Histone Adduction by 4-Oxo-2-nonenal: A Potential Link between Oxidative Stress and Epigenetics. J. Am. Chem. Soc. 2014, 136, 11864–11866. 10.1021/ja503604t. PubMed DOI PMC

Dai L.; Peng C.; Montellier E.; Lu Z.; Chen Y.; Ishii H.; Debernardi A.; Buchou T.; Rousseaux S.; Jin F.; Sabari B. R.; Deng Z.; Allis C. D.; Ren B.; Khochbin S.; Zhao Y. Lysine 2-hydroxyisobutyrylation is a widely distributed active histone mark. Nat. Chem. Biol. 2014, 10, 365–370. 10.1038/nchembio.1497. PubMed DOI

Anderson K. A.; Huynh F. K.; Fisher-Wellman K.; Stuart J. D.; Peterson B. S.; Douros J. D.; Wagner G. R.; Thompson J. W.; Madsen A. S.; Green M. F.; Sivley R. M.; Ilkayeva O. R.; Stevens R. D.; Backos D. S.; Capra J. A.; Olsen C. A.; Campbell J. E.; Muoio D. M.; Grimsrud P. A.; Hirschey M. D. SIRT4 Is a Lysine Deacylase that Controls Leucine Metabolism and Insulin Secretion. Cell Metabol. 2017, 25, 838–855. 10.1016/j.cmet.2017.03.003. PubMed DOI PMC

Wagner G. R.; Bhatt D. P.; O’Connell T. M.; Thompson J. W.; Dubois L. G.; Backos D. S.; Yang H.; Mitchell G. A.; Ilkayeva O. R.; Stevens R. D.; Grimsrud P. A.; Hirschey M. D. A Class of Reactive Acyl-CoA Species Reveals the Non-enzymatic Origins of Protein Acylation. Cell Metabol. 2017, 25, 823–837. 10.1016/j.cmet.2017.03.006. PubMed DOI PMC

Moellering R. E.; Cravatt B. F. Functional lysine modification by an intrinsically reactive primary glycolytic metabolite. Science 2013, 341, 549–553. 10.1126/science.1238327. PubMed DOI PMC

Huang H.; Zhang Di.; Wang Y.; Perez-Neut M.; Han Z.; Zheng Y. G.; Hao Q.; Zhao Y. Lysine benzoylation is a histone mark regulated by SIRT2. Nat. Commun. 2018, 9, 337410.1038/s41467-018-05567-w. PubMed DOI PMC

Stevenson F. T.; Bursten S. L.; Locksley R. M.; Lovett D. H. Myristyl acylation of the tumor necrosis factor alpha precursor on specific lysine residues. J. Exp. Med. 1992, 176, 1053–1062. 10.1084/jem.176.4.1053. PubMed DOI PMC

Liu W.; Zhou Y.; Peng T.; Zhou P.; Ding X.; Li Z.; Zhong H.; Xu Y.; Chen S.; Hang H. C.; Shao F. Nε-fatty acylation of multiple membrane-associated proteins by Shigella IcsB effector to modulate host function. Nat. Microbiol. 2018, 3, 996–1009. 10.1038/s41564-018-0215-6. PubMed DOI PMC

Choudhary C.; Weinert B. T.; Nishida Y.; Verdin E.; Mann M. The growing landscape of lysine acetylation links metabolism and cell signalling. Nat. Rev. Mol. Cell Biol. 2014, 15, 536–550. 10.1038/nrm3841. PubMed DOI

Sabari B. R.; Zhang Di.; Allis C. D.; Zhao Y. Metabolic regulation of gene expression through histone acylations. Nat. Rev. Mol. Cell Biol. 2017, 18, 90–101. 10.1038/nrm.2016.140. PubMed DOI PMC

Seto E.; Yoshida M. Erasers of histone acetylation: the histone deacetylase enzymes. Cold Spring Harbor Perspect. Biol. 2014, 6, a01871310.1101/cshperspect.a018713. PubMed DOI PMC

Kutil Z.; Novakova Z.; Meleshin M.; Mikesova J.; Schutkowski M.; Barinka C. Histone Deacetylase 11 Is a Fatty-Acid Deacylase. ACS Chem. Biol. 2018, 13, 685–693. 10.1021/acschembio.7b00942. PubMed DOI

Moreno-Yruela C.; Galleano I.; Madsen A. S.; Olsen C. A. Histone Deacetylase 11 Is an ε-N-Myristoyllysine Hydrolase. Cell Chem. Biol. 2018, 25, 849–856. 10.1016/j.chembiol.2018.04.007. PubMed DOI

Cao J.; Sun L.; Aramsangtienchai P.; Spiegelman N. A.; Zhang X.; Huang W.; Seto E.; Lin H. HDAC11 regulates type I interferon signaling through defatty-acylation of SHMT2. Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 5487–5492. 10.1073/pnas.1815365116. PubMed DOI PMC

McCullough C. E.; Marmorstein R. Molecular Basis for Histone Acetyltransferase Regulation by Binding Partners, Associated Domains, and Autoacetylation. ACS Chem. Biol. 2016, 11, 632–642. 10.1021/acschembio.5b00841. PubMed DOI PMC

Sun L.; Marin de Evsikova C.; Bian K.; Achille A.; Telles E.; Pei H.; Seto E. Programming and Regulation of Metabolic Homeostasis by HDAC11. EBioMedicine 2018, 33, 157–168. 10.1016/j.ebiom.2018.06.025. PubMed DOI PMC

Huang J.; Wang L.; Dahiya S.; Beier U. H.; Han R.; Samanta A.; Bergman J.; Sotomayor E. M.; Seto E.; Kozikowski A. P.; Hancock W. W. Histone/protein deacetylase 11 targeting promotes Foxp3+ Treg function. Sci. Rep. 2017, 7, 862610.1038/s41598-017-09211-3. PubMed DOI PMC

Martin M. W.; Lee J. Y.; Lancia D. R.; Ng P. Y.; Han B.; Thomason J. R.; Lynes M. S.; Marshall C. G.; Conti C.; Collis A.; Morales M. A.; Doshi K.; Rudnitskaya A.; Yao L.; Zheng X. Discovery of novel N-hydroxy-2-arylisoindoline-4-carboxamides as potent and selective inhibitors of HDAC11. Bioorg. Med. Chem. Lett. 2018, 28, 2143–2147. 10.1016/j.bmcl.2018.05.021. PubMed DOI

in Son S.; Cao J.; Zhu C.-L.; Miller S. P.; Lin H. Activity-Guided Design of HDAC11-Specific Inhibitors. ACS Chem. Biol. 2019, 29210.1021/acschembio.9b00292. PubMed DOI PMC

Fan Y.; Scriba G. K. E. Electrophoretically mediated microanalysis assay for sirtuin enzymes. Electrophoresis 2010, 31, 3874–3880. 10.1002/elps.201000336. PubMed DOI

Ohla S.; Beyreiss R.; Scriba G. K. E.; Fan Y.; Belder D. An integrated on-chip sirtuin assay. Electrophoresis 2010, 31, 3263–3267. 10.1002/elps.201000220. PubMed DOI

Blackwell L.; Norris J.; Suto C. M.; Janzen W. P. The use of diversity profiling to characterize chemical modulators of the histone deacetylases. Life Sci. 2008, 82, 1050–1058. 10.1016/j.lfs.2008.03.004. PubMed DOI

Liu Y.; Gerber R.; Wu J.; Tsuruda T.; McCarter J. D. High-throughput assays for sirtuin enzymes: a microfluidic mobility shift assay and a bioluminescence assay. Anal. Biochem. 2008, 378, 53–59. 10.1016/j.ab.2008.02.018. PubMed DOI

Khan A. N.; Lewis P. N. Unstructured conformations are a substrate requirement for the Sir2 family of NAD-dependent protein deacetylases. J. Biol. Chem. 2005, 280, 36073–36078. 10.1074/jbc.M508247200. PubMed DOI

Du J.; Zhou Y.; Su X.; Yu J. J.; Khan S.; Jiang H.; Kim J.; Woo J.; Kim J. H.; Choi B. H.; He B.; Chen W.; Zhang S.; Cerione R. A.; Auwerx J.; Hao Q.; Lin H. Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase. Science 2011, 334, 806–809. 10.1126/science.1207861. PubMed DOI PMC

Marcotte P. A.; Richardson P. L.; Richardson P. R.; Guo J.; Barrett L. W.; Xu N.; Gunasekera A.; Glaser K. B. Fluorescence assay of SIRT protein deacetylases using an acetylated peptide substrate and a secondary trypsin reaction. Anal. Biochem. 2004, 332, 90–99. 10.1016/j.ab.2004.05.039. PubMed DOI

Tanner K. G.; Landry J.; Sternglanz R.; Denu J. M. Silent information regulator 2 family of NAD- dependent histone/protein deacetylases generates a unique product, 1-O-acetyl-ADP-ribose. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14178–14182. 10.1073/pnas.250422697. PubMed DOI PMC

Jackson M. D.; Denu J. M. Structural identification of 2′- and 3′-O-acetyl-ADP-ribose as novel metabolites derived from the Sir2 family of beta -NAD+-dependent histone/protein deacetylases. J. Biol. Chem. 2002, 277, 18535–18544. 10.1074/jbc.M200671200. PubMed DOI

Khan A. N.; Lewis P. N. Use of substrate analogs and mutagenesis to study substrate binding and catalysis in the Sir2 family of NAD-dependent protein deacetylases. J. Biol. Chem. 2006, 281, 11702–11711. 10.1074/jbc.M511482200. PubMed DOI

Borra M. T.; Denu J. M.. Quantitative Assays for Characterization of the Sir2 Family of NAD+-Dependent Deacetylases. In Chromatin and Chromatin Remodeling Enzymes; Allis C. D., Ed.; Elsevier: Amsterdam, 2004; pp 171–187. PubMed

McDonagh T.; Hixon J.; DiStefano P. S.; Curtis R.; Napper A. D. Microplate filtration assay for nicotinamide release from NAD using a boronic acid resin. Methods 2005, 36, 346–350. 10.1016/j.ymeth.2005.03.005. PubMed DOI

Hoffmann K.; Heltweg B.; Jung M. Improvement and validation of the fluorescence-based histone deacetylase assay using an internal standard. Arch. Pharm. 2001, 334, 248–252. 10.1002/1521-4184(200107)334:7<248::AID-ARDP248>3.0.CO;2-K. PubMed DOI

Rye P. T.; Frick L. E.; Ozbal C. C.; Lamarr W. A. Advances in label-free screening approaches for studying sirtuin-mediated deacetylation. J. Biomol. Screening 2011, 16, 1217–1226. 10.1177/1087057111420291. PubMed DOI

Fischer F.; Gertz M.; Suenkel B.; Lakshminarasimhan M.; Schutkowski M.; Steegborn C. Sirt5 deacylation activities show differential sensitivities to nicotinamide inhibition. PLoS One 2012, 7, e4509810.1371/journal.pone.0045098. PubMed DOI PMC

Gurard-Levin Z. A.; Kilian K. A.; Kim J.; Bähr K.; Mrksich M. Peptide arrays identify isoform-selective substrates for profiling endogenous lysine deacetylase activity. ACS Chem. Biol. 2010, 5, 863–873. 10.1021/cb100088g. PubMed DOI PMC

Gurard-Levin Z. A.; Kim J.; Mrksich M. Combining mass spectrometry and peptide arrays to profile the specificities of histone deacetylases. ChemBioChem 2009, 10, 2159–2161. 10.1002/cbic.200900417. PubMed DOI PMC

Kuo H.-Y.; DeLuca T. A.; Miller W. M.; Mrksich M. Profiling deacetylase activities in cell lysates with peptide arrays and SAMDI mass spectrometry. Anal. Chem. 2013, 85, 10635–10642. 10.1021/ac402614x. PubMed DOI PMC

Castaneda C. A.; Lopez J. E.; Joseph C. G.; Scholle M. D.; Mrksich M.; Fierke C. A. Active Site Metal Identity Alters Histone Deacetylase 8 Substrate Selectivity: A Potential Novel Regulatory Mechanism. Biochemistry 2017, 56, 5663–5670. 10.1021/acs.biochem.7b00851. PubMed DOI PMC

Kutil Z.; Skultetyova L.; Rauh D.; Meleshin M.; Snajdr I.; Novakova Z.; Mikesova J.; Pavlicek J.; Hadzima M.; Baranova P.; Havlinova B.; Majer P.; Schutkowski M.; Barinka C. The unraveling of substrate specificity of histone deacetylase 6 domains using acetylome peptide microarrays and peptide libraries. FASEB J. 2019, 33, 4035–4045. 10.1096/fj.201801680R. PubMed DOI

Rauh D.; Fischer F.; Gertz M.; Lakshminarasimhan M.; Bergbrede T.; Aladini F.; Kambach C.; Becker C. F. W.; Zerweck J.; Schutkowski M.; Steegborn C. An acetylome peptide microarray reveals specificities and deacetylation substrates for all human sirtuin isoforms. Nat. Commun. 2013, 4, 232710.1038/ncomms3327. PubMed DOI

Robers M. B.; Loh C.; Carlson C. B.; Yang H.; Frey E. A.; Hermanson S. B.; Bi K. Measurement of the cellular deacetylase activity of SIRT1 on p53 via LanthaScreen technology. Mol. BioSyst. 2011, 7, 59–66. 10.1039/C0MB00026D. PubMed DOI

Dudek J. M.; Horton R. A. TR-FRET biochemical assays for detecting posttranslational modifications of p53. J. Biomol. Screening 2010, 15, 569–575. 10.1177/1087057110365898. PubMed DOI

Machleidt T.; Robers M. B.; Hermanson S. B.; Dudek J. M.; Bi K. TR-FRET cellular assays for interrogating posttranslational modifications of histone H3. J. Biomol. Screening 2011, 16, 1236–1246. 10.1177/1087057111422943. PubMed DOI

Degorce F.; Card A.; Soh S.; Trinquet E.; Knapik G. P.; Xie B. HTRF: A technology tailored for drug discovery - a review of theoretical aspects and recent applications. Curr. Chem. Genomics 2009, 3, 22–32. 10.2174/1875397300903010022. PubMed DOI PMC

Inoue A.; Fujimoto D. Enzymatic deacetylation of histone. Biochem. Biophys. Res. Commun. 1969, 36, 146–150. 10.1016/0006-291X(69)90661-5. PubMed DOI

Kölle D.; Brosch G.; Lechner T.; Lusser A.; Loidl P. Biochemical methods for analysis of histone deacetylases. Methods 1998, 15, 323–331. 10.1006/meth.1998.0636. PubMed DOI

Taunton J.; Hassig C. A.; Schreiber S. L. A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p. Science 1996, 272, 408–411. 10.1126/science.272.5260.408. PubMed DOI

Milne J. C.; Lambert P. D.; Schenk S.; Carney D. P.; Smith J. J.; Gagne D. J.; Jin L.; Boss O.; Perni R. B.; Vu C. B.; Bemis J. E.; Xie R.; Disch J. S.; Ng P. Y.; Nunes J. J.; Lynch A. V.; Yang H.; Galonek H.; Israelian K.; Choy W.; Iffland A.; Lavu S.; Medvedik O.; Sinclair D. A.; Olefsky J. M.; Jirousek M. R.; Elliott P. J.; Westphal C. H. Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes. Nature 2007, 450, 712–716. 10.1038/nature06261. PubMed DOI PMC

Wolfson N. A.; Pitcairn C. A.; Sullivan E. D.; Joseph C. G.; Fierke C. A. An enzyme-coupled assay measuring acetate production for profiling histone deacetylase specificity. Anal. Biochem. 2014, 456, 61–69. 10.1016/j.ab.2014.03.012. PubMed DOI PMC

Fatkins D. G.; Monnot A. D.; Zheng W. Nepsilon-thioacetyl-lysine: a multi-facet functional probe for enzymatic protein lysine Nepsilon-deacetylation. Bioorg. Med. Chem. Lett. 2006, 16, 3651–3656. 10.1016/j.bmcl.2006.04.075. PubMed DOI

Heltweg B.; Dequiedt F.; Verdin E.; Jung M. Nonisotopic substrate for assaying both human zinc and NAD+-dependent histone deacetylases. Anal. Biochem. 2003, 319, 42–48. 10.1016/S0003-2697(03)00276-8. PubMed DOI

Toro T. B.; Watt T. J. KDAC8 substrate specificity quantified by a biologically relevant, label-free deacetylation assay. Protein science: a publication of the Protein. Society 2015, 24, 2020–2032. 10.1002/pro.2813. PubMed DOI PMC

Baba R.; Hori Y.; Mizukami S.; Kikuchi K. Development of a fluorogenic probe with a transesterification switch for detection of histone deacetylase activity. J. Am. Chem. Soc. 2012, 134, 14310–14313. 10.1021/ja306045j. PubMed DOI

Baba R.; Hori Y.; Kikuchi K. Intramolecular long-distance nucleophilic reactions as a rapid fluorogenic switch applicable to the detection of enzymatic activity. Chem. - Eur. J. 2015, 21, 4695–4702. 10.1002/chem.201406093. PubMed DOI

Rooker D. R.; Klyubka Y.; Gautam R.; Tomat E.; Buccella D. Peptide-Based Fluorescent Probes for Deacetylase and Decrotonylase Activity: Toward a General Platform for Real-Time Detection of Lysine Deacylation. ChemBioChem 2018, 19, 496–504. 10.1002/cbic.201700582. PubMed DOI

Liu X.; Xiang M.; Tong Z.; Luo F.; Chen W.; Liu F.; Wang F.; Yu R.-Q.; Jiang J.-H. Activatable Fluorescence Probe via Self-Immolative Intramolecular Cyclization for Histone Deacetylase Imaging in Live Cells and Tissues. Anal. Chem. 2018, 90, 5534–5539. 10.1021/acs.analchem.8b00709. PubMed DOI

Xie Y.; Ge J.; Lei H.; Peng B.; Zhang H.; Wang D.; Pan S.; Chen G.; Chen L.; Wang Y.; Hao Q.; Yao S. Q.; Sun H. Fluorescent Probes for Single-Step Detection and Proteomic Profiling of Histone Deacetylases. J. Am. Chem. Soc. 2016, 138, 15596–15604. 10.1021/jacs.6b07334. PubMed DOI

Yu C.; Wu Y.; Zeng F.; Li X.; Shi J.; Wu S. Hyperbranched polyester-based fluorescent probe for histone deacetylase via aggregation-induced emission. Biomacromolecules 2013, 14, 4507–4514. 10.1021/bm401548u. PubMed DOI

Dhara K.; Hori Y.; Baba R.; Kikuchi K. A fluorescent probe for detection of histone deacetylase activity based on aggregation-induced emission. Chem. Commun. 2012, 48, 11534–11536. 10.1039/c2cc36591j. PubMed DOI

Minoshima M.; Matsumoto T.; Kikuchi K. Development of a fluorogenic probe based on a DNA staining dye for continuous monitoring of the histone deacetylase reaction. Anal. Chem. 2014, 86, 7925–7930. 10.1021/ac501881s. PubMed DOI

Han Y.; Li H.; Hu Y.; Li P.; Wang H.; Nie Z.; Yao S. Time-resolved luminescence biosensor for continuous activity detection of protein acetylation-related enzymes based on DNA-sensitized terbium(III) probes. Anal. Chem. 2015, 87, 9179–9185. 10.1021/acs.analchem.5b01338. PubMed DOI

Halley F.; Reinshagen J.; Ellinger B.; Wolf M.; Niles A. L.; Evans N. J.; Kirkland T. A.; Wagner J. M.; Jung M.; Gribbon P.; Gul S. A bioluminogenic HDAC activity assay: validation and screening. J. Biomol. Screening 2011, 16, 1227–1235. 10.1177/1087057111416004. PubMed DOI

Dose A.; Jost J. O.; Spieß A. C.; Henklein P.; Beyermann M.; Schwarzer D. Facile synthesis of colorimetric histone deacetylase substrates. Chem. Commun. 2012, 48, 9525–9527. 10.1039/c2cc34422j. PubMed DOI

Riester D.; Hildmann C.; Grünewald S.; Beckers T.; Schwienhorst A. Factors affecting the substrate specificity of histone deacetylases. Biochem. Biophys. Res. Commun. 2007, 357, 439–445. 10.1016/j.bbrc.2007.03.158. PubMed DOI

Wegener D.; Hildmann C.; Riester D.; Schober A.; Meyer-Almes F.-J.; Deubzer H. E.; Oehme I.; Witt O.; Lang S.; Jaensch M.; Makarov V.; Lange C.; Busse B.; Schwienhorst A. Identification of novel small-molecule histone deacetylase inhibitors by medium-throughput screening using a fluorigenic assay. Biochem. J. 2008, 413, 143–150. 10.1042/BJ20080536. PubMed DOI

Ciossek T.; Julius H.; Wieland H.; Maier T.; Beckers T. A homogeneous cellular histone deacetylase assay suitable for compound profiling and robotic screening. Anal. Biochem. 2008, 372, 72–81. 10.1016/j.ab.2007.07.024. PubMed DOI

Wegener D.; Hildmann C.; Riester D.; Schwienhorst A. Improved fluorogenic histone deacetylase assay for high-throughput-screening applications. Anal. Biochem. 2003, 321, 202–208. 10.1016/S0003-2697(03)00426-3. PubMed DOI

Wegener D.; Wirsching F.; Riester D.; Schwienhorst A. A fluorogenic histone deacetylase assay well suited for high-throughput activity screening. Chem. Biol. 2003, 10, 61–68. 10.1016/S1074-5521(02)00305-8. PubMed DOI

Bradner J. E.; West N.; Grachan M. L.; Greenberg E. F.; Haggarty S. J.; Warnow T.; Mazitschek R. Chemical phylogenetics of histone deacetylases. Nat. Chem. Biol. 2010, 6, 238–243. 10.1038/nchembio.313. PubMed DOI PMC

Lahm A.; Paolini C.; Pallaoro M.; Nardi M. C.; Jones P.; Neddermann P.; Sambucini S.; Bottomley M. J.; Lo Surdo P.; Carfí A.; Koch U.; Francesco R.; de; Steinkühler C.; Gallinari P. Unraveling the hidden catalytic activity of vertebrate class IIa histone deacetylases. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 17335–17340. 10.1073/pnas.0706487104. PubMed DOI PMC

Toro T. B.; Bryant J. R.; Watt T. J. Lysine Deacetylases Exhibit Distinct Changes in Activity Profiles Due to Fluorophore Conjugation of Substrates. Biochemistry 2017, 56, 4549–4558. 10.1021/acs.biochem.7b00270. PubMed DOI PMC

Kawaguchi M.; Ikegawa S.; Ieda N.; Nakagawa H. A Fluorescent Probe for Imaging Sirtuin Activity in Living Cells, Based on One-Step Cleavage of the Dabcyl Quencher. ChemBioChem 2016, 17, 1961–1967. 10.1002/cbic.201600374. PubMed DOI PMC

Schuster S.; Roessler C.; Meleshin M.; Zimmermann P.; Simic Z.; Kambach C.; Schiene-Fischer C.; Steegborn C.; Hottiger M. O.; Schutkowski M. A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions. Sci. Rep. 2016, 6, 2264310.1038/srep22643. PubMed DOI PMC

Jiang H.; Khan S.; Wang Y.; Charron G.; He B.; Sebastian C.; Du J.; Kim R.; Ge E.; Mostoslavsky R.; Hang H. C.; Hao Q.; Lin H. SIRT6 regulates TNF-α secretion through hydrolysis of long-chain fatty acyl lysine. Nature 2013, 496, 110–113. 10.1038/nature12038. PubMed DOI PMC

Neelarapu R.; Holzle D. L.; Velaparthi S.; Bai H.; Brunsteiner M.; Blond S. Y.; Petukhov P. A. Design, synthesis, docking, and biological evaluation of novel diazide-containing isoxazole- and pyrazole-based histone deacetylase probes. J. Med. Chem. 2011, 54, 4350–4364. 10.1021/jm2001025. PubMed DOI PMC

Yu C.-W.; Chang P.-T.; Hsin L.-W.; Chern J.-W. Quinazolin-4-one derivatives as selective histone deacetylase-6 inhibitors for the treatment of Alzheimer’s disease. J. Med. Chem. 2013, 56, 6775–6791. 10.1021/jm400564j. PubMed DOI

Salvador L. A.; Park H.; Al-Awadhi F. H.; Liu Y.; Kim B.; Zeller S. L.; Chen Q.-Y.; Hong J.; Luesch H. Modulation of Activity Profiles for Largazole-Based HDAC Inhibitors through Alteration of Prodrug Properties. ACS Med. Chem. Lett. 2014, 5, 905–910. 10.1021/ml500170r. PubMed DOI PMC

Yao Y.; Tu Z.; Liao C.; Wang Z.; Li S.; Yao H.; Li Z.; Jiang S. Discovery of Novel Class I Histone Deacetylase Inhibitors with Promising in Vitro and in Vivo Antitumor Activities. J. Med. Chem. 2015, 58, 7672–7680. 10.1021/acs.jmedchem.5b01044. PubMed DOI

Chen Y.; Wang X.; Xiang W.; He L.; Tang M.; Wang F.; Wang T.; Yang Z.; Yi Y.; Wang H.; Niu T.; Zheng L.; Lei L.; Li X.; Song H.; Chen L. Development of Purine-Based Hydroxamic Acid Derivatives: Potent Histone Deacetylase Inhibitors with Marked in Vitro and in Vivo Antitumor Activities. J. Med. Chem. 2016, 59, 5488–5504. 10.1021/acs.jmedchem.6b00579. PubMed DOI

Spiegelman N. A.; Price I. R.; Jing H.; Wang M.; Yang M.; Cao J.; Hong J. Y.; Zhang X.; Aramsangtienchai P.; Sadhukhan S.; Lin H. Direct Comparison of SIRT2 Inhibitors: Potency, Specificity, Activity-Dependent Inhibition, and On-Target Anticancer Activities. ChemMedChem 2018, 13, 1890–1894. 10.1002/cmdc.201800391. PubMed DOI PMC

Kannan S.; Melesina J.; Hauser A.-T.; Chakrabarti A.; Heimburg T.; Schmidtkunz K.; Walter A.; Marek M.; Pierce R. J.; Romier C.; Jung M.; Sippl W. Discovery of inhibitors of Schistosoma mansoni HDAC8 by combining homology modeling, virtual screening, and in vitro validation. J. Chem. Inf. Model. 2014, 54, 3005–3019. 10.1021/ci5004653. PubMed DOI

Riester D.; Wegener D.; Hildmann C.; Schwienhorst A. Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates. Biochem. Biophys. Res. Commun. 2004, 324, 1116–1123. 10.1016/j.bbrc.2004.09.155. PubMed DOI

Roessler C.; Nowak T.; Pannek M.; Gertz M.; Nguyen G. T. T.; Scharfe M.; Born I.; Sippl W.; Steegborn C.; Schutkowski M. Chemical probing of the human sirtuin 5 active site reveals its substrate acyl specificity and peptide-based inhibitors. Angew. Chem., Int. Ed. 2014, 53, 10728–10732. 10.1002/anie.201402679. PubMed DOI

Zhang J. H. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J. Biomol. Screening 1999, 4, 67–73. 10.1177/108705719900400206. PubMed DOI

Auzzas L.; Larsson A.; Matera R.; Baraldi A.; Deschênes-Simard B.; Giannini G.; Cabri W.; Battistuzzi G.; Gallo G.; Ciacci A.; Vesci L.; Pisano C.; Hanessian S. Non-natural macrocyclic inhibitors of histone deacetylases: design, synthesis, and activity. J. Med. Chem. 2010, 53, 8387–8399. 10.1021/jm101092u. PubMed DOI

Zhou N.; Moradei O.; Raeppel S.; Leit S.; Frechette S.; Gaudette F.; Paquin I.; Bernstein N.; Bouchain G.; Vaisburg A.; Jin Z.; Gillespie J.; Wang J.; Fournel M.; Yan P. T.; Trachy-Bourget M.-C.; Kalita A.; Lu A.; Rahil J.; MacLeod A. R.; Li Z.; Besterman J. M.; Delorme D. Discovery of N-(2-aminophenyl)-4-(4-pyridin-3-ylpyrimidin-2-ylamino)methylbenzamide (MGCD0103), an orally active histone deacetylase inhibitor. J. Med. Chem. 2008, 51, 4072–4075. 10.1021/jm800251w. PubMed DOI

Arts J.; King P.; Mariën A.; Floren W.; Beliën A.; Janssen L.; Pilatte I.; Roux B.; Decrane L.; Gilissen R.; Hickson I.; Vreys V.; Cox E.; Bol K.; Talloen W.; Goris I.; Andries L.; Du Jardin M.; Janicot M.; Page M.; van Emelen K.; Angibaud P. JNJ-26481585, a novel “second-generation” oral histone deacetylase inhibitor, shows broad-spectrum preclinical antitumoral activity. Clin. Cancer Res. 2009, 15, 6841–6851. 10.1158/1078-0432.CCR-09-0547. PubMed DOI

Novotny-Diermayr V.; Sangthongpitag K.; Hu C. Y.; Wu X.; Sausgruber N.; Yeo P.; Greicius G.; Pettersson S.; Liang A. L.; Loh Y. K.; Bonday Z.; Goh K. C.; Hentze H.; Hart S.; Wang H.; Ethirajulu K.; Wood J. M. SB939, a novel potent and orally active histone deacetylase inhibitor with high tumor exposure and efficacy in mouse models of colorectal cancer. Mol. Cancer Ther. 2010, 9, 642–652. 10.1158/1535-7163.MCT-09-0689. PubMed DOI

Yang Z.; Wang T.; Wang F.; Niu T.; Liu Z.; Chen X.; Long C.; Tang M.; Cao D.; Wang X.; Xiang W.; Yi Y.; Ma L.; You J.; Chen L. Discovery of Selective Histone Deacetylase 6 Inhibitors Using the Quinazoline as the Cap for the Treatment of Cancer. J. Med. Chem. 2016, 59, 1455–1470. 10.1021/acs.jmedchem.5b01342. PubMed DOI

Santo L.; Hideshima T.; Kung A. L.; Tseng J.-C.; Tamang D.; Yang M.; Jarpe M.; van Duzer J. H.; Mazitschek R.; Ogier W. C.; Cirstea D.; Rodig S.; Eda H.; Scullen T.; Canavese M.; Bradner J.; Anderson K. C.; Jones S. S.; Raje N. Preclinical activity, pharmacodynamic, and pharmacokinetic properties of a selective HDAC6 inhibitor, ACY-1215, in combination with bortezomib in multiple myeloma. Blood 2012, 119, 2579–2589. 10.1182/blood-2011-10-387365. PubMed DOI PMC

Muthyala R.; Shin W. S.; Xie J.; Sham Y. Y. Discovery of 1-hydroxypyridine-2-thiones as selective histone deacetylase inhibitors and their potential application for treating leukemia. Bioorg. Med. Chem. Lett. 2015, 25, 4320–4324. 10.1016/j.bmcl.2015.07.065. PubMed DOI

Marek L.; Hamacher A.; Hansen F. K.; Kuna K.; Gohlke H.; Kassack M. U.; Kurz T. Histone deacetylase (HDAC) inhibitors with a novel connecting unit linker region reveal a selectivity profile for HDAC4 and HDAC5 with improved activity against chemoresistant cancer cells. J. Med. Chem. 2013, 56, 427–436. 10.1021/jm301254q. PubMed DOI

Cincinelli R.; Musso L.; Giannini G.; Zuco V.; Cesare M.; de; Zunino F.; Dallavalle S. Influence of the adamantyl moiety on the activity of biphenylacrylohydroxamic acid-based HDAC inhibitors. Eur. J. Med. Chem. 2014, 79, 251–259. 10.1016/j.ejmech.2014.04.021. PubMed DOI

Yung-Chi C.; Prusoff W. H. Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharm. 1973, 22, 3099–3108. 10.1016/0006-2952(73)90196-2. PubMed DOI

Comparative Structure-Based Virtual Screening Utilizing Optimized AlphaFold Model Identifies Selective HDAC11 Inhibitor

Comprehensive Mechanistic View of the Hydrolysis of Oxadiazole-Based Inhibitors by Histone Deacetylase 6 (HDAC6)

Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines

Synthesis, Molecular Docking and Biological Characterization of Pyrazine Linked 2-Aminobenzamides as New Class I Selective Histone Deacetylase (HDAC) Inhibitors with Anti-Leukemic Activity