Continuous Activity Assay for HDAC11 Enabling Reevaluation of HDAC Inhibitors

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F86652036%3A_____%2F19%3A00519605" target="_blank" >RIV/86652036:_____/19:00519605 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/68378050:_____/19:00579099

Výsledek na webu

<a href="https://pubs.acs.org/doi/10.1021/acsomega.9b02808" target="_blank" >https://pubs.acs.org/doi/10.1021/acsomega.9b02808</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1021/acsomega.9b02808" target="_blank" >10.1021/acsomega.9b02808</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Continuous Activity Assay for HDAC11 Enabling Reevaluation of HDAC Inhibitors

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

Continuous Activity Assay for HDAC11 Enabling Reevaluation of HDAC Inhibitors

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2019

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

ACS Omega

ISSN

2470-1343

e-ISSN

—

Svazek periodika

4

Číslo periodika v rámci svazku

22

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

10

Strana od-do

19895-19904

Kód UT WoS článku

000499133200042

EID výsledku v databázi Scopus

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