Continuous Sirtuin/HDAC (histone deacetylase) activity assay using thioamides as PET (Photoinduced Electron Transfer)-based fluorescence quencher

0548916 - BTÚ 2022 RIV NL eng J - Journal Article
Zessin, M. - Meleshin, M. - Simic, Z. - Kalbas, D. - Arbach, M. - Gebhardt, P. - Melesina, J. - Liebscher, S. - Bordusa, F. - Sippl, W. - Bařinka, Cyril - Schutkowski, M.
Continuous Sirtuin/HDAC (histone deacetylase) activity assay using thioamides as PET (Photoinduced Electron Transfer)-based fluorescence quencher.
Bioorganic Chemistry. Roč. 117, DEC 2021 (2021). ISSN 0045-2068. E-ISSN 1090-2120
Institutional support: RVO:86652036
Keywords : Sirtuin assay * HDAC11 assay * Thioamide
OECD category: Organic chemistry
Impact factor: 5.307, year: 2021
Method of publishing: Open access
https://www.sciencedirect.com/science/article/pii/S0045206821008026?via%3Dihub

Histone deacylase 11 and human sirtuins are able to remove fatty acid-derived acyl moieties from the epsilon-amino group of lysine residues. Specific substrates are needed for investigating the biological functions of these enzymes. Additionally, appropriate screening systems are required for identification of modulators of enzymatic activities of HDAC11 and sirtuins. We designed and synthesized a set of activity probes by incorporation of a thioamide quencher unit into the fatty acid-derived acyl chain and a fluorophore in the peptide sequence. Systematic variation of both fluorophore and quencher position resulted super-substrates with catalytic constants of up to 15,000,000 M(-1)s(-1) for human sirtuin 2 (Sirt2) enabling measurements using enzyme concentrations down to 100 pM in microtiter plate-based screening formats. It could be demonstrated that the stalled intermediate formed by the reaction of Sirt2-bound thiomyristoylated peptide and NAD(+) has IC50 values below 200 pM.
Permanent Link: http://hdl.handle.net/11104/0324965