title: A Fluorescent Probe for Imaging Sirtuin Activity in Living Cells, Based on One-Step Cleavage of the Dabcyl Quencher.
abstract: Sirtuins (SIRTs) are a family of NAD+-dependent histone deacetylases. In mammals, dysfunction of SIRTs is associated with age-related metabolic diseases and cancers, so SIRT modulators are considered attractive therapeutic targets. However, current screening methodologies are problematic, and no tools for imaging endogenous SIRT activity in living cells have been available until now. In this work we present a series of simple and highly sensitive new SIRT activity probes. Fluorescence of these probes is activated by SIRT-mediated hydrolytic release of a 4-(4-dimethylaminophenylazo)benzoyl (Dabcyl)-based FRET quencher moiety from the ϵ-amino group of lysine in a nonapeptide derived from histone H3K9 and bearing a C-terminal fluorophore. The probe SFP3 detected activities of SIRT1, -2, -3, and -6, which exhibit deacylase activities towards long-chain fatty acyl groups. We then truncated the molecular structure of SFP3 in order to improve both its stability to peptidases and its membrane permeability, and developed probe KST-F, which showed specificity for SIRT1 over SIRT2 and SIRT3. We show that KST-F can visualize endogenous SIRT1 activity in living cells.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi, 467-8603, Japan.
authors.forename: Mitsuyasu
authors.initials: M
authors.lastname: Kawaguchi
authors.affiliation: Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi, 467-8603, Japan.
authors.forename: Shohei
authors.initials: S
authors.lastname: Ikegawa
authors.affiliation: Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi, 467-8603, Japan.
authors.forename: Naoya
authors.initials: N
authors.lastname: Ieda
authors.affiliation: Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi, 467-8603, Japan. deco@phar.nagoya-cu.ac.jp.
authors.forename: Hidehiko
authors.initials: H
authors.lastname: Nakagawa
mesh_terms: D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000032:analysis*; D057809:HEK293 Cells; D006801:Humans; D015394:Molecular Structure; D056564:Sirtuin 1 / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; C447939:SIRT1 protein, human; D056564:Sirtuin 1
keywords: FRET; enzymatic reaction; fluorescent probes; imaging agents; quencher dyes; sirtuins