Development of ratiometric sirtuin fluorescence probes via cleavage of a coumarin dye

Dysfunction of sirtuins (SIRTs), a family of NAD⁺-dependent histone deacetylases involved in epigenetic modulation of protein function and gene expression, is associated with both age-related metabolic diseases and cancers in mammals, and SIRT modulators are considered attractive therapeutic targets. However, although various SIRT detection methodologies have been developed, especially focusing on other enzymatic activities of SIRTs, such as long chain defatty-acylase activity, there has been little progress in developing methodologies capable of quantitatively evaluating SIRT activity. Herein, we present the first Förster resonance energy transfer (FRET)-based ratiometric fluorescence probes for SIRT1, containing fluorescein isothiocyanate (FITC) as a FRET acceptor, coumarin as a FRET donor and an H3K9 SIRT recognition peptide sequence. Cleavage by SIRT of firstly designed probe released the coumarin fluorophore and structural modification of the latter units yielded compounds with high SIRT1 selectivity and reactivity. We confirmed that these probes enabled the quantitative measurement of SIRT1 enzyme activity. They are expected to be useful tools in studies of epigenetic regulation mechanisms, and in high-throughput screening for drug discovery targeting SIRT.