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.