A Novel Continuous Assay for the Deacylase Sirtuin 5 and Other Deacetylases

Abstract

Sirtuins are NAD(+) dependent lysine deacylases involved in many regulatory processes like control of metabolic pathways, DNA repair, and stress response. Modulators of sirtuin activity are needed as tools for uncovering the biological function of these enzymes and as potential therapeutics. Systematic discovery of such modulators is hampered by the lack of efficient and simple continuous activity assays running at low sirtuin concentrations in microtiter plates. Here we describe an improved continuous sirtuin 5 assay based on the coupling of the sirtuin reaction to a proteolytic cleavage using internally fluorescence-quenched substrates. Systematic optimization of a carbamoyl phosphate synthetase 1 derived, glutarylated peptide yielded a Sirt5 substrate with k(cat)/K(M) value of 337,000 M(-1) s(-1), which represents the best sirtuin substrate described so far. These extraordinary substrate properties allowed reliable determination of Ki values for different inhibitors in the presence of only 10 nM sirtuin in microtiter plate format. Assay conditions could be transferred effectively to other lysine deacetylases, like sirtuin 2 and sirtuin 3, which now enables more efficient development of sirtuin targeting drugs.

Design of modular dual enzyme‐responsive peptides

“…Trypsin is a serine protease that cleaves at the C‐terminus of positively charged amino acids such as lysine but does not cleave if the ɛ‐amine of lysine is acetylated, or masked . Previously, trypsin has been used to indirectly measure histone deacetylase (HDAC) activity by monitoring trypsin digestion of lysine after deacetylation by HDAC . We designed our system to be dual enzyme‐responsive by modifying the ɛ‐amine of lysine with substrates for four different proteases: chymotrypsin, papain, and caspases 3 and 8 (Figure ).…”

Peptide‐Based Fluorescent Probes for Deacetylase and Decrotonylase Activity: Toward a General Platform for Real‐Time Detection of Lysine Deacylation

“…In light of the observed substrate promiscuity of HDACs, a number of fluorescent deacetylase activity assays have been modified for the assessment of deacylase activity toward other lysine modifications . In particular, the modular design of the current commercially available trypsin‐coupled deacetylation assays presents a useful starting point for the development of more general fluorogenic deacylation assays . These assays have many attractive attributes, including continuous detection, easy fluorescence quantification, and high‐throughput screening compatibility.…”

Identification of sirtuin 5 inhibitors by ultrafast microchip electrophoresis using nanoliter volume samples

“…While these assays are amenable to HTS, the close proximity of dye molecule and lysine residue has resulted in artifactual results during screening [20,28,29]. To avoid these limitations, groups have developed alternative assays for SIRT5 screening based on optical detection of nicotinamide formation [30], inclusion of fluorophore-quencher pairs in the substrate [31], high-performance liquid chromatrography-mass spectrometry (HPLC-MS) [5,32,33], and fluorescence-resonance energy transfer [34]. However, the number of compounds screened has been limited and the throughput required for large-scale screening has not been demonstrated.…”