A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions

Abstract

Sirtuins are NAD+ dependent lysine deacylases involved in many regulatory processes such as control of metabolic pathways, DNA repair and stress response. Modulators of sirtuin activity are required as tools for uncovering the biological function of these enzymes and as potential therapeutic agents. Systematic discovery of such modulators is hampered by the lack of direct and continuous activity assays. The present study describes a novel continuous assay based on the increase of a fluorescence signal subsequent to sirtuin mediated removal of a fluorescent acyl chain from a modified TNFα-derived peptide. This substrate is well recognized by human sirtuins 1–6 and represents the best sirtuin 2 substrate described so far with a kcat/KM-value of 176 000 M−1s−1. These extraordinary substrate properties allow the first determination of Ki-values for the specific Sirt2 inhibitory peptide S2iL5 (600 nM) and for the quasi-universal sirtuin inhibitor peptide thioxo myristoyl TNFα (80 nM).

Lysine Deacetylases Exhibit Distinct Changes in Activity Profiles Due to Fluorophore Conjugation of Substrates

“…Other recently developed substrates allow deacetylation to be monitored in a single-step. 24,50,51 For some of these substrates, the dyes are positioned further away from the acetylated lysine than for the AMC substrates; 52,53 however, it will be important to determine how the configuration of these substrates affects KDAC activity. An assumption that AMC or other fluorophores might mimic large non-polar residues after the acetyllysine is clearly incorrect, as tryptophan in the +1 position causes distinctly different activity effects than AMC.…”

Sirtuin activators and inhibitors: Promises, achievements, and challenges

“…A potent Sirtuin inhibitor that exploits the C-site and which shows some isoform selectivity is Ex-527 (Table 1). It exploits the unique Sirtuin deacylation mechanism to inhibit SIRT1 strongly, SIRT2/3/6 more moderately, and not SIRT5 (Gertz et al, 2013; Napper et al, 2005; Schuster et al, 2016), and it entered a clinical trial for Huntington’s disease treatment (see below) (Sussmuth et al, 2015). The compound binds to the C-site and a hydrophobic patch next to it (“extended C-site”, ECS; Figure 3c) (Gertz et al, 2013; Zhao et al, 2013).…”

A Fluorescent Probe for Imaging Sirtuin Activity in Living Cells, Based on One‐Step Cleavage of the Dabcyl Quencher

“…Recently,S chutkowskia nd co-workersr eportedasimple methodology for detecting SIRT activity in which longchain acyl groups bearings mall quencher moieties-2-aminobenzoylamide groups-at the other end of the chain are directly cleaved by SIRT2. [15] We believe the discovery that Dabcyl lysinei saSIRT substrate further expands the substrate scope of SIRTsa nd that the longer excitation wavelengths of Dabcyl dyes should also be advantageous for activity measurements.…”