Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines

Abstract

Lysine deacetylases, like histone deacetylases (HDACs) and sirtuins (SIRTs), are involved in many regulatory processes such as control of metabolic pathways, DNA repair, and stress responses. Besides robust deacetylase activity, sirtuin isoforms SIRT2 and SIRT3 also show demyristoylase activity. Interestingly, most of the inhibitors described so far for SIRT2 are not active if myristoylated substrates are used. Activity assays with myristoylated substrates are either complex because of coupling to enzymatic reactions or time-consuming because of discontinuous assay formats. Here we describe sirtuin substrates enabling direct recording of fluorescence changes in a continuous format. Fluorescence of the fatty acylated substrate is different when compared to the deacylated peptide product. Additionally, the dynamic range of the assay could be improved by the addition of bovine serum albumin, which binds the fatty acylated substrate and quenches its fluorescence. The main advantage of the developed activity assay is the native myristoyl residue at the lysine side chain avoiding artifacts resulting from the modified fatty acyl residues used so far for direct fluorescence-based assays. Due to the extraordinary kinetic constants of the new substrates (KM values in the low nM range, specificity constants between 175,000 and 697,000 M−1s−1) it was possible to reliably determine the IC50 and Ki values for different inhibitors in the presence of only 50 pM of SIRT2 using different microtiter plate formats.

Development of First-in-Class Dual Sirt2/HDAC6 Inhibitors as Molecular Tools for Dual Inhibition of Tubulin Deacetylation

“…To confirm the inhibition of Sirt2-mediated demyristoylation for our lead structure 33 , we also used a recently published biochemical activity assay that relies on the conversion of a peptide-based myristoylated substrate. 48 With this assay, which is known to be very sensitive, 48 we detected an IC 50 value of 0.88 ± 0.09 μM (SI, Figure S1 ), thus corroborating the inhibition of Sirt2-mediated demyristoylation for our dual Sirt2/HDAC6 inhibitor 33 .…”

“…As a second method to confirm the inhibition of Sirt2-mediated demyristoylation by 33 , we used a continuous Sirt2 demyristoylation assay as reported by Zessin et al 48 In brief, this assay was performed in a black 384-well fluorescence plate in a total volume of 40 μL of Sirt assay buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mg/mL MgCl 2 , 2 mg/mL BSA, pH 7.8). Dilution series of the inhibitor was done in DMSO.…”

“…Dilution series of the inhibitor was done in DMSO. A total of 2 μL of the inhibitor dilutions were incubated with 14 μL of the peptide substrate, referred to as F4 in Zessin et al 48 (final concentration 40 nM) and 14 μL of Sirt2 solution (final concentration 1 nM) for 5 min at room temperature. In addition to the inhibitor-containing samples, a sample without inhibitors was measured as a positive control, and a sample without enzymes was measured as a negative control.…”

Does Selection for Longevity in Acheta domesticus Involve Sirtuin Activity Modulation and Differential Response to Activators (Resveratrol and Nanodiamonds)?

“…Thus, the life history of this strain, including selection with the delay in reproduction, likely induced profound changes that support longevity at the level of regulatory proteins. Additional analysis of SIRT1 activity concerning its substrates, such as histones and transcription factors (e.g., p53, NF-κB, and FOXO), may help elucidate the observed changes [4,32,33]. Based on the information provided by Xuan et al (2017), the fluorescence studies involving SIRT1 with the EGFP-K85AcK probe illuminate its role in enhancing fluorescence in E. coli ∆cobB, particularly in response to the acetylated lysine modification (HibK) at position K85 [34].…”

Untitled

“…Previously reported high-throughput screens targeting SIRT2 focused on inhibiting its deacetylase activity [28][29][30] or identifying ligands that bind to a SIRT2-decanoyl-peptide complex [25]. New assays that are compatible with screening have been developed to detect inhibition of SIRT2's defatty-acylase activity [24,26,27,31]. Assays developed to study other defatty-acylases including other sirtuin isoforms and classical histone deacetylases (HDACs) may also be adapted to identify SIRT2 inhibitors [32].…”