Substrate-Dependent Modulation of SIRT2 by a Fluorescent Probe, 1-Aminoanthracene

Abstract

Sirtuin isoform 2 (SIRT2) is an enzyme that catalyzes the removal of acyl groups from lysine residues. SIRT2's catalytic domain has a hydrophobic tunnel where its substrate acyl groups bind. Here, we report that the fluorescent probe 1-aminoanthracene (AMA) binds within SIRT2's hydrophobic tunnel in a substrate-dependent manner. AMA's interaction with SIRT2 was characterized by its enhanced fluorescence upon protein binding (>10-fold). AMA interacted weakly with SIRT2 alone in solution (K d = 37 μM). However, when SIRT2 was equilibrated with a decanoylated peptide substrate, AMA's affinity for SIRT2 was enhanced ∼10-fold (K d = 4 μM). The peptide's decanoyl chain and AMA co-occupied SIRT2's hydrophobic tunnel when bound to the protein. In contrast, binding of AMA to SIRT2 was competitive with a myristoylated substrate whose longer acyl chain occluded the entire tunnel. AMA competitively inhibited SIRT2 demyristoylase activity with an IC 50 of 21 μM, which was significantly more potent than its inhibition of other deacylase activities. Finally, binding and structural analysis suggests that the AMA binding site in SIRT2's hydrophobic tunnel was structurally stabilized when SIRT2 interacted with a decanoylated or 4-oxononanoylated substrate, but AMA's binding site was less stable when SIRT2 was bound to an acetylated substrate. Our use of AMA to explore changes in SIRT2's hydrophobic tunnel that are induced by interactions with specific acylated substrates has implications for developing ligands that modulate SIRT2's substrate specificity.

High‐Throughput Screening Identifies Ascorbyl Palmitate as a SIRT2 Deacetylase and Defatty‐Acylase Inhibitor

“…Compared to its intensity in the absence of protein, AMA's fluorescence increased 7‐fold when equilibrated with 12 μM SIRT2 (Figure 1A). We determined a K d of ∼35 μM for SIRT2’s interaction with AMA based on the binding data generated in the 384‐well plate format, which matched our published K d of 37 μM from a cuvette‐based assay [9] …”

“…We miniaturized a cuvette‐based SIRT2‐AMA binding assay for use in high‐throughput screening to identify new compounds that interact with SIRT2. AMA has high fluorescence when bound to SIRT2, and its fluorescence significantly reduces when the probe is displaced from SIRT2 [9] . For SIRT2‐AMA binding assays, our buffer was PBS, and the final assay volume was 10 μl in 384‐well plates.…”

“…Most early SIRT2 inhibitors only affected its deacetylase activity without inhibiting its ability to remove longer acyl groups such as myristoyl from substrates [6,7] . More recent efforts identified compounds that also inhibit SIRT2’s demyristoylase/defatty‐acylase activity, but these compounds often lack SIRT2 selectivity or conventional drug‐like properties [4,8–10] . The synergistic benefit of inhibiting SIRT2 deacetylase and defatty‐acylase activities is largely unexplored due to the scarcity of defatty‐acylase inhibitors.…”

“…This allows detection of AMA binding to SIRT2 using simple fluorescence measurements. Because AMA's fluorescence becomes reduced when it is displaced from SIRT2, the probe is also useful in competition assays to identify other molecules that bind SIRT2 [9] . Interestingly, AMA is a competitive SIRT2 demyristoylase inhibitor and an allosteric deacetylase inhibitor [9] .…”

“…Because AMA's fluorescence becomes reduced when it is displaced from SIRT2, the probe is also useful in competition assays to identify other molecules that bind SIRT2 [9] . Interestingly, AMA is a competitive SIRT2 demyristoylase inhibitor and an allosteric deacetylase inhibitor [9] . This predicts that AMA competition assays should discover other molecules that also compete with long fatty acyl modifications for SIRT2 binding and act as defatty‐acylase inhibitors, and additionally, these same molecules might retain potency as deacetylase inhibitors.…”