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

Abstract

Small‐molecule inhibitors of the human sirtuin SIRT2 are being developed because of their therapeutic potential in a variety of diseases. Here, we developed a high‐throughput screen to identify novel SIRT2 inhibitors using a fluorescent SIRT2 probe, 1‐aminoanthracene (AMA). AMA has high fluorescence when bound to SIRT2, and its fluorescence reduces >10‐fold when it is displaced from SIRT2 by other ligands. We used this property of AMA to screen a library of known bioactive compounds for SIRT2 binding and discovered two known pharmaceutical compounds that bind SIRT2 with Kd values in the low μM range, ascorbyl palmitate and pictilisib. Both compounds inhibit the deacetylase and defatty‐acylase activities of SIRT2. While pictilisib has selectivity for SIRT2, ascorbyl palmitate also inhibits the enzymatic activities of SIRT1 and SIRT6. Finally, we show that ascorbyl palmitate inhibits SIRT2 deacetylase and defatty‐acylase activities in cells, and SIRT2 inhibition by ascorbyl palmitate contributes to the cytotoxicity of the compound. Our work discovered novel SIRT2 deacylase inhibitors and presents a screening approach that can be applied on a larger scale.

Effects of Dimerization on the Deacylase Activities of Human SIRT2

“…13mer synthetic peptides containing the histone H4 sequence with Lys16 modified were used in cross-linking reactions and were described previously (H4K16 peptide sequence: KGLGKGGAK­(Acylation)­RHRK) . 16mer synthetic peptides containing an identical sequence with additional residues on the C-terminus were used in MALDI-based enzyme activity assays and were also described previously (sequence: KGLGKGGAK­(Acylation)­RHRKGWW). , The FAM-myristoyl-H4K16 peptide used in cross-linking, binding, and crystallography experiments contained the 13mer sequence; a fluorescein (FAM) group was attached to the N-terminal amine of the peptide with a PEG4 linker in between, and the peptide had a C-terminal carboxylic acid. The 28mer SIRT2 peptide corresponding to amino acids 286–313 contained the sequence NKEKAGQSDPFLGMIMGLGGGMDFDSKK with no modifications to the peptide.…”

“…The final concentrations of SIRT2 inhibitor added during initial cross-linking experiments were chosen based on their IC 50 values for inhibiting SIRT2 deacetylase activity in vitro to ensure that a large fraction of SIRT2 was bound to ligand during cross-linking. The IC 50 values for inhibiting SIRT2 deacetylase activity are as follows: thiomyristoyl (TM), 0.03–0.04 μM; , SirReal2, 0.14–0.16 μM; , ascorbyl palmitate, 3–17 μM; pictilisib, ∼3 μM; propofol, 140 μM . For this work, TM and SirReal2 were obtained from Selleck Chemicals, and our use of ascorbyl palmitate, pictilisib, and propofol was reported previously. , …”

“…The IC 50 values for inhibiting SIRT2 deacetylase activity are as follows: thiomyristoyl (TM), 0.03–0.04 μM; , SirReal2, 0.14–0.16 μM; , ascorbyl palmitate, 3–17 μM; pictilisib, ∼3 μM; propofol, 140 μM . For this work, TM and SirReal2 were obtained from Selleck Chemicals, and our use of ascorbyl palmitate, pictilisib, and propofol was reported previously. , …”

“…The cells grew for another 6 h before the media was removed, the cells were washed with PBS, then fixed with paraformaldehyde and processed as described above. We note that a 6 h treatment with 200 μM ascorbyl palmitate was previously shown to inhibit SIRT2 deacetylase and defatty-acylase activities in other cancer cell lines with minimal toxicity at that time point . Because ascorbyl palmitate was originally dissolved in DMSO, the cells were also exposed to 0.2% DMSO during the 6 h treatment; DMSO alone at this concentration was also added to control cells during the experiment.…”

“…By catalyzing the removal of protein acyl modifications, SIRT2 regulates diverse processes including cancer growth, neurodegeneration, metabolism, and inflammation. − The acyl substrates of SIRT2 are chemically diverse and range from small modifications such as acetyl-lysine to much larger, fatty modifications such as myristoyl-lysine . Numerous studies have characterized the substrate-specific enzymology of SIRT2. − Small molecules are being developed to selectively modulate SIRT2’s deacylase activities to understand how different acylations affect disease states and to explore SIRT2’s potential as a therapeutic target. ,− Additionally, novel acyl modifications on lysines continue to be identified as SIRT2 substrates, ,− which increases the challenge of modulating select SIRT2 activities and increases our need for tools that can probe specific SIRT2 functions.…”