Mechanism‐Based Inhibitors of the Human Sirtuin 5 Deacylase: Structure–Activity Relationship, Biostructural, and Kinetic Insight

Abstract

The sirtuin enzymes are important regulatory deacylases in a variety of biochemical contexts and may therefore be potential therapeutic targets through either activation or inhibition by small molecules. Here, we describe the discovery of the most potent inhibitor of sirtuin 5 (SIRT5) reported to date. We provide rationalization of the mode of binding by solving co-crystal structures of selected inhibitors in complex with both human and zebrafish SIRT5, which provide insight for future optimization of inhibitors with more “drug-like” properties. Importantly, enzyme kinetic evaluation revealed a slow, tight-binding mechanism of inhibition, which is unprecedented for SIRT5. This is important information when applying inhibitors to probe mechanisms in biology.

An NAD+-Dependent Sirtuin Depropionylase and Deacetylase (Sir2La) from the Probiotic Bacterium Lactobacillus acidophilus NCFM

“…The two substrate-derived compounds, thioacetamide 6 (65) and thiourea 7 (Fig. 4a), contain chemotypes that form stalled intermediates in the active site of sirtuins (65,83,84,85,86). In accordance with previous strong inhibition of sirtuins by thioamidebased inhibitors, thioacetamide 6 was the most potent of the present investigated inhibitors.…”

“…The remaining substrates from series 2 and substrates from series 3-5 were reported earlier (44,60,61,70,104,105,106) and synthesized using a combination of solution-phase and solid-phase peptide synthesis (see the Supporting Information for references). Compound 6 has previously been reported (65) and compound 7 was synthesized from bis(1benzotriazolyl)methanethione, methyl amine, and Cbz-Lys-Trp-NHiPr•TFA using a protocol reported in the same paper (see the Supporting Information for full experimental details).…”

Sirtuin 5 Deficiency Does Not Compromise Innate Immune Responses to Bacterial Infections

“…Thus, SIRT5 is a potential therapeutic target for several pathological conditions. Efforts are currently devoted to the generation of SIRT5 inhibitors such as thiosuccinyl peptides, cyclic pentapeptide harboring a central N(ε)-carboxyethyl-thiocarbamoyl-lysine residue and 3-arylthiosuccinylated and 3-benzylthiosuccinylated peptide derivatives ( 19 – 21 ) for specific cancer types ( 18 , 22 ).…”

Kinetic characterization of inhibitors of histone deacetylases (HDACs) and sirtuins

“…9,12,14,15 Standard substrates are Ac-Leu-Gly-Lys(Ac)-AMC (LGKac, for HDACs 1, 2, 4 AMC (RHKacKac, for HDAC8), 6,16 Ac-Glu-Thr-Asp-Lys(Myr)-AMC (ETDKmyr, for HDAC11 and SIRT2), 9,17 Ac-Glu-Thr-Asp-Lys(Ac)-AMC (ETDKac, for SIRT2), 9 and Ac-Leu-Gly-Lys(Suc)-AMC or Ac-Leu-Gly-Lys(Glu)-AMC (LGKsuc or LGKglu, for SIRT5). 10 Recombinant enzymes: 4. Plot data from duplicate wells as discrete data points, in order to identify outliers and experimental errors ("New Graph of Existing Data…", with the option "Create a new graph for each data set").…”

“…This assay helped identify previously unreported noncovalent irreversible properties of known HDAC inhibitors. 6,7 Moreover, this assay format has also been adapted for the kinetic characterization of both inhibitors and substrates of the sirtuins, 9,10 which are class III HDACs involved in metabolic control and oncogene regulation. 11 To characterize inhibition and evaluate potential time-dependent changes in inhibition, deacylase enzyme activity is recorded in a continuous manner with the use of peptide substrates containing a Cterminal ε-N-acyllysine residue coupled to the 7-amino-4-methylcoumarin (AMC) fluorophore.…”