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.

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We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34 + cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype agnostic and extends to RAS-and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML.SIgnIfIcAnce: Reducing SIRT5 activity is detrimental to the survival of AML cells regardless of genotype, yet well tolerated by healthy hematopoietic cells. In mouse models, disrupting SIRT5 inhibits AML progression. SIRT5 controls several metabolic pathways that are required for leukemia cell survival. These results identify SIRT5 as a therapeutic target in AML.

The sirtuin enzymes are potential drug targets for intervention in a series of diseases. Efforts to inhibit enzymes of this class with thioamide-and thiourea-containing, substrate-mimicking entities have produced a number of high-affinity binders. However, less attention has been dedicated to the investigation of the stability of these inhibitors under various conditions. Here, we provide evidence of unprecedented degree of cleavage of short-chain e-N-thioacyllysine modifications meant to target these sirtuins and further provide insights into the serum stability of compounds containing both thioamides and thioureas. Our study questions the utility short-chain thioamide-based inhibitors of sirtuins for drug development and points to mono-alkylated thiourea-based chemotypes as being more stable in human serum.

Sirtuin 2 (SIRT2) is a protein deacylase enzyme that removes acetyl groups and longer chain acyl groups from post-translationally modified lysine residues. Here, we developed small peptide-based inhibitors of its activity in living cells in culture.

Sirtuin 2 (SIRT2) is a protein deacylase enzyme that has been reported to remove both acetyl groups and longer chain acyl groups from lysine residues in post-translationally modified proteins. It affects diverse biological functions in the cell and has been considered a drug target in relation to both neurodegenerative diseases and cancer. Therefore, access to good chemical tool compounds are essential for the continued investigation of the complex function of this enzyme. Here, we report a collection of probes that are potent, selective, stable in serum, water soluble, amenable to cell culture experiments, and inhibit both SIRT2 deacetylation and demyristoylation. Compared to the current landscape of SIRT2 inhibitors, this is a unique ensemble of features built into a single compound.We expect the developed chemotypes to find broad applications in the interrogation of SIRT2 functions in both healthy and diseased cells to provide a foundation for the development of new therapeutics in the future.

Sirtuin 5 (SIRT5) is a protein lysine deacylase enzyme that regulates diverse biology by hydrolyzing ϵ‐N‐carboxyacyllysine posttranslational modifications in the cell. Inhibition of SIRT5 has been linked to potential treatment of several cancers but potent compounds with activity in cells have been lacking. Here we developed mechanism‐based inhibitors that incorporate isosteres of a carboxylic acid residue that is important for high‐affinity binding to the enzyme active site. By masking of the tetrazole moiety of the most potent candidate from our initial SAR study, we achieved potent and cytoselective growth inhibition for the treatment of SIRT5‐dependent leukemic cancer cell lines in culture. Thus, we provide an efficient, cellularly active small molecule that targets SIRT5, which can help elucidate its function and potential as a future drug target. This work shows that masked isosteres of carboxylic acids are viable chemical motifs for the development of inhibitors that target mitochondrial enzymes, which may have applications beyond the sirtuin field.

Sirtuins, a group of NAD-dependent deacylases, have emerged as the key connection between NAD metabolism and aging. This class of enzymes hydrolyzes a range of ε- N-acyllysine PTMs, and determining the repertoire of catalyzed deacylation reactions is of high importance to fully elucidate the roles of a given sirtuin. Here we have identified and produced two potential sirtuins from the probiotic bacterium Lactobacillus acidophilus NCFM. Screening more than 80 different substrates, covering 26 acyl groups on five peptide scaffolds, demonstrated that one of the investigated proteins, Sir2La, is a bona fide NAD-dependent sirtuin, catalyzing hydrolysis of acetyl-, propionyl-, and butyryllysine. Further substantiating the identity of Sir2La as a sirtuin, known sirtuin inhibitors, nicotinamide and suramin, as well as a thioacetyllysine compound inhibit the deacylase activity in a concentration-dependent manner. On the basis of steady-state kinetics, Sir2La showed a slight preference for propionyllysine (Kpro) over acetyllysine (Kac). For nonfluorogenic peptide substrates, the preference is driven by a remarkably low K (280 nM vs 700 nM, for Kpro and Kac, respectively), whereas k was similar (21 × 10 s). Moreover, while NAD is a prerequisite for Sir2La-mediated deacylation, Sir2La has a very high K for NAD compared to the expected levels of the dinucleotide in L. acidophilus. Sir2La is the first sirtuin from Lactobacillales and of the Gram-positive bacterial subclass of sirtuins to be functionally characterized. The ability to hydrolyze propionyl- and butyryllysine emphasizes the relevance of further exploring the role of other short-chain acyl moieties as PTMs.