Highlights d SIRT3 is highly expressed and linked to unfavorable outcome in DLBCL d SIRT3 is required for anaplerotic metabolism in DLBCL by enhancing GDH activity d Loss of Sirt3 impairs lymphomagenesis and prolongs survival of VavP-Bcl2 mice d Selective inhibition of SIRT3 by YC8-02 kills DLBCLs in vitro and in vivo

Sirtuin inhibitors have attracted much interest due to the involvement of sirtuins in various biological processes. Several SIRT2-selective inhibitors have been developed, and some exhibit anticancer activities. To facilitate the choice of inhibitors in future studies and the development of better inhibitors, we directly compared several reported SIRT2-selective inhibitors: AGK2, SirReal2, Tenovin-6, and TM. In vitro, TM is the most potent and selective inhibitor, and only TM could inhibit the demyristoylation activity of SIRT2. SirReal2, Tenovin-6, and TM all showed cytotoxicity in cancer cell lines, with Tenovin-6 being the most potent, but only TM showed cancer-cell-specific toxicity. All four compounds inhibited the anchorage-independent growth of HCT116 cells, but the effect of TM was most significantly affected by SIRT2 overexpression, suggesting that the anticancer effect of TM depends more on SIRT2 inhibition. These results not only provide useful guidance about choosing the right SIRT2 inhibitor in future studies, but also suggest general practices that should be followed for small-molecule inhibitor development activities.

SIRT2, a member of the sirtuin family of protein lysine deacylases, has been identified as a promising therapeutic target for treating cancer. In addition to catalyzing deacetylation, SIRT2 has recently been shown to remove fatty acyl groups from K‐Ras4a and promote its transforming activity. Among the SIRT2‐specific inhibitors, only the thiomyristoyl lysine compound TM can weakly inhibit the demyristoylation activity of SIRT2. Therefore, more potent small‐molecule SIRT2 inhibitors are needed to further evaluate the therapeutic potential of SIRT2 inhibition, and to understand the function of protein lysine defatty‐acylation. Herein we report a SIRT2 inhibitor, JH‐T4, which can increase K‐Ras4a lysine fatty acylation. This is the first small‐molecule inhibitor that can modulate the lysine fatty acylation levels of K‐Ras4a. JH‐T4 also inhibits SIRT1 and SIRT3 in vitro. The increased potency of JH‐T4 is likely due to the formation of hydrogen bonding between the hydroxy group and SIRT1, SIRT2, and SIRT3. This is further supported by in vitro studies with another small‐molecule inhibitor, NH‐TM. These studies provide useful insight for future SIRT2 inhibitor development.

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.

SIRT5 is a member of the sirtuin family of NAD +-dependent protein lysine deacylases implicated in a variety of physiological processes. SIRT5 removes negatively charged malonyl, succinyl, and glutaryl groups from lysine residues and thereby regulates multiple enzymes involved in cellular metabolism and other biological processes. SIRT5 is overexpressed in human breast cancers and other malignancies, but little is known about the therapeutic potential of SIRT5 inhibition for treating cancer. Here we report that genetic SIRT5 disruption in breast cancer cell lines and mouse models caused increased succinylation of IDH2 and other metabolic enzymes, increased oxidative stress, and impaired transformation and tumorigenesis. We, therefore, developed potent, selective, and cell-permeable small-molecule SIRT5 inhibitors. SIRT5 inhibition suppressed the transformed properties of cultured breast cancer cells and significantly reduced mammary tumor growth in vivo, in both genetically engineered and xenotransplant mouse models. Considering that Sirt5 knockout mice are generally normal, with only mild phenotypes observed, these data establish SIRT5 as a promising target for treating breast cancer. The new SIRT5 inhibitors provide useful probes for future investigations of SIRT5 and an avenue for targeting SIRT5 as a therapeutic strategy.