Pharmacological Advantage of SIRT2-Selective versus pan-SIRT1-3 Inhibitors.

Because of their involvement in various biological pathways, the sirtuin enzyme family members SIRT1, SIRT2, and SIRT3 play both tumor-promoting and tumor-suppressing roles, based on the context and experimental conditions. Thus, an interesting question is whether inhibiting one of them or inhibiting all of them would be better for treating cancers. Pharmacologically, this is difficult to address, due in part to potential off-target effects of different compounds. Compounds with almost identical properties but differing in SIRT1-3 selectivity will be useful for addressing this question. Here, we have developed a pan SIRT1-3 inhibitor (NH4-6) and a SIRT2-selective inhibitor (NH4-13) with very similar chemical structures, with the only difference being the substitution of an ester bond to an amide bond. Such a minimal difference allows us to accurately compare the anticancer effect of pan SIRT1-3 inhibition and SIRT2-selective inhibition in cellular and mouse models. NH4-6 showed stronger cytotoxicity than NH4-13 in cancer cell lines. In mice, both inhibitors showed similar anticancer efficacy. However, NH4-6 is toxic to mice, which hinders the use of higher dosages. These results highlight the advantage of SIRT2-selective inhibitors as potential anticancer therapeutics.

Pharmacological and genetic perturbation establish SIRT5 as a promising target in breast cancer.

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.

Simultaneous Inhibition of SIRT2 Deacetylase and Defatty-Acylase Activities via a PROTAC Strategy.

As a member of the sirtuin family of enzymes, SIRT2 promotes tumor growth and regulates various biological pathways through lysine deacetylation and defatty-acylation. In the past few years, many SIRT2-selective small molecule inhibitors have been developed, but none have demonstrated simultaneous inhibition of both SIRT2 activities in cells. To further scrutinize the physiological importance and significance of SIRT2 deacetylase and defatty-acylase activities, small molecules that can selectively inhibit both activities of SIRT2 in living cells are needed. Here, we have applied the Proteolysis Targeting Chimera (PROTAC) strategy and synthesized a new SIRT2 inhibitor (TM-P4-Thal) to degrade SIRT2 selectively, which led to simultaneous inhibition of its deacetylase and defatty-acylase activities in living cells. Additionally, this compound exemplifies the advantage of the PROTAC strategy that allows complete eradication of an enzyme and its activity in biological settings.

A unifying paradigm for transcriptional heterogeneity and squamous features in pancreatic ductal adenocarcinoma.

Pancreatic cancer expression profiles largely reflect a classical or basal-like phenotype. The extent to which these profiles vary within a patient is unknown. We integrated evolutionary analysis and expression profiling in multiregion-sampled metastatic pancreatic cancers, finding that squamous features are the histologic correlate of an RNA-seq-defined basal-like subtype. In patients with coexisting basal and squamous and classical and glandular morphology, phylogenetic studies revealed that squamous morphology represented a subclonal population in an otherwise classical and glandular tumor. Cancers with squamous features were significantly more likely to have clonal mutations in chromatin modifiers, intercellular heterogeneity for MYC amplification and entosis. These data provide a unifying paradigm for integrating basal-type expression profiles, squamous histology and somatic mutations in chromatin modifier genes in the context of clonal evolution of pancreatic cancer.

A Glycoconjugated SIRT2 Inhibitor with Aqueous Solubility Allows Structure-Based Design of SIRT2 Inhibitors.

Small molecule inhibitors for SIRT2, a member of the sirtuin family of nicotinamide adenine dinucleotide-dependent protein lysine deacylases, have shown promise in treating cancer and neurodegenerative diseases. Developing SIRT2-selective inhibitors with better pharmacological properties is key to further realize the therapeutic potential of targeting SIRT2. One of the best SIRT2-selective inhibitors reported is a thiomyristoyl lysine compound called TM, which showed promising anticancer activity in mouse models without much toxicity to normal cells. The main limitations of TM, however, are the low aqueous solubility and lack of X-ray crystal structures to aid future drug design. Here, we designed and synthesized a glucose-conjugated TM (glucose-TM) analog with superior aqueous solubility. Although glucose-TM is not cell permeable, the excellent aqueous solubility allowed us to obtain a crystal structure of SIRT2 in complex with it. The structure enabled us to design several new TM analogs, one of which, NH4-6, showed superior water solubility and better anticancer activity in cell culture. The results of these studies provided important insights that will further fuel the future development of improved SIRT2 inhibitors as promising therapeutics for treating cancer and neurodegeneration.

Novel Lysine-Based Thioureas as Mechanism-Based Inhibitors of Sirtuin 2 (SIRT2) with Anticancer Activity in a Colorectal Cancer Murine Model.

Sirtuin 2 (SIRT2) is a protein lysine deacylase that has been indicated as a therapeutic target for cancer. To further establish the role of SIRT2 in cancers, it is necessary to develop selective and potent inhibitors. Here, we report the facile synthesis of novel lysine-derived thioureas as mechanism-based SIRT2 inhibitors with anticancer activity. Compounds AF8, AF10, and AF12 selectively inhibited SIRT2 with IC50 values of 0.06, 0.15, and 0.08 µM, respectively. Compounds AF8 and AF10 demonstrated broad cytotoxicity amongst cancer cell lines, but minimal toxicity in noncancerous cells. AF8 and AF10 inhibited the anchorage-independent growth of human colorectal cancer cell line HCT116 with GI50 values of ∼7 µM. Furthermore, AF8 potently inhibited tumor growth in a HCT116 xenograft murine model, supporting that SIRT2 is a viable therapeutic target for colorectal cancer.