Sirtuin functions and modulation: from chemistry to the clinic

Many SIRT modulators, used alone or in combination with other epigenetic modulators or known drugs, have been described as having beneficial effects against neurodegeneration and cancer [41]. Identified in 2001 from a yeast-based screening for inhibitors of Sir2, splitomicin (1, Table 2), despite being substantially inactive against hSIRTs, has been the starting point for the development of several SIRT1/2 inhibitors [42]. Among them, the compound with a phenyl ring on 2-position and a bromine on 8-position indicated as HR-73 (2, Table 2) is a single-digit micromolar SIRT1i that inhibits HIV transcription through affecting the Tat protein acetylation [43]. In a series of splitomicin analogs carrying a phenyl ring on 3-position and different substituents on 8-position, compounds 3 and 4 (Table 2), respectively, revealed to be low micromolar SIRT2i able to exert antiproliferative effects and cause ?-tubulin hyperacetylation in MCF-7 cells [44]. Discovered in 2005, EX-527 (selisistat, 5, Table 2) was the first potent, selective (over SIRT2/3), and cell-permeable SIRT1i [45, 46]. Despite increasing p53 acetylation, EX-527 has no major effect on viability and proliferation in various tumors [47]. EX-527 is being developed for HD. A phase II clinical study recently demonstrated that EX-527 is safe and well tolerated in HD patients at plasma concentrations, providing benefit in non-clinical HD models [48]. Recently, EX-527 was also found to block the amplification of human papillomavirus via a SIRT1 inhibition-dependent mechanism [49]. Identified in 2006, cambinol (6, Table 2) is a moderate SIRT1/2i that induces hyperacetylation of p53, ?-tubulin, FOXO3a, and Ku70 in NCI-H460 and HeLa cancer cells; promotes apoptosis in BCL-6-expressing Burkitt lymphoma cells; and reduces tumor growth in a xenograft model [50]. It is also endowed with anticancer effects on hepatocellular carcinoma tumor models in vitro and in vivo [51] and reduces neuroblastoma formation in N-Myc transgenic mice [52]. These promising results stimulated various optimization efforts, leading to the identification of derivatives/analogs with increased potency/selectivity for either SIRT1 or SIRT2 [53–55]. Discovered through a focused library screening, AGK2 (7, Table 2) is a single-digit micromolar SIRT2i selective over SIRT1/3. Able to inhibit the deacetylation of ?-tubulin in HeLa cells, AGK2 rescues dopaminergic neurons from ?-synuclein toxicity and protects against PD in different PD models [25]. Likely through SIRT2 inhibition, AGK2 also induces caspase-3-dependent death in glioma cells [56]. A cell-based screen designed to detect p53 activators identified tenovin-1 (8, Table 2) and its water-soluble analog tenovin-6 (9, Table 2) as SIRTi. Tenovin-6 is a micromolar SIRT1-3i that increases the level of p53K382ac; shows cytotoxic effects on melanoma cells, delaying the growth of ARN8-derived xenograft tumors [57]; and induces apoptosis in gastric cancer cells [58] and in chronic myeloid leukemia cells, decelerating the disease progression in mice models [59]. Discovered in 2001 by a high-throughput cell-based screening, sirtinol (10, Table 2) is a micromolar ySir2 and hSIRT1/2 inhibitor endowed with various anticancer activities [60]. It induces senescence-like growth arrest in human MCF-7 and H1299 cells [61]; inhibits the growth of PC3, DU145, and HeLa cells, enhancing their chemosensitivity to cisplatin and camptothecin [62, 63]; and promotes significant growth inhibition or apoptosis in cells from adult T cell leukemia-lymphoma (ATL) [64]. Salermide (11, Table 2), which resulted from the med-chem optimization of sirtinol, is a moderate SIRT1/2i that induces cancer-specific pro-apoptotic effects on different tumor cells (mainly MOLT4, KG1A, SW480, and Raji) through reactivation of pro-apoptotic genes (CASP8, TNF, TNFRSF10B, and PUMA) previously repressed by SIRT1-mediated H4K16ac deacetylation [65] and through the upregulation of DR5 [66]. Salermide and its analogs also show potent antiproliferative effects on cancer stem cells [67] and exert cell protection effects on a C. elegans model of muscular dystrophy [68]. In infectious diseases (Schistosoma mansoni), salermide was the most potent among the tested SIRTi in inducing apoptosis and death of schistosomula, in separation of adult worm pairs and in reduction in egg laying, through inhibition of the S. mansoni specific sirtuin [69]. Identified in 2010 as result of cambinol manipulation, MC2141 (12, Table 2) is the prototype of a series of benzodeazaoxaflavins active as low micromolar SIRT1/2i. MC2141 displays significant pro-apoptotic and antiproliferative properties in different cancer cell lines, including cancer stem cells [70, 71]. Identified in 2012 in a virtual screening for inhibitors of the p53-MDM2/MDMX interaction, inauhzin (13, Table 2) is a (sub)micromolar SIRT1i selective over SIRT2/3 able to exert antiproliferative and tumor-specific pro-apoptotic effects on various cancer lines by activating and stabilizing p53. Inauhzin represses the growth of xenograft tumors derived from p53-harboring H460 and HCT116 cells [72]. The most potent SIRTi reported to date are thieno[3,2-d]pyrimidine-6-carboxamides that resulted from a SAR analysis of the hit compounds deriving from a DNA-encoded small molecule library screen [73]. Although the most potent compound in the series (14, Table 2) is active in the single-digit nanomolar range against SIRT1–3 and its binding mode has been elucidated by X-ray crystallography, no biological activities are currently known for these compounds. Very recently, SirReal2 (15, Table 2) was reported as a submicromolar SIRT2i with 1000-fold selectivity over SIRT1/3/4/5/6 [74]. Its potency and selectivity, as revealed by X-ray crystallography, are based on a ligand-induced structural rearrangement of the SIRT2 active site revealing a previously unexploited binding pocket. The SIRT2 inhibition capability of SirReal2 has been confirmed in HeLa cells by the induction of ?-tubulin hyperacetylation and the destabilization of the SIRT2 substrate BubR1. A subsequent SAR investigation on the prototype led to the development of some derivatives (16–20, Table 2) with improved SIRT2i potency [75].

An elegant fragment-based approach inspired by the structures of the SIRTi suramin and nicotinamide recently identified a nanomolar SIRT2-selective (over SIRT1/3) inhibitor (21, Table 2), which is able to induce clear time-dependent and dose-dependent hyperacetylation of ?-tubulin in MCF-7 cells and shows cytotoxic effects on some cancer cell lines [76]. In recent years, a series of chroman-4-one derivatives as low micromolar SIRT2i selective over SIRT1/3 has been reported [77, 78]. Among them, compounds 22 and 23 (Table 2) are the most attractive in terms of their overall pharmacological profile as they display good dose-dependent ?-tubulin hyperacetylation and significant antiproliferative effects on cancer cells (MCF-7 and A549). From the screening of a panel of polyglutamine aggregate inhibitors, AK-7 (24, Table 2) was identified in 2011 as a brain-permeable micromolar SIRT2 selective (over SIRT1/3) inhibitor able to reduce neuronal cholesterol biosynthesis [79]. In addition, AK-7 displays other SIRT2 inhibition-dependent neuroprotective effects on various models of HD [80] and PD [81], supporting the development of SIRT2i as potential therapeutics for these disorders. Very recently, by high-throughput screening employing self-assembled monolayer desorption/ionization mass spectrometry, the first submicromolar small molecule inhibitor of SIRT3, SDX-437 (25, Table 2), was identified. SDX-437 is 100-fold selective over SIRT1 and could be a useful tool for studying SIRT3 biology and a promising lead for future med-chem optimization campaigns [82]. By a high-throughput molecular docking screen, compound 26 (Table 2) was recently identified as the first micromolar small molecule inhibitor of SIRT6 with eightfold selectivity over SIRT1/2 [83]. Although this molecule needs optimization, it can be considered as a promising lead for the functional annotation of SIRT6 and the development of potential SIRT6-based therapeutics due to its ability to induce hyperacetylation of the SIRT6 substrate H3K9, to increase glucose uptake through the upregulation of GLUT-1, and to reduce TNF-? secretion (BxPC-3 cells).