SIRTUIN 2 AND SIRTUIN 6 MODULATORS AS CANCER THERAPEUTICS

Sirtuins (SIRTs) are a family of 7 NAD+-dependant enzymes with a deacetylase, deacylase and/or ADP-ribosyl transferase activity. They are involved in several physiological and pathological processes, including metabolism, inflammation and cancer. SIRT-2 and SIRT-6 have been linked to carcinogenesis either as tumour promoters or suppressors and their pharmacological modulation can be considered a promising strategy to modify cancer initiation and progression. Known SIRT-2 and SIRT-6 inhibitors face several issues, such as water solubility and selectivity for the intended sirtuin over the other isoforms. Therefore, it is still crucial to seek novel SIRT-2 and SIRT-6 inhibitors, that are potent and selective. In cancer, sirtuins often possess a dual function in tumorigenesis, behaving as oncopromoter or oncosuppressor, depending on the sirtuin and on the cancer type. In cutaneous squamous cell carcinoma (cSCC), both SIRT-2 and SIRT-6 present a controversial role. Further work therefore is still needed to clarify their functions in this type of cancer. The main goals of my PhD work were: - The discovery of novel SIRT-2 and SIRT-6 inhibitors; - The comparison of the pharmacological effects of the inhibition and activation of SIRT-6 in a skin cancer mouse model. In this thesis, the following stages of the PhD research are described: the development of an HPLC-based method for the evaluation of the enzymatic activity of different sirtuins; the screening of compounds for the discovery of novel SIRT-2 and SIRT-6 inhibitors, and the evaluation of their biological effects on recombinant sirtuins, and subsequently on SCC cells; the development of a stable formulation containing a SIRT-6 inhibitor or a SIRT-6 activator for topical use; the evaluation of the effects of SIRT-6 pharmacological modulation by topical application in a 2-step carcinogenesis mouse model. Our results indicate that thiazoles, pyrazolopyrimidines and benzimidazoles represent good scaffolds for the development of SIRT-2 inhibitors, with the discovery of several SIRT-2 and dual SIRT-1/SIRT-2 inhibitors. A selection of these compounds had also a cytotoxic effect on cSCC cells, with minor effects on healthy keratinocytes, hinting the oncogenic role of SIRT-2 in this type of cancer. In addition, few compounds with a quinazoline-2,4(1H,3H)-dione structure were identified as novel SIRT-6 inhibitors. These proved to be cell-membrane permeable and induced keratinocyte differentiation in cSCC cells, reproducing the pro-differentiating effects of SIRT-6 silencing on keratinocytes, observed by Lefort and colleagues. Last, by inhibiting pharmacologically SIRT-6 in a 7,12-dimethylbenz[a]anthracene (DMBA) - 12-O-tetradecanoylphorbol-13-acetate (TPA) induced skin cancer mouse model, skin cancer progression was delayed. In particular, by applying topically a SIRT-6 inhibitor (compound 2,4-Dioxo-N-(4-(pyridin-3-yloxy)phenyl)-1,2,3,4-tetrahydroquinazoline-6-sulfonamide, renamed in this thesis “S6”) with a preventive approach at the promotion stage, epithelial-mesenchymal transition (EMT), and thus carcinogenesis, resulted less advanced compared to untreated mice.