Design, Synthesis And Activity Evaluation Of Novel FTO Small Molecule Inhibitors

Obesity is a major health problem worldwide currently. The prevalence of obesity is increasing in both developed and developing countries. Obesity is not a single disease, it is associated with many pathological complications including respiratory, endocrine, cardiovascular disease, type 2 diabetes and several cancers.The fat mass and obesity associated protein(FTO) has been identified to be associated with obesity in several genome-wide-association studies, However, whether it is a valid drug target remains ambiguous. Thus, developing FTO-specific inhibitors is of great importance to evaluate the therapeutic potential of FTO. Following the FTO crystal structure resolution in 2010, the development of FTO-specific inhibitors has drawn much attention from pharmaceutical chemists. By now, FTO inhibitors such as rhein, 2-OG analogues, meclofenamic acid and other nonspecific inhibitors have been reported, and structural studies indicated that most of these inhibitors target the conserved 3-me U- or 2-OG-recognizing site of FTO nonspecificly. The identification of the new binding site offers new opportunities for further development of specific inhibitors of FTO.In this dissertation, we designed and synthesized 51 compounds containing the structural motif of benzene-1,3-diol based on the lead compound 3-me T. All these new synthesized compounds with purities of at least 95% were characterized by NMR, 13 C NMR and HRMS. In addition, we studied the inhibition effect on the enzyme activity of FTO.The cytotoxicity and activity of the new synthetic compounds were examined firstly, and compounds 9a and 22 were selected for further activity evaluation. Then, the inhibitory activity of compounds 9a and 22 on FTO demethylation of the 15-mer ss RNA were tested, their IC50 values are 4.95 μM and 39.24 μM respectively.The dose-dependent inhibition of FTO by compounds 9a and 22 suggests they are competitive inhibitors of FTO. To further validate the interaction between FTO and compounds, we determined the binding affinity of compounds 9a and 22 using the isothermal titration calorimetry(ITC) technique. The ITC results showed that compounds 9a and 22 interact with FTO with dissociation constants of 13 μM and 1.0 μM respectively, suggesting that compounds 9a and 22 can inhibit the enzyme activity of FTO. In addition, the inhibitory activity of compounds 9a and 22 on FTO demethylation of m6 A Modified m RNA in Cells were tested. The results showed that compounds 9a and 22 can interact with FTO specificly, suggesting that compounds 9a and 22 can inhibit the enzyme activity of FTO. To analyze the structural basis for their interaction, we solved the crystal structures of 9a-FTO and 22-FTO. The strictures revealed novel binding sites for compounds 9a and 22, suggesting that compounds 9a and 22 can interact with FTO specificly. The studies show that compounds 9a and 22 are potential FTO-specific inhibitors.