Development Of RNA Demethylase FTO Inhibitors And Preliminary Crystallographic Of The ALKBH5

N6-methyladenosine (m6A) is a ubiquitous modification found in mammalian mRNA and long non-coding RNA. M6A modification can affect diverse biological processes including mRNA splicing, nuclear exportation, stability, and translational efficiency. The m6A in RNA may be involved in adipogenesis, spermatogenesis, development, carcinogenesis, as well as other yet unidentified life processes.The fat mass and obesity-associated FTO gene is the first and most robust obesity-risk gene discovered in genome-wide association studies. In addition, FTO is reported to be involved in various disease processes, including cardiovascular diseases, type Ⅱ diabetes, Alzheimer’s disease, and breast cancer. Such discoveries make FTO an increasingly interesting target. FTO is able to catalyse the demethylation of3-methylthymine (3-meT) and3-methyluracil (3-meU) in ssDNA and ssRNA. The latest research results show that m6A in nuclear RNA may be a physiological substrate of FTO.The crystal structure of FTO in complex with3-meT (PDB code3LFM) was used as a docking target. Based on docking results of virtual screening,120compounds were synthesized or bought.Initially, we evaluate the inhibition of FTO demethylation on3-meT to screen120compounds. Three compounds (C1, C2, and C3) were found to be active for FTO. Two new compounds (C4and C5) were designed with chemical modification. A restriction endonuclease digestion assay was adopted to evaluate the inhibition of FTO demethylation on m6A in ssDNA by compounds. FTO repair activity was inhibited by5compounds in a concentration-dependent manner. We have determined the dose-dependent response of3compounds inhibition of FTO demethylation on an m6A-containing15-mer ssRNA. To evaluate the enzyme selectivity of inhibitors, we first examined its activity against several other AlkB human isoforms in vitro. We found that compound1and compound2can inhibited ALKBH2activity. The compound1also inhibited ALKBH5, another demethylase of m6A. We established a stable FTO over-expression293cell line and found that FTO over-expression decreased m6A content in mRNA in293cells, revealing that FTO also regulated m6A modification in293cells in an FTO activity dependent manner. After FTO over-expression293cells were respectively treated with compound1, compound3and compound4at a concentration of40μ M for24h, and the mRNA isolated from cells showed a notable increase of the modifying m6A level compared to untreated cells.Similar to FTO, ALKBH5has been identified as a mammalian m6A RNA demethylase in vitro and in vivo. ALKBH5is ubiquitously expressed in testicles and lung. The ALKBH5significantly affects nuclear mRNA export and RNA metabolism. ALKBH5-deficiency results in testis atrophy and reduction of sperm number and motility.The available data have unambiguously established the link between ALKBH5and RNA demethylation. But how ALKBH5specifically selects its substrates remains unknown. Structural study will provide insight into the underlying mechanism. Here, the ALKBH5protein was purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to2.4A resolution sing synchrotron radiations. The crystals belonged to the space group P212121, and one molecule in the asymmetric unit. Structural determination using Se-MAD is underway.