Development of Cell-Active N⁶-Methyladenosine RNA Demethylase FTO Inhibitor

Abstract

The direct nucleic acid repair dioxygenase FTO is an enzyme that demethylates N(6)-methyladenosine (m(6)A) residues in mRNA in vitro and inside cells. FTO is the first RNA demethylase discovered that also serves a major regulatory function in mammals. Together with structure-based virtual screening and biochemical analyses, we report the first identification of several small-molecule inhibitors of human FTO demethylase. The most potent compound, the natural product rhein, which is neither a structural mimic of 2-oxoglutarate nor a chelator of metal ion, competitively binds to the FTO active site in vitro. Rhein also exhibits good inhibitory activity on m(6)A demethylation inside cells. These studies shed light on the development of powerful probes and new therapies for use in RNA biology and drug discovery.

Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage

“…What these studies lack, however, is 2-fold: one, a profile of the target engagement of inhibitor and two, a full elucidation of the inhibitor mode of action, which should provide a better understanding of the biological consequences of the replication-blocking m 1 A and m 3 C lesions (30). As we reported in previous studies, the natural product rhein inhibits FTO demethylation of N 6 -methyladenine in vitro and elevates the level of N 6 -methyladenine within mRNA in HeLa cells (31,32). In this paper, we provide significant new data that describe the in vitro and in vivo effects of rhein as an inhibitor of AlkB repair enzymes.…”

“…BA is a moderate inhibitor of FTO (Fig. 1B) (31,49). AlkB repair remained intact, however, even in the presence of 100-fold excess BA (Fig.…”

Assignment of 2′-O-Methyltransferases to Modification Sites on the Mammalian Mitochondrial Large Subunit 16 S Ribosomal RNA (rRNA)

“…It is possible that modification of either U 1369 or G 1370 may be sufficient to support mitoribosome assembly and translation, so that there may be some redundancy in the retention of both enzymes. We have not ruled out the possibility that some of the methylations may be dynamic (reversible) as with m 6 A in cytoplasmic mRNA (57)(58)(59). Our in vitro methylation assays with purified protein and in vitro-synthesized RNA have not been successful (38).…”

Influence of the Ring Size on the Binding Ability of FTO Investigated by Fluorescence Spectroscopy

“…FTO protein, belonging to the Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase family, is an N-methyl nucleic acid demethylase acting on both single-stranded DNA and RNA substrates. In addition, it is reported that FTO involved in various disease, such as obesity, cardiovascular diseases, type II diabetes, heart disease and so on [2][3][4]. The recently reported crystal structure of FTO has offered insights into cofactors and substrate binding sites.…”

“…The recently reported crystal structure of FTO has offered insights into cofactors and substrate binding sites. Taken together with the extensive structural and mechanistic studies of AlkB family proteins, these studies enable the rational design and development of inhibitors targeting RNA demethylases [2]. There is an unmet medical need for new approaches to treating obesity.…”