Identification of Candidate Inhibitor of m6a-RNA Demethylase ALKBH5

Genetic information is preserved in DNA and expressed in the form of protein via messenger RNA (mRNA). Although recent studies showed that chemical modifications of DNA and RNA play an important role, the effects in details have not been elucidated yet. N6-methyladenosine (m⁶A), one of the most prevalent modifications in eukaryotic mRNA, was discovered in the 1970s. Enzymes and proteins which bind to chemically modified sites write, erase, and recognize m6A in DNA and RNA nucleotides, and thus affects many biological processes, such as cellular differentiation, body mass, and metabolism, indicating the role of m⁶A in obesity, cancer, and other human diseases. As erasers, ALKBH5 and FTO from the AlkB family have demethylase activities against m⁶A. FTO is known to be associated with obesity, and some specific inhibitors have already been reported. In contrast, although ALKBH5 was found to be related to the abnormal spermatozoa in mice, proliferation of ZIKA virus and HIV, specific inhibitors of this enzyme have been reported.

In this study, our aim was to identify compounds that can effectively inhibit ALKBH5, in order to contribute to treatment of infertility, ZIKA virus, HIV.

A pharmacologically/physiologically active substance library (120 compounds) derived from plants was used. ALKBH5, together with candidate compounds were added to react with single-stranded DNA containing m⁶A. Consequently, complementary strands were added in order to anneal, and then cleaved with a restriction enzyme. When ALKBH5 was inhibited, it was not demethylated and DNA was not cleaved, so the inhibition rate of the candidate compound was measured from the cleavage rate.

As a result, we selected 26 compounds with inhibition rate of 50% or more, and similar experiments of these selections were carried out for FTO. Finally, four compounds were hit as the candidates of ALKBH5 inhibitors with no inhibitory activities against FTO.