Abstracts

Rationale: MicroRNA (miRNA) is a class of small non-coding RNA that silence target genes by degradation and/or translation inhibition of mRNA via a mechanism that involves binding to a specific sequence in the 3 UTR of the target mRNA. miRNA are highly expressed in the CNS and implicated in the regulation of neuronal differentiation and in the pathology of epilepsy. The most prevalent mRNA modification in eukaryotes is N-6-methyladenosine (m6A). m6A is enriched in brain tissue and is preferentially found in mature neurons and in adult brains. Many genes encoding m6A-containing RNA are linked to neurodevelopmental and neurological disorders. There is an association between the binding sites of miRNA and m6A residues in the 3 UTR. FTO is an enzyme that demethylates m6A in RNA substrates, is highly expressed in neurons, is essential for normal CNS development and is likely involved in the regulation of splicing and other processing of nuclear RNAs. We have recently synthesized a family of small molecules designed to inhibit FTO and related enzymes. One, 7d, was shown to inhibit purified FTO. We reasoned that inhibiting FTO may modulate specific miRNA that are involved in epilepsyMethods: 7d was screened for anticonvulsant activity (MES, scMET, 6Hz models) and toxicity (rotarod method) in animals by the NIH/NINDS/ASP. 7d was tested for ability to inhibit purified FTO and to increase m6A-RNA in HeLa cells. To assess for modulation of miRNA, total RNA was isolated from control and 7d-treated HeLa cells and analyzed by microarray (LC Sciences, LLC).Results: 7d had anticonvulsant activity in the 6Hz model with an anticonvulsant EC50 of 18 mg/kg and a toxic EC50 of 347 mg/kg, resulting in a safety ratio of 19.3. 7d inhibited purified FTO with an IC50 of 8.7 M. It also produced a 9.3% increase in m6A of total HeLa cell RNA, the expected result of FTO inhibition. Microarray analysis indicated that 7d modulated specific miRNA; some were up-regulated (p < 0.05 and p < 0.1) and some were down-regulated (p < 0.1).Conclusions: We have synthesized a CNS-penetrating small molecule that has anticonvulsant activity in animals, inhibits FTO and produces the expected increase in cellular m6A-RNA, and modulates levels of specific miRNA. This is the first description of an FTO inhibitor that penetrates the blood brain barrier. While we cannot state at this time that the inhibition of FTO is the cause of the anticonvulsant and/or miRNA modulating activities, these results are nevertheless compatible with the considerable emerging discussion of the connection between miRNA and epilepsy. Funding: JR and MO acknowledge support from Midwestern University, CPG.