Abstract
FTO catalyzes the Fe(II) and 2-oxoglutarate (2OG)-dependent modification of nucleic acids, including the demethylation of N6-methyladenosine (m6A) in mRNA. FTO is a proposed target for anti-cancer therapy. Using information from crystal structures of FTO in complex with 2OG and substrate mimics, we designed and synthesized two series of FTO inhibitors, which were characterized by turnover and binding assays, and by X-ray crystallography with FTO and the related bacterial enzyme AlkB. A potent inhibitor employing binding interactions spanning the FTO 2OG and substrate binding sites was identified. Selectivity over other clinically targeted 2OG oxygenases was demonstrated, including with respect to the hypoxia-inducible factor prolyl and asparaginyl hydroxylases (PHD2 and FIH) and selected JmjC histone demethylases (KDMs). The results illustrate how structure-based design can enable the identification of potent and selective 2OG oxygenase inhibitors and will be useful for the development of FTO inhibitors for use in vivo.
Highlights
The fat mass and obesity associated protein (FTO) is an Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the oxidation of N-methyl groups in nucleic acids, and, in particular, of N6-methyladenosine (m6A) in RNA.[1−3] FTO is a potential medicinal chemistry target due to its roles in obesity and cancer
nucleic acid oxygenases (NAOXs) include eight AlkB homologues (ALKBH 1−8), some of which are involved in DNA damage repair (e.g., ALKBH2) and RNA oxidation (e.g., FTO and ALKBH5) and the Ten Eleven Translocation enzymes (TET1-3), which catalyze sequential oxidations of the methyl group of 5-methylcytosine.[15,16]
FTO catalyzes hydroxylation which can lead to demethylation of N-methyl groups on single-stranded RNA and DNA substrates (Figure 1A), including N3-methylthymidine (m3T), N3-methyluridine (m3U), m6A, and N6,2′O-dimethyladenosine (m6Am).[1−3,17] The lifetime of the nascent N-hydroxymethylated products/ intermediates varies depending on the nucleobase undergoing oxidation, leading to the proposal that in some circumstances they may be biologically relevant.[17−19]
Summary
The fat mass and obesity associated protein (FTO) is an Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the oxidation of N-methyl groups in nucleic acids, and, in particular, of N6-methyladenosine (m6A) in RNA.[1−3] FTO is a potential medicinal chemistry target due to its roles in obesity and cancer. Single nucleotide polymorphisms in an intron of the f to gene were reported to be associated with body weight and type II diabetes in genome-wide association studies.[4,5] More recent studies have reported roles for FTO in m6A regulation and cancers, including acute myeloid leukemia and glioblastoma.[6−11] Precisely how RNA oxidation by FTO is linked to disease is uncertain. There are some reports of FTO inhibition, including by known inhibitors of 2OG oxygenases, such as PHD inhibitors (Figure S1).[6,7,20−26] We have reported crystallographic and in vitro inhibition studies of FTO with known 2OG oxygenase inhibitors including 2OG competitors/analogues (2−4) and Received: July 6, 2021 Published: November 11, 2021
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