Small-Molecule Inhibitors of the RNA M6A Demethylases FTO Potently Support the Survival of Dopamine Neurons

Abstract

The fat mass and obesity-associated protein (FTO), an RNA N6-methyladenosine (m6A) demethylase, is an important regulator of central nervous system development, neuronal signaling and disease. We present here the target-tailored development and biological characterization of small-molecule inhibitors of FTO. The active compounds were identified using high-throughput molecular docking and molecular dynamics screening of the ZINC compound library. In FTO binding and activity-inhibition assays the two best inhibitors demonstrated Kd = 185 nM; IC50 = 1.46 µM (compound 2) and Kd = 337 nM; IC50 = 28.9 µM (compound 3). Importantly, the treatment of mouse midbrain dopaminergic neurons with the compounds promoted cellular survival and rescued them from growth factor deprivation induced apoptosis already at nanomolar concentrations. Moreover, both the best inhibitors demonstrated good blood-brain-barrier penetration in the model system, 31.7% and 30.8%, respectively. The FTO inhibitors demonstrated increased potency as compared to our recently developed ALKBH5 m6A demethylase inhibitors in protecting dopamine neurons. Inhibition of m6A RNA demethylation by small-molecule drugs, as presented here, has therapeutic potential and provides tools for the identification of disease-modifying m6A RNAs in neurogenesis and neuroregeneration. Further refinement of the lead compounds identified in this study can also lead to unprecedented breakthroughs in the treatment of neurodegenerative diseases.

Regulation of N6-methyladenosine (m6A) RNA methylation in microglia-mediated inflammation and ischemic stroke

“…Moreover, increased FTO expression promoted m6A demethylation and induced apoptosis. These findings indicate that m6A modification and its regulatory proteins can control the survival of dopaminergic neurons and influence the incidence of PD ( Selberg et al, 2021 ).…”

Epitranscriptomics of Ischemic Heart Disease—The IHD-EPITRAN Study Design and Objectives

“…Considering the multileveled entanglement of epitranscriptomics with IHD pathophysiology, that is even suggested as a driver of some crucial steps of its pathogenesis [ 75 ], these modifications could also provide a novel source of therapeutic drug targets for IHD. Indeed, small molecule ligands for METTL3 writer complex [ 116 ], FTO [ 170 ], and ALKBH5 [ 171 ] erasers have already been described by the members of the IHD-EPITRAN Consortium. Remarkably, just recently, a potent METTL3 inhibitor has also been reported with leukemia-repressing effects in vivo in mice, providing simultaneously an enchanting proof-of-principle and a seminal endeavor to thrust the door ajar into the yet uncharted realm of epitranscriptomics-based pharmacology in vivo—ultimately shifting eyes also increasingly towards the clinic [ 172 ].…”

Recent Advances of m6A Demethylases Inhibitors and Their Biological Functions in Human Diseases

“…Since FTO and ALKBH5 rely on cofactors 2OG and Fe 2+ for their m6A demethylation activity, early studies focused on screening a series of 2OG analogues and related compounds as their inhibitors [ 97 ]. Structure-based virtual screening of different compound libraries was an important way to obtain potent FTO/ALKBH5 inhibitors [ 30 , 98 , 99 , 100 , 101 , 102 ]. Interestingly, a high-throughput fluorescence polarization (FP) assay was performed for compounds that competed with FTO/ALKBH5 for binding to m6A-containing single-stranded nucleic acids, and meclofenamic acid (MA) was found to be a selective inhibitor of FTO over ALKBH5 [ 103 ].…”

“…The structural model of FTO- 12 revealed that compound 12 coordinated with Fe 2+ in a bidentate manner, which was further stabilized by a combination of hydrogen-bonding and salt bridge interactions with Arg96, Arg319, Tyr295, and Ser318 of side chains from FTO. Two compounds 13a (IC 50 = 1.46 µM, Figure 3 ) and 13b (IC 50 = 28.9 µM, Figure 3 ) were defined as FTO inhibitors through a virtual screening on the ZINC compound library [ 99 ]. Molecular docking calculations revealed specific interactions between the amino acid residues of the FTO proteins Asp233, Tyr106, Glu234, Arg96, and Arg322, as well as two compounds.…”