Highlights d High-binding-efficiency ligands of METTL3-14-WTAP are identified in silico d The binding mode of compounds overlaps with the METTL3-14-WTAP active site d The compounds are experimentally characterized as METTL3-14-WTAP activators d The compounds affect m 6 A level in mRNA and rRNA in cells

The RNA 6-N-methyladenosine (m6A) demethylase ALKBH5 has been shown to be oncogenic in several cancer types, including leukemia and glioblastoma. We present here the target-tailored development and first evaluation of the antiproliferative effects of new ALKBH5 inhibitors. Two compounds, 2-[(1-hydroxy-2-oxo-2-phenylethyl)­sulfanyl]­acetic acid (3) and 4-{[(furan-2-yl)­methyl]­amino}-1,2-diazinane-3,6-dione (6), with IC50 values of 0.84 μM and 1.79 μM, respectively, were identified in high-throughput virtual screening of the library of 144 000 preselected compounds and subsequent verification of hits in an m6A antibody-based enzyme-linked immunosorbent assay (ELISA) enzyme inhibition assay. The effect of these compounds on the proliferation of selected target cancer cell lines was then measured. In the case of three leukemia cell lines (HL-60, CCRF-CEM, and K562) the cell proliferation was suppressed at low micromolar concentrations of inhibitors, with IC50 ranging from 1.38 to 16.5 μM. However, the effect was low or negligible in the case of another leukemia cell line, Jurkat, and the glioblastoma cell line A-172. These results demonstrate the potential of ALKBH5 inhibition as a cancer-cell-type-selective antiproliferative strategy.

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.

The N6-methyladenosine (m6A) modifications in both viral and host cell RNAs play an important role in HIV-1 virus genome transcription and virus replication. We demonstrate here that activators of the METTL3/METTL14/WTAP RNA methyltransferase complex enhance the production of virus particles in cells harboring HIV-1 provirus. In parallel, the amount of m6A residues in the host cell mRNA was increased in the presence of these activator compounds. Importantly, the m6A methylation of the HIV-1 RNA was also enhanced significantly (about 18%). The increase of virus replication by the small-molecule activators of the METTL3/METTL14/WTAP complex excludes them as potential anti-HIV-1 drug candidates. However, the compounds may be of large interest as activators for the latent HIV-1 provirus copies deposited in host cells’ genome and the subsequent virus eradication by an antiviral compound.

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N6-Methyladenosine (m6A) is the most common mRNA base modification in eukaryotes. Methylation of adenosine residues to m6A contributes to the regulation of splicing, transport, stability, and translation of mRNA and two main classes of enzymes regulate it. The formation of m6A is catalysed by a methyltransferase complex containing methyltransferase-like 3 (METTL3), METTL14, and Wilms' tumour 1-associated protein (WTAP) as well as monomeric METTL16. Demethylation of m6A is catalysed by the fat mass and obesity-associated protein FTO and the RNA demethylase AlkB homolog 5 (ALKBH5). The m6A mRNA methylation dysregulation occurs in the nervous system and in Parkinson's disease (PD), but it remains poorly studied. Moreover, the role of m6A mRNA methylation in neuronal survival, neuroprotection, and neuroregeneration is unclear. We have earlier used high-throughput virtual screening of large compound libraries and identified four unique small-molecule ligands that activate m6A mRNA methylation by binding to the METTL3/14/WTAP complex and enhancing the binding of the methylation substrate SAM to nanomolar concentrations. Following this, we now discovered that two methyltransferase activators at 10 nM concentrations supported the survival and protected dopamine (DA) neurons in culture in growth factor deprivation and 6-hydroxydopamine (6-OHDA) neurotoxin models. In contrast, METTL3/14 inhibitor STM2457 triggered death of DA neurons. For clinical translation we also tested the most efficient compound C4 on induced pluripotent stem cell-derived human DA neurons and in animal model of Parkinson's disease (PD). C4 compound protected human DA neurons from 6-OHDA-induced cell death and increased neurite outgrowth and the number of processes demonstrating that it has both neuroprotective and neurorestorative properties. METTL3/14 activator C4 improved motor behaviour and protected DA neurons and their fibres faster and much more efficiently than GDNF in the rat 6-OHDA model of PD. These are the first specific activators of METTL3/14/WTAP and first demonstration that m6A regulators can protect and regenerate neurons. These data demonstrate that m6A mRNA methylation is a novel pathway regulating neuronal survival and regeneration.

The in situ scanning tunneling microscopy (STM) and electrochemical impedance spectroscopy studies of electrochemically polished Cd(0001) electrode have been performed in the 1-ethyl-3-methylimidazolium tetrafluoroborate (EMImBF4) ionic liquid within electrode potential region from -1.6 V to –0.9 V (vs. Ag|AgCl|EMImBF4). The in situ STM data show that there are no quick surface reconstruction processes and the ideal surface structure of Cd(0001), very similar to that previously observed in aqueous electrolyte, has been established.

The fat mass and obesity-associated protein (FTO), an RNA N6-methyladenosine (m6A) demethylase, is an important regulator of central nervous system development, neuronal signalling 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 mkM (compound 2) and Kd = 337 nM; IC50 = 28.9 mkM (compound 3). Importantly, the treatment of mouse midbrain dopamine neurons with the compounds promoted cellular survival and rescued them from growth factor deprivation induced apoptosis already at nanomolar concentrations. Moreover, these inhibitors demonstrated good blood-brain-barrier penetration in the model system, 31.7% and 30.8%, respectively. The compounds 2 and 3 protected dopamine neurons with greater potency than our recently developed alkylation repair homolog protein 5 (AlkBH5) m6A demethylase inhibitors. 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.