Specific RNA sequences modified by a methylated adenosine, N⁶-methyladenosine (m⁶A), contribute to the post-transcriptional regulation of gene expression. The quantity of m⁶A in RNA is orchestrated by enzymes that write and erase it, while its effects are mediated by proteins that bind to read this modification. Dysfunction of this post-transcriptional regulatory process has been linked to human disease. Although the initial focus has been on pharmacological targeting of the writer and eraser enzymes, interest in the reader proteins has been challenged by a lack of clear understanding of their functional roles and molecular mechanisms of action. Readers of m⁶A-modified RNA (m⁶A-RNA) - the YTH (YT521-B homology) domain-containing protein family paralogs 1-3 (YTHDF1-3, referred to here as DF1-DF3) - are emerging as therapeutic targets as their links to pathological processes such as cancer and inflammation and their roles in regulating m⁶A-RNA fate become clear. We provide an updated understanding of the modes of action of DF1-DF3 and review their structures to unlock insights into drug design approaches for DF paralog-selective inhibition.

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 K_d = 185 nM; IC₅₀ = 1.46 µM (compound 2) and K_d = 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.

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 overexpression of RNA 6-N-methyladenosine (m ⁶ A) methyltransferase METTL16 has oncogenic role in the case of several cancer types, including leukemia, but efficient small-molecule inhibitors are not available. Initially identified by high-throughput virtual screening of the ZINC15 database in vivo subset, but then confirmed by measuring catalytic activity, two nanomolar-active METTL16 inhibitors, compounds 1 (IC ₅₀ = 25.82 ± 17.19 nM) and 2 (IC ₅₀ = 60.91 ± 2.75 nM) were found. The inhibitory activity of the compounds was measured using the m ⁶ A antibody-based ELISA assay. We also present the results on the effect of these inhibitors on the viability of promyeloblast HL-60 and lymphoblast CCRF-CEM leukemia cell lines. In unstressed growth conditions, both identified METTL16 inhibitors reduced the viability of HL-60 cells by up to 40%. The effect on the viability of CCRF-CEM cells was smaller with no dose dependency observed. In parallel, the level of the m ⁶ A as compared to unmodified adenosine in the HL-60 cell mRNAs was significantly reduced by the inhibitor 1 . Collectively, we herein demonstrate novel METTL16 inhibitors that exert tumor cell-lineage-selective antiproliferative effects.