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Alzheimer’s disease (AD) is characterized by a progressive cognitive decline involving a multifactorial pathophysiology, including epigenetic dysregulation. Here, we report the discovery and preclinical validation of FLAV-27, a first-in-class, S-adenosyl-l-methionine (SAM)-competitive, brain-penetrant, and selective inhibitor of the histone methyltransferase G9a. Unlike prior G9a/GLP inhibitors, FLAV-27 exhibits subnanomolar potency, over 30-fold selectivity, and robust central nervous system bioavailability. Structural studies confirm a unique SAM-binding mode that confers superior specificity and avoids off-target effects. FLAV-27 reduces amyloid beta (Aβ) and p-tau aggregation and restores neuritic complexity in vitro. In Caenorhabditis elegans, it improves mobility, lifespan, and mitochondrial respiration. In mouse models of both late-onset AD (SAMP8) and early-onset AD (5xFAD), FLAV-27 rescues memory performance, social behavior, and synaptic structure. Multi-omics analyses reveal a global reprogramming of H3K9me2/H3K18me-mediated repression, reduced ferroptosis vulnerabilities, and normalization of AD-linked biomarkers, including SMOC1, H3K9me2, and p-Tau181, in the plasma and brain. Our findings position FLAV-27 as a promising epigenetic therapeutic with disease-modifying potential and translational biomarker alignment in AD.

One of the most abundant RNA modifications is N6-methyladenosine (m6A), which is present in RNA from all forms of life, including viruses. This modification has been detected in many types of RNAs, such as mRNA, ribosomal RNA, long non-coding RNAs, small nuclear RNAs, and microRNAs. Diverse sets of proteins have been characterized that methylate, demethylate, and specifically bind to this modification in different organisms. Caenorhabditis elegans is a unique model organism with abundant m6A modification, although its genome does not code for orthologs of the well-characterized m6A methyltransferase METTL3/METTL14 complex or the demethylases FTO and ALKBH5. Furthermore, orthologs of the YTH family of m6A reader proteins seem to be absent from the worm genome as well. To gain insights into how this modification is installed in this organism, we set out to identify enzymes that contribute to the abundant level of m6A in C. elegans.

mRNA modifications play important roles in regulating gene expression. One of the most abundant mRNA modifications is N6,2-O-dimethyladenosine (m6Am). Here, we demonstrate that m6Am is an evolutionarily conserved mRNA modification mediated by the Phosphorylated CTD Interacting Factor 1 (PCIF1), which catalyzes m6A methylation on 2-O-methylated adenine located at the 5′ ends of mRNAs. Furthermore, PCIF1 catalyzes only 5′ m6Am methylation of capped mRNAs but not internal m6A methylation in vitro and in vivo. To study the biological role of m6Am, we developed a robust methodology (m6Am-Exo-Seq) to map its transcriptome-wide distribution, which revealed no global crosstalk between m6Am and m6A under assayed conditions, suggesting that m6Am is functionally distinct from m6A. Importantly, we find that m6Am does not alter mRNA transcription or stability but negatively impacts cap-dependent translation of methylated mRNAs. Together, we identify the only human mRNA m6Am methyltransferase and demonstrate a mechanism of gene expression regulation through PCIF1-mediated m6Am mRNA methylation.