First-in-Class SAM-Competitive G9a Inhibitor FLAV-27 as a Disease-Modifying Therapy for Alzheimer’s Disease

First-in-Class SAM-Competitive G9a Inhibitor FLAV-27 as a Disease-Modifying Therapy for Alzheimer’s Disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article First-in-Class SAM-Competitive G9a Inhibitor FLAV-27 as a Disease-Modifying Therapy for Alzheimer’s Disease Christian Griñán-Ferré, Aina Bellver-Sanchis, David Valle-Garcia, and 35 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6940373/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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, 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. Biological sciences/Drug discovery/Medicinal chemistry/Drug discovery and development Biological sciences/Neuroscience/Epigenetics in the nervous system/Epigenetics and behaviour Alzheimer’s disease epigenetics cognition neuroprotection SAM-competitive inhibitor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Alzheimer’s disease (AD) is one of the most pressing and unresolved medical challenges of our time. Collectively affecting hundreds of millions of individuals worldwide, this neurodegenerative disorder is characterized by progressive neuronal loss and cognitive decline, without any effective disease-modifying treatments. 1 – 3 Despite intensive research, clinical progress has been limited by the multifactorial nature of the neurodegeneration, an incomplete understanding of its molecular underpinnings, and the formidable barrier posed by the blood–brain barrier (BBB) that restricts CNS drug delivery. 4 Emerging evidence implicates epigenetic dysregulation as a central contributor to the pathogenesis of neurodegenerative diseases. Unlike irreversible genetic mutations, epigenetic marks such as histone methylation are dynamic and potentially reversible, making them attractive therapeutic targets. 5 – 7 In particular, two histone methyltransferases (HMTs), GLP (EHMT2) and G9a (EHMT2), have attracted increasing attention due to their role in catalyzing the dimethylation of histone H3 at lysine 9 (H3K9me2), a repressive mark associated with transcriptional silencing. G9a/GLP-mediated epigenetic repression has been shown to influence critical processes such as neuronal development, synaptic plasticity, and memory consolidation. 8 – 11 Intriguingly, an aberrant upregulation of G9a activity has been linked to increased oxidative stress (OS), neuroinflammation, and neuronal dysfunction, which are hallmarks of AD and other neurodegenerative conditions. However, translating G9a inhibition into a viable therapeutic strategy has proven to be difficult. Most known G9a inhibitors, including BIX-01294, UNC0638, and A-366, suffer from poor selectivity, high cytotoxicity, and inadequate BBB permeability, which are limitations that are less critical in oncology but represent major obstacles for CNS applications. 12 Consequently, the therapeutic potential of G9a inhibition in neurodegeneration remains largely untapped. Here, we report the discovery and characterization of FLAV-27, a brain-penetrant, subnanomolar inhibitor of G9a with exceptional selectivity for G9a over the closely related GLP and other methyltransferases. Unlike previously reported G9a inhibitors, FLAV-27 exhibits favorable CNS drug-like properties, including excellent BBB permeability and a strong safety profile. Structural studies revealed a unique SAM-competitive binding mode, as demonstrated by the first X-ray co-crystal structure of FLAV-27 bound to human G9a. To evaluate its therapeutic potential, we employed a comprehensive, cross-species strategy incorporating primary neuronal and microglial cultures, the SAMP8 mouse model of AD, and Caenorhabditis elegans ( C. elegans ) models of neurodegeneration. The results showed that FLAV-27 reduced H3K9me2 levels, attenuated neuroinflammation, mitigated pathological protein aggregation, and restored cognitive and motor functions. In SAMP8 mice, FLAV-27 treatment led to marked improvements in memory performance, the reactivation of neuroprotective gene expression, and the suppression of the pro-inflammatory cytokine IL-1β. These effects were supported by converging evidence from behavioral, molecular, and epigenetic profiling. In C. elegans , FLAV-27 decreased Aβ aggregation, enhanced mitochondrial respiration, and extended the lifespan. Building on these promising findings, we further assessed the disease-modifying potential of FLAV-27 in the 5xFAD transgenic mouse model of early-onset AD, observing that FLAV-27 reversed the established cognitive and social deficits, reduced plaque burden, normalized markers of ferroptosis, and reprogrammed pathological proteomic signatures. Importantly, to assess clinical relevance, we analyzed human post-mortem brain tissue, cerebrospinal fluid (CSF), and plasma from AD patients. We found that the key epigenetic and molecular markers altered in animal models, namely H3K9me2, H3K18 methylation, and SMOC1, were significantly elevated in human AD samples. Furthermore, SMOC1 and H3K9me2 plasma levels correlated with tau pathology, neuroinflammatory markers, and cognitive decline, supporting their potential use as peripheral biomarkers of disease progression and therapeutic response. Together, our results position FLAV-27 as a first-in-class selective G9a inhibitor with disease-modifying potential for treating AD and related neurodegenerative disorders, highlighting the translational value of integrated epigenetics- and biomarker-based therapeutic strategies. RESULTS Building on prior efforts to identify novel epigenetic modulators, we undertook a structure-based design strategy to discover new G9a inhibitors. Interestingly, a promising chemotype emerged, featuring a central 1,4-oxazepane scaffold flanked by a piperidine ring and an aryl moiety. This scaffold served as the foundation for a medicinal chemistry campaign to optimize potency, selectivity, and brain penetration (see SI for details on synthesis, characterization, and structure-activity relationships, Tables S1-S5, Figure S1 A-E). In the initial evaluations, all compounds were analyzed as racemic mixtures. In the initial phase, modifications were introduced to the aryl ring attached to the piperidine. A wide range of substituents at the meta, para, and ortho positions were tested (Table S1 ). The majority of the analogs displayed weak or no activity (IC₅₀ > 1 µM), but several compounds, such as 2 (m-NH₂), 4 (m-CO₂CH₃), 10 (p-OH), 12 (p-Cl), and 17 (o-OH), showed inhibition in the low nanomolar range. These results pointed to the critical role of the m -hydroxyl group in achieving potent inhibition. Subsequently, the m -hydroxyphenyl group was replaced by various heterocycles (Table S2 ), but none of the resulting analogs retained nanomolar activity. Similarly, ring contraction of the piperidine to pyrrolidine abolished activity entirely, likely due to conformational changes and the generation of a new stereocenter. Further efforts focused on the oxazepane core. Replacement with a morpholine ring (Table S3) yielded analogs such as 32 and 35 with reduced but measurable activity. Methylation of the nitrogen atom within the oxazepane ring (Table S4) significantly diminished potency, as did modifications of the benzylic moiety. Among the latter, only compound 47 (p-chlorobenzyl) showed activity close to that of the reference compounds (IC₅₀ = 1.7 ± 0.1 nM; Table S5). Ultimately, the most promising compound emerged as a result of this iterative process. Compound 1, herein referred to as FLAV-27, featuring an m -hydroxyphenyl ring, a piperidine linker, a 1,4-oxazepane core, and a benzylic substituent, exhibited the highest potency (IC₅₀ = 0.6 ± 0.001 nM) and excellent selectivity over GLP (3.2% inhibition at 1 µM). FLAV-27 also outperformed standard G9a inhibitors such as UNC0638 (IC₅₀ = 11.1 ± 8 nM) and UNC0642 (IC₅₀ = 6.6 ± 4.7 nM), being more selective for G9a. It should be noted that neither UNC0642 nor UNC0638 are selective as it has been proven that they inhibit both G9a and GLP. 13 , 14 Moreover, an enantiomeric separation using semi-preparative chiral HPLC of FLAV-27 was performed, although no significant differences in ADME properties were observed (see SI for details on synthesis, characterization, and structure-activity relationships). A further relevant finding was the impressive selectivity profile. FLAV-27 demonstrated high selectivity for G9a, exhibiting < 15% inhibition at 1 µM across a panel of 32 HMTs, including H3K9 (SUV39H1, SUV39H2, and SUV420H1), H3K27 (EZH2), H3K4 (SETD7 and MLL), H3K79 (DOT1L), and H4K20 (SETD8), as well as various protein arginine methyltransferases (PRMTs) (Figure S1 F). In addition, FLAV-27 showed no inhibitory activity at 10 µM against a panel of 50 off-target kinases, supporting its clean selectivity profile (Figure S1 G). This result is highly relevant as other compounds that are considered selective inhibitors of methyl transferases, such as UNC0638 and UNC0642, inhibit both G9a and GLP. 13 X-ray crystallography examining the binding of FLAV-27 at the human G9a SAM-site G9a inhibitors can interfere with either the lysine-binding site or the binding site of S-adenosylmethionine (SAM), the methyl donor. Inhibitors targeting the latter are referred to as SAM-competitive. Enzyme kinetics conducted at a fixed concentration of the lysine-containing H3 peptide and increasing levels of SAM (0–7 µM) revealed a significant reduction of the maximum velocity (V max ) by FLAV-27 (Fig. 1 A). Consistent with its low IC 50 , the decrease in V max was already significant even at the lowest FLAV-27 concentration tested (1 nM) (Fig. 1 A). UNC0642, a non-selective G9a inhibitor, did not reduce the V max of the enzyme (Fig. 1 B), suggesting that its interaction with G9a differs mechanistically from that of FLAV-17. This implies that UNC0642 and FLAV-17 bind to distinct sites on the enzyme. Moreover, co-treatment with FLAV-27 and UNC0642 did not result in a synergistic inhibition of G9a activity (Figure S6C–D), further supporting the hypothesis of non-overlapping binding sites. To elucidate the precise binding mode of FLAV-27, we employed a structural biology approach and successfully resolved the crystal structure of the human G9a enzyme in complex with FLAV-27. Provided that FLAV-27 binds to the SAM-binding site, it would confer unprecedented selectivity and position FLAV-27 as the first G9a inhibitor to directly block co-substrate binding, thereby inhibiting both methyl transfer and overall enzymatic activity. Protein–inhibitor crystals were obtained under the conditions detailed in the methods (see SI for more details). Subsequent X-ray crystallographic analyses revealed a homodimeric architecture, with the inhibitor occupying the binding pocket of each protomer (Fig. 1 C-E, Table S6, Figure S2 ). The active site in the structure reported in Fig. 1 C is similar to the structure reported for the heterodimer formed by the suppressor of variegation 3 − 9, enhancer of zeste, and trithorax (SET) domain of G9a (residues 913 to 1193 of the human sequence) and the SET domain of GLP (residues 982 to 1266 of the human sequence) (PDB 5TTF). 15 In the reported G9a SET–SAM complex structure (PDB: 5TTF), the position of the SAM co-substrate is defined in Xiong et al. (2017). 15 Structural comparison between the G9a–FLAV-27 complex and the 5TTF crystal structure revealed that FLAV-27 occupies the same binding pocket as SAM (Fig. 1 D). The binding mode was, however, different as FLAV-27 binding is primarily driven by hydrophobic interactions (Figure S2 D-E), in contrast to SAM, which establishes a more extensive hydrogen bonding network (Fig. 1 E). Notably, the nitrogen atom of the piperidine ring in FLAV-27 forms hydrogen bonds with the hydroxyl group of Tyr1154 and the backbone carbonyl of Ser1084 (Fig. 1 D). These interactions are absent in the SAM-bound structure and may contribute to the enhanced stability of FLAV-27 binding. Additionally, the hydroxyl group of the phenol ring in FLAV-27 forms a hydrogen bond with the side chain hydroxyl of Tyr1085, an interaction that is also observed in the SAM complex. The phenol ring of FLAV-27 is further stabilized by hydrophobic contacts with Trp1050 and Phe1110 (Fig. 1 C), which are interactions that are not present in the G9a SET–SAM structure. These contacts may underlie the stronger binding of FLAV-27. Finally, the benzyl ring of FLAV-27 is deeply buried within a hydrophobic pocket in G9a, forming significant hydrophobic interactions. This increased hydrophobic contribution in the G9a–FLAV-27 complex likely explains its enhanced binding affinity and may underlie its greater inhibitory potency. In fact, Fig. 1 F-H and Figure S2 F-J explain the difference between G9a and GLP. The active sites of the G9a and GLP proteins look very similar, but the surface charge distribution is different. The surface charge distribution (SCD) of G9a clearly suggests that the SAM-binding site is hydrophobic with little negative charge distribution, while the GLP SCD for SAM binding is mostly positively charged. This could be a possible reason for why the hydrophobic FLAV-27 compound might favor binding to G9a instead of GLP. In vivo assessment of FLAV-27 efficacy In our previous experiments with the SAMP8 senescence model, we detected epigenetic alterations related to the methylation of histones. To better characterize the epigenetic landscape and simultaneously evaluate the in vivo efficacy of FLAV-27, we first assessed global histone methylation profiles in cortical tissue. As expected, SAMP8 mice treated with FLAV-27 exhibited markedly reduced levels of H3K9me2 in the brain (Fig. 2 A-B), confirming effective inhibition of the primary enzymatic activity of G9a. Given that G9a has also been implicated in catalyzing methylation at additional histone sites, we extended our analysis to other epigenetic marks. FLAV-27 treatment selectively reversed the aberrant levels of H3K18me1/2/3, modifications that have been previously linked to G9a function, 16 without having detectable effects on H3K23me1/2/3 (Fig. 2 A-B). To gain a genome-wide perspective on these epigenetic changes, ChIP-seq analysis for H3K9me2 was performed in the cortical tissue of control SAMP8 mice and SAMP8 mice treated with FLAV-27. This approach enabled us to map the epigenetic changes induced by FLAV-27 and to identify the genomic regions where the G9a-regulated repressive histone modification H3K9me2 is dynamically altered in the context of aging. Our results revealed a clear global decrease in H3K9me2 levels in FLAV-27-treated samples, as illustrated in Fig. 2 C. Furthermore, across different false discovery rate (FDR) thresholds during peak calling, we observed that FLAV-27-treated samples consistently presented fewer H3K9me2 peaks (Figure S3A), with the remaining peaks showing significantly lower H3K9me2 enrichment (Fig. 2 C-D). While the majority of the peaks exhibited reduced methylation after FLAV-27 treatment (Fig. 2 E), we focused specifically on the subset of peaks that showed the strongest H3K9me2 decrease (Fig. 2 F-G). To investigate the functional impact of these changes, we identified the genes whose promoters were closest to these downregulated H3K9me2 peaks (Fig. 2 H). We then performed gene set enrichment and ontology analyses on this gene set. Validating our approach, these genes showed overlap with known repressive histone modifications (H3K9me3 and H3K27me3) from the ENCODE datasets (Figure S3B), supporting the notion that FLAV-27 broadly affects heterochromatin organization. Strikingly, there was a notable enrichment of GO terms associated with synaptic structure and function after FLAV-27 treatment (Fig. 2 I). Pathways linked to synaptic plasticity and structural synapse development, such as "synapse assembly”, "regulation of synapse structure or activity", and "positive regulation of synapse assembly”, were significantly upregulated in the FLAV-27-treated SAMP8 mice, according to the GO enrichment analysis. These pathways, which regulate dendritic growth, receptor trafficking, and synaptic potentiation, involve important genes like Gria1 , Shank3 , Snap25 , Slitrk5/6 , and Dlgap1 . An enrichment was also observed of pre-synapse and post-synapse organization terms highlighting a coordinated reconstruction of both ends of the synaptic cleft that is necessary for effective neurotransmission. The presence of other GO terms like "cognition" and "regulation of synaptic plasticity" that directly align with higher-order neurological processes in the FLAV-27-treated group suggested possible behavioral and memory-level rescue (Fig. 2 I). Conversely, maladaptive neuroplasticity and the suppression of inhibitory synaptic signaling were found in the GO pathways downregulated in the FLAV-27-treated group. The terms "inhibitory synapse assembly", "GABAergic synaptic transmission", and "GABA signaling", which involve genes like Gabra1 , Gad1 , Gabrg2 , and Slc6a1 , were significantly enriched. This indicates that FLAV-27 improves synaptic plasticity and the excitatory/inhibitory rebalance by suppressing dysfunctional inhibitory circuits. Indirect epigenetic silencing of neuroinflammatory pathways by FLAV-27 is suggested by the repression of several genes associated with inflammation and oxidative stress. The suppression of redox-sensitive genes indicates possible dampening of pro-degenerative oxidative pathways, even though ferroptosis itself was not enriched. The downregulation of the terms associated with inhibitory and inflammatory signals represents a neuroprotective response (Fig. 2 J). Furthermore, many sequences near the hypomethylated regions are transcribed into non-coding RNAs (Fig. 2 K), suggesting that FLAV-27 may impact the expression of regulatory RNA species whose functions are still largely unknown. Strikingly, the genes associated with regions of H3K9me2 loss were enriched in functions related to serotonin signaling, the regulation of nervous system processes, anxiety, and behavioral responses to fear (Fig. 2 L-M), indicating that the central impairments previously reported in this senescence-accelerated SAMP8 model correlate with epigenetic traits. Complementary to these findings, western blot analysis of histone modifications in human post-mortem brain tissue was performed. Increased levels of H3K9me2 and H3K18 mono-, di-, and tri-methylation were observed in the AD brains, with no significant changes detected in H3K23 methylation states (Fig. 2 N-O). Modulation of Aβ₁₋₄₂, tau, and p-tau aggregation and neuritic integrity in primary neuronal cultures AD is characterized by the accumulation of toxic protein aggregates, including Aβ and hyperphosphorylated tau (p-tau), which disrupt neuronal function and contribute to synaptic degeneration and cognitive decline. To initially explore the potential neuroprotective properties of FLAV-27, in vitro experiments were conducted using mixed neuronal and microglial primary cultures treated with Aβ 1−42 (500 nM), tau (1 µM), or p-tau (1 µM) protein aggregates for 48 hours, before exposure to FLAV-27 (1 µM) for 24 h. Results demonstrated a significant reduction in Aβ 1−42 , tau, and p-tau aggregation after FLAV-27 treatment (Fig. 3 A-C). Additionally, neurite patterning was analyzed by immunocytochemistry in primary cortical neuronal cultures under the same treatment conditions. The data revealed a marked loss of neurite formation after exposure to Aβ 1−42 , tau, or p-tau aggregates, which was largely reversed by FLAV-27 treatment (Fig. 3 D-F). Reversion of cognitive impairments in a worm and a mouse model Reversion of cognitive impairments in a worm and a mouse model We investigated whether the selective inhibition of G9a would be efficacious in restoring central impairments by using FLAV-27 and non-mammalian and mammalian models of AD that are widely accepted as tools to test the efficacy of potential anti-AD drugs. First, FLAV-27 was tested on the CL2006 transgenic strain of C. elegans , which develops age-related paralysis upon expression of human Aβ 1−42 in the muscle cells. 17 , 18 FLAV-27 treatment reduced locomotor impairment (Fig. 3 G), indicating its potential to alleviate Aβ-associated phenotypes. Notably, CL2006 worms exhibit elevated H3K9me2 levels and increased expression of set-25 , the ortholog of human G9a/EHMT2, linking epigenetic dysregulation to Aβ pathology. 19 Treatment with FLAV-27 (1 µM) significantly reduced the H3K9me2/H3 ratio compared to vehicle, while the reference inhibitor UNC0638 showed only a non-significant trend towards reduction (Fig. 3 H-I). Moreover, FLAV-27 significantly reduced Aβ aggregation by approximately 45% relative to the vehicle group, surpassing the effect of UNC0638 (~ 26% reduction) (Fig. 3 J-K). To evaluate the potential impact on organismal longevity, we conducted survival analyses in CL2006 worms. Although no significant differences in the overall lifespan were observed, FLAV-27 extended the mean lifespan by up to 22% in the CL2006 strain (Fig. 3 L-M). FLAV-27 was also tested in the senescence-accelerated SAMP8 model, which is considered a model of late-onset AD in which non-selective methyltransferase inhibitors have shown efficacy. 20 – 23 These experiments were essential to refine and standardize drug administration protocols, ensuring optimal delivery and reproducibility. Drug administration commenced at 4 months of age, as this time point corresponds to the early symptomatic phase of age-related cognitive decline in the SAMP8 mouse model. 20 , 23 FLAV-27 was administered orally at a dose of 5 mg/kg body weight once daily for one month (Fig. 4 A). To assess the effects of FLAV-27 on working memory, the novel object recognition test (NORT) was performed. During the familiarization phase (Figure S3C), FLAV-27 had no effect on the total exploration time. Importantly, FLAV-27 treatment resulted in a significant improvement in both short-term (measured at 2 hours post-training) and long-term (measured at 24 hours post-training) object recognition memory, as evidenced by the NORT performance (Fig. 4 B-C). Similarly, exploration during the habituation phase remained unchanged by FLAV-27 treatment in the object location test (OLT) (Figure S3D). Additionally, a significant enhancement in the discrimination index (DI) was observed during the OLT (Fig. 4 D). To gain a deeper insight into the mechanisms by which selective G9a inhibition might enhance cognitive function in the SAMP8 model, cortical brain sections from vehicle-treated and FLAV-27-treated animals were processed using the Golgi staining method. This technique allowed for a detailed morphological analysis of individual neurons, focusing on two key indicators of synaptic plasticity: dendritic spine density and neuronal branching complexity. Dendritic complexity was quantified using the Sholl analysis, a well-established neuroanatomical method that involves centering concentric circles on the neuronal soma and counting the number of dendritic intersections at increasing radial distances. This approach provides a robust measure of dendritic arborization, which is closely linked to the capacity for synaptic integration of a neuron (Fig. 4 E-F). Remarkably, animals treated with FLAV-27 exhibited a significant increase in both dendritic spine density and the number of neuronal intersections compared to controls (Fig. 4 E-G). These findings support the hypothesis, previously proposed by the in vitro studies, that selective G9a inhibition enhances neuronal plasticity. Importantly, the observed improvements in the dendritic architecture correlated with the reversal of cognitive deficits in the SAMP8 model, reinforcing the therapeutic potential of G9a inhibitors for age-related neurodegenerative disorders. Aβ levels are associated with behavioral abnormalities and cognitive decline; thus, we evaluated the Aβ 42 /Aβ 40 ratio in the brain. After FLAV-27 treatment, brain levels of the Aβ 42 /Aβ 40 ratio was reduced in the SAMP8 mice (Fig. 4 H). Levels of Aβ 42 were also reduced after FLAV-27 treatment (Fig. 4 I), while Aβ 40 levels remained unaltered (Fig. 4 J). FLAV-27 treatment also induced significant changes in the expression of genes associated with synaptic plasticity and neuroinflammation (Fig. 4 K-L). Specifically, FLAV-27 administration increased the expression of Syt4 and Arc (Fig. 4 K), two genes that are critically involved in synaptic function, neurotransmitter release, and activity-dependent neuronal remodeling. 24 – 27 The increase in these markers in the hippocampus suggests a potential enhancement of synaptic integrity and cognitive resilience. In parallel, FLAV-27 treatment led to a marked reduction in the expression of Il-6 and Tnf-α (Fig. 4 L), which are key pro-inflammatory cytokines known to be elevated in neurodegenerative conditions and implicated in AD progression. These findings suggest that FLAV-27 supports synaptic gene programs and exerts anti-inflammatory effects in the aging AD brain. Furthermore, to explore the effects of G9a inhibition on synaptic transmission, we measured AMPAR-mediated spontaneous miniature excitatory postsynaptic currents (mEPSCs) in primary cortical neurons from knock-in (KI) mice after exposure to FLAV-27 (Fig. 4 M-N). To isolate AMPAR-mEPSCs, D-(-)-2-amino-5-phosphonopentanoic acid and picrotoxin were added to the perfusion to block NMDA and GABA A receptors, respectively. FLAV-27 treatment for 24 hours significantly increased mEPSC amplitudes, suggesting an enhancement in postsynaptic AMPAR expression (Fig. 4 M). By contrast, the mEPSC frequency remained unchanged, indicating no presynaptic alterations (Fig. 4 M). These findings indicate that FLAV-27 promotes neuronal plasticity (Fig. 4 N). Reversal of neuronal, glial, and ferroptosis dysregulation in AD To further characterize the molecular effects of FLAV-27 in the SAMP8 AD mouse model, we began by performing bulk RNA sequencing on samples of hippocampal tissue from vehicle-treated and FLAV-27–treated mice. Differential gene expression (DEG) analysis revealed a pattern of de-repression consistent with G9a inhibition (Fig. 5 A). Gene Ontology (GO) enrichment analysis of the upregulated genes indicated a strong association with immune-related pathways (Figure S3E). Additionally, we applied the RNA-Age Calculator tool 14 to estimate the molecular age of the treated and control animals based on transcriptomic data. Although differences were not statistically significant due to sample size limitations, FLAV-27–treated mice exhibited a trend towards a reduced molecular age relative to controls (Figure S3F). To further dissect the cell-type-specific mechanisms underlying these transcriptomic changes, we employed single-cell RNA sequencing (scRNA-seq). Unlike bulk RNA-seq, which captures average gene expression across heterogeneous tissue, scRNA-seq enables the examination of transcriptional dynamics at the individual cell level. This approach allowed us to characterize cellular heterogeneity and identify distinct populations that are particularly responsive to FLAV-27 treatment. Through this analysis, we aimed to pinpoint key cell types and molecular pathways affected by selective G9a inhibition, thereby advancing our understanding of the therapeutic potential of FLAV-27 in AD. Gene expression analyses revealed important transcriptional changes in response to FLAV-27 treatment. We successfully sequenced 6,316 cells for SAMP8 controls, with a median of 1,215 UMIs per cell, and 11,919 cells for FLAV-27-treated mice, with a median of 1,050 UMIs per cell. Subsequent clustering analysis based on transcriptional profiles identified distinct cell types in both the control and FLAV-27 groups (Fig. 5 B-C). Notably, while analyzing cell type composition, we observed a two-fold decrease in microglia, a known disease driver, upon FLAV-27 treatment (Fig. 5 D). Notably, neurons in FLAV-27-treated mice displayed a transcriptional profile consistent with enhanced synaptic function, which aligns with our previous findings indicating that FLAV-27 supports neuronal activity. Interestingly, in the control samples, cluster 1 exhibited signatures of microglial and astrocytic activation, reflecting the characteristic pro-inflammatory environment seen in AD. By contrast, cluster 1 in FLAV-27-treated samples showed a gene expression profile associated with pathways related to synaptic transmission and nervous system development (Figure S3G-H). In fact, the most prominent upregulated pathways were those that regulated vesicle-mediated transport in the synapse, synapse assembly, post-synapse organization, synapse structure or activity, and signal release from the synapse. Furthermore, GO terms associated with the modulation of neuroinflammatory responses, such as gliogenesis, regulation of cytokine production, and the response to interleukin-1, were enriched, with an upregulation of genes like Serpine1 , Nfkbia , and Ccl3 . Changes in the genes related to the redox balance and iron-related stress responses indicated that FLAV-27 could indirectly inhibit ferroptosis, contributing to its neuroprotective action, even though ferroptosis-related GO terms were not directly enriched. Together, these findings highlight how FLAV-27 triggers a transcriptional program that supports synaptic integrity, cognitive function, and inflammatory control, while potentially reducing the onset of neurodegenerative diseases. The potential of FLAV-27 to preserve or restore excitatory neurotransmission and memory function in an aging brain is strongly supported by the inclusion of genes related to glutamatergic synapses (Figure S3I-J). Accordingly, analysis of differentially expressed genes (DEGs) revealed that FLAV-27-treated samples uniquely expressed genes implicated in glutamatergic and serotonergic synapses, as well as long-term potentiation, supporting the notion that FLAV-27 enhances processes critical for learning and memory (Figure S3K). GO enrichment analysis indicated that FLAV-27-induced genes were associated with neuronal development, regulation of neuronal differentiation, and neuronal migration (Figure S3L), consistent with our previous observations of FLAV-27 playing a role in promoting dendritic growth and improving memory. To refine our understanding of cell-type-specific transcriptional responses, we analyzed the top expressed genes within each identified cell population (Fig. 5 E-F, Figure S3G-H). While the overall abundance of microglial markers was similar between the groups (as expected in the single-cell analyses), the expression levels of these markers were reduced in FLAV-27-treated samples compared to controls, indicating that FLAV-27 modulates microglial gene expression without altering the population size. To validate these transcriptomic findings, we selected several DEGs from key cell types, particularly neurons, that are relevant to AD pathology and evaluated their expression following FLAV-27 treatment. All the selected genes exhibited increased expression upon FLAV-27 administration, confirming the robustness of our scRNA-seq results (Fig. 5 G, Figure S3M). Given the observed modulation of microglial gene expression, we further investigated the functional impact of FLAV-27 on human iPSC-derived microglia. Cells treated with FLAV-27 (1 µM) were assessed for cytokine secretion and phagocytic activity (Fig. 5 H). Under basal conditions, neither FLAV-27 nor the reference G9a inhibitor UNC0642 significantly altered cytokine secretion across most targets. Interestingly, UNC0642 induced a significant increase in IL-1β secretion, an effect not observed with FLAV-27 (Fig. 5 H), suggesting distinct mechanisms of action that could lead to different microglial responses. Notably, since Aβ 1−42 uptake by microglia is a key event in AD pathology, we evaluated this process using flow cytometry. FLAV-27 significantly enhanced the uptake of Aβ 1−42 oligomers by iPSC-derived microglia (Fig. 5 I). This effect was not observed with UNC0642 pre-treatment, further supporting the unique potential of FLAV-27 to modulate microglial function and its promise as a therapeutic candidate. To further assess the anti-inflammatory effects of FLAV-27, we tested its impact on glial cells exposed to LPS. FLAV-27 pre-treatment preserved viability in LPS-challenged HEK-293T cells (Figure S7A) and significantly reduced nitrite production in primary astrocyte and microglia cultures (9.65 µM and 7.15 µM, respectively), outperforming UNC0642 (Figure S7B-C). These results reinforce the anti-inflammatory and neuroprotective potential of FLAV-27. Finally, among the DEGs validated in our scRNA-seq dataset, some of them were associated with ferroptosis pathways, prompting us to explore this regulated form of iron-dependent cell death that has been increasingly implicated in AD. Supporting its relevance, ferroptosis markers were significantly elevated in SAMP8 mice compared to the senescence-resistant SAMR1 strain (Figure S7D-H), indicating enhanced susceptibility to ferroptosis in this AD model. In line with this, FLAV-27 treatment in SAMP8 mice led to a significant upregulation of Gpx4 and Fsp1 , two key ferroptosis suppressor genes acting through glutathione-dependent and -independent mechanisms, respectively (Fig. 5 J). Additionally, FLAV-27 restored the redox balance, as evidenced by a higher GSH:GSSG ratio and reduced levels of ROS, Fe²⁺, and MDA when compared to vehicle-treated controls (Fig. 5 K-N). To evaluate the impact of FLAV-27 on mitochondrial function and oxidative stress, we turned to the C. elegans CL2006 model, which enables high-resolution metabolic profiling in vivo (Fig. 5 O, Figure S7I-L). Seahorse assays revealed that FLAV-27 significantly improved mitochondrial respiration, increasing basal and mitochondrial oxygen consumption rates by up to 36% in CL2006 worms (Fig. 5 P-S). FLAV-27 also conferred protection against oxidative stress induced by tert-butyl hydroperoxide, with survival rates approaching those of vitamin C–treated controls (Figure S7M). Furthermore, expression of the oxidative stress–responsive gene sod-1 , which is elevated in the AD model, was normalized by FLAV-27 treatment (Figure S7N), suggesting improved redox homeostasis. Finally, to assess translational relevance, we examined post-mortem brain tissue from AD patients. While GPX4 and FSP1 transcript levels did not differ significantly from controls (Figure S7O), markers of oxidative stress and ferroptosis vulnerabilities were evident in the tissue from AD patients, including a reduced GSH:GSSG ratio and increased levels of ROS, Fe²⁺, and MDA (Fig. 5 T-W). FLAV-27 as a promising disease-modifying therapy for AD To assess whether selectivity in the inhibition of G9a could lead to novel properties in the way to combat AD, we aimed to test the effects of FLAV-27 on both cognition and the disease course. The effects of FLAV-27 were evaluated in the 5xFAD transgenic mouse model, a well-established model of early-onset AD. Two treatment regimens were implemented: (i) an early intervention schedule (treatment 1 + 0; weeks 20–24) designed to mimic preventive therapy and (ii) a late intervention schedule (treatment 0 + 1; weeks 24–28) simulating treatment during an established disease state. The design in Fig. 6 A allowed us to assess both the symptomatic effects on cognition (using standard behavioral assays) and the potential of FLAV-27 as a disease-modifying agent in a transgenic mouse model of early-onset hereditary AD. The NORT was used to assess cognition in these animals. The results revealed that FLAV-27 treatment significantly improved both short-term and long-term memory (Fig. 6 B-C, Figure S8A). Both FLAV-27 regimens provided cognitive benefits. Despite a lack of significance, the earlier treatment showed a trend towards preventing cognitive deficits. Importantly, FLAV-27 did not affect locomotor activity, as the total distance traveled in the open field (OF) test remained unchanged (Fig. 6 D, Figure S8B-H). The amount of rearing behavior decreased only in the animals treated with FLAV-27 (Fig. 6 E), while the amount of grooming did not change after treatment, with significant differences only observed between the two control groups (WT vs. 5xFAD) (Figure S8I). To examine spatial memory, we performed the OLT (Fig. 6 F, Figure S8J), which showed a significant enhancement in the discrimination index following all pharmacological interventions. Notably, both FLAV-27 treatment regimens resulted in higher discrimination index values than those observed in the WT controls, suggesting a robust cognitive-enhancing effect of FLAV-27. Furthermore, sociability was assessed using the three-chamber test (TCT) by recording the time spent sniffing (Fig. 6 G, Figure S8K-L). The WT control group and both 5xFAD groups treated with FLAV-27 exhibited greater sniffing time, suggesting a social improvement compared to the 5xFAD control group (Fig. 6 G). Histopathological analysis of Aβ (Fig. 6 H-J) showed that FLAV-27 significantly reduced Aβ plaque numbers in both the hippocampus (Fig. 6 H) and cortex (Fig. 6 I). In addition, FLAV-27 treatment significantly increased dendritic spine length (Fig. 6 K, M) and, recapitulating the effect observed in SAMP8 mice, FLAV-27 significantly increased spine density (Fig. 6 L, N). Astrogliosis increases progressively in 5xFAD mice as they age, aligning with the onset of cognitive impairments. 31 Accordingly, GFAP immunolabeling was conducted to ascertain the effect of the different treatments on the course of astrogliosis. This evaluation was performed using an immunofluorescence assay against GFAP (Fig. 6 O). The results confirmed a significant increase in GFAP immunostaining in 5XFAD mice compared to WT mice in the entire hippocampus (Fig. 6 P, Figure S8M). This increase was also observed in specific regions, including the dentate gyrus (DG), CA1, and CA3 regions (Fig. 6 O, Figure S8N-P). Quantification of the GFAP levels in the whole hippocampus revealed a decrease after both FLAV-27 treatment regimens (Fig. 6 P). Interestingly, while the beneficial effects were negligible in the DG and CA1 regions (Figure S8N-P), both the early and late FLAV-27 treatments demonstrated a significant reduction in GFAP immunoreactivity in the hippocampal CA3 region (see Fig. 6 O). FLAV-27 restored the expression of the immediate-early gene Arc , a key regulator of synaptic plasticity, particularly in the 1 + 0 group (Figure S8Q), indicating enhanced neuronal activity and cognitive function. Moreover, FLAV-27 significantly reduced the expression of the pro-inflammatory cytokine Il-6 (Figure S8R), while upregulating Ftl-1 (Figure S8S), suggesting a reduction in neuroinflammatory signaling and a potential activation of ferroptosis-related protective mechanisms. To validate the molecular alterations underlying key pathological events in AD, such as synaptic plasticity, neuroinflammation, and ferroptosis, comprehensive proteomic analyses were performed following FLAV-27 treatment. WT mice were clearly separated from the 5xFAD mice in a principal component analysis (PCA), with treated groups clustering in intermediate positions, suggesting partial reversal of disease-associated proteomic changes by the treatments (Figure S9A). The heatmap confirmed distinct expression patterns among the groups, with a characteristic proteomic signature in 5xFAD mice and notable differences between the treatment conditions (Fig. 6 Q). Furthermore, global enrichment analysis revealed that several biological processes were significantly altered when all the groups were considered together, indicating broad functional differences driven by disease state and modulated by treatment (Figure S9B). Next, we compared the 5xFAD and WT proteomes. As shown in Figure S9C, ~ 40% of the differentially expressed proteins were nuclear or plasma membrane proteins, while 6.5% were mitochondrial, underscoring the potential contribution of mitochondrial dysfunction to AD pathogenesis. Pathway analysis revealed a reduction in Hippo signaling (Figure S9D), which is involved in cell proliferation and apoptosis regulation, 33 and an upregulation of glycosphingolipid biosynthesis and glycosaminoglycan degradation (Figure S9E), both of which may disrupt membrane composition and promote inflammation and aggregation in AD. 34 Finally, to assess treatment impact, we compared each group to untreated 5xFAD mice. All treatments significantly altered the proteomic profile (Fig. 6 R-S, Figure S9F), indicating disease-modifying potential. A detailed analysis of the 5xFAD proteome compared to the WT proteome revealed widespread alterations in the pathways related to synaptic function, energy metabolism, and protein homeostasis (Fig. 7 , Figure S9G-J). Notably, 5xFAD mice showed a downregulation of the proteins involved in mitochondrial function, ATP synthesis, ubiquitination, and RNA processing, alongside an upregulation of the proteins linked to the innate immunity, synaptic pruning, and inflammatory responses. Additional changes were observed in membrane trafficking, cytoskeletal organization, and proteasome function, indicating broader disturbances in neuronal structure and intracellular signaling. Metabolic imbalances, including disruptions in sphingolipid metabolism, glycolysis, lipid transport, and mitochondrial translation, were also evident, suggesting deficits in energy production and the biosynthetic capacity. In the context of the molecular alterations observed in 5xFAD mice, the tested treatments modulated protein expression in key disease-related pathways. The FLAV-27 early treatment (1 + 0) upregulated Grid2, Ist1, and Gng3, suggesting restoration of synaptic signaling and vesicle trafficking mechanisms that are both impaired in 5xFAD mice. 35 The late treatment (0 + 1) additionally increased the expression of Pigt, Cpped1, Cox14, Atp5mpl, Nf2, Ints8, Mkln1, Ube2v1, Smpd3, and Ndufa3, pointing to improvements in mitochondrial bioenergetics (e.g., Cox14, Atp5mpl, and Ndufa3), protein homeostasis and ubiquitination (e.g., Ube2v1), and RNA metabolism (e.g., Ints8), which are all commonly dysregulated in AD. 36 In addition to upregulating beneficial proteins, the FLAV-27 treatments reversed the increases in several proteins previously found to be upregulated in 5xFAD mice, thereby targeting pathways implicated in AD pathology. The early treatment (1 + 0) reduced the levels of Dhcr24, Smarcc2, Slc12a2, Ints1, Josd2, and Abcd1, which are associated with lipid metabolism, redox homeostasis, synaptic gene expression, and ion transport. The late treatment (0 + 1) led to a broader downregulation of disease-elevated proteins, including Ptpn6, Golga2, Pik3ca, Igtp, Ubb, Ctsh, Supt4a, Dhcr24, Ncbp2, Pak1ip1, B3galnt1, and Ptcd1, suggesting a widespread therapeutic impact. The reduced expression of inflammatory regulators (Ptpn6 and Igtp) aligns with the prior findings in the single-cell RNA-seq analysis of attenuated microglial activation. The downregulation of proteostasis-related proteins (Ubb and Ctsh) suggests an improved protein degradation capacity, 37 while decreased Golga2 and Pak1ip1 may indicate recovery of vesicle trafficking and cytoskeletal structure, which are both essential for proper APP processing and synaptic function. 38 Furthermore, the downregulation of Pik3ca, a PI3K/Akt signaling component, may reflect rebalanced cell survival and autophagy pathways, while the decreased levels of Supt4a and Ncbp2 (involved in RNA processing) may indicate a partial reversal of transcriptional dysregulation. A lower Dhcr24 expression points to corrected cholesterol biosynthesis and OS regulation, 34 while B3galnt1 downregulation may modulate glycosylation processes that influence protein aggregation and synaptic signaling. 39 Together, these changes suggest that FLAV-27 exerts multi-target neuroprotective effects, modulating inflammation, metabolism, vesicle trafficking, and neuronal architecture in a manner consistent with a disease-modifying therapeutic profile. Modulation of the plasma biomarkers of AD and of plasma epigenetic-, synaptic plasticity-, and neuroinflammatory-related parameters We next aimed to correlate the effects of FLAV-27 with the levels of biomarkers associated with AD pathology that are commonly measured in clinical settings and are detectable in the plasma of 5xFAD mice. 40 – 47 All the data presented below were obtained from plasma samples collected from different experimental groups. Given that H3K9me2 levels are known to be elevated in the brains of 5xFAD mice and given its role in transcriptional repression, 48 , 49 we conducted a preliminary experiment to determine whether H3K9me2 is detectable in the plasma and whether FLAV-27 alters its levels. As shown in Fig. 7 A, untreated 5xFAD mice displayed higher plasma levels of H3K9me2 compared to control animals. Notably, FLAV-27 treatment resulted in a significant reduction of H3K9me2 levels, consistent with its anticipated activity as a lysine methyltransferase. Next, we assessed the plasma levels of SMOC1, an Aβ plaque-associated synaptic protein identified as a biomarker of early-stage AD. 50 , 51 – 53 Plasma concentrations of SMOC1 were significantly elevated in untreated 5xFAD mice compared to controls. Importantly, treatment with FLAV-27 reduced these elevated levels, restoring them to values comparable to those observed in control animals (Fig. 7 B). The levels of GFAP, a marker of astrocytic activation and neuroinflammation, as well as TNF-α, a proinflammatory cytokine, were also assessed, given that both are elevated in the plasma of AD patients. 54 As expected, the plasma levels of both inflammatory markers were higher in the 5xFAD mice compared to wild-type controls (Figure S10A-B). Notably, plasma TNF-α levels were only normalized following the early (1 + 0) FLAV-27 treatment regimen (Figure S10B), while a reduction in GFAP plasma levels was observed exclusively with the late (0 + 1) treatment (Figure S10A). We then aimed to correlate the potential benefits of the early and late FLAV-27 treatments with the plasma biomarkers associated with AD. Specifically, we focused on Aβ peptides, p-tau, and neurofilament light chain (NF-L), which are key indicators of disease pathology and progression. These correlations are critical to determine whether FLAV-27 acts as a disease-modifying therapy rather than offering merely symptomatic relief. To this end, we measured the plasma levels of Aβ peptides, two p-tau forms recognized as clinical biomarkers of AD, and NF-L, a well-established marker of neuroaxonal damage in neurological disorders. 61 Notably, the early (1 + 0) treatment regimen showed no significant changes in Aβ 1−40 or Aβ 1−42 levels, nor in the Aβ 1−42 /Aβ 1−40 ratio, suggesting that the preventive benefits of FLAV-27 do not stem from reducing the amyloid burden (Figure S10C-E). Regarding p-Tau181 and p-Tau217 (phosphorylated tau forms identified by antibodies specific to tau phosphorylated at Thr181 and Thr217, respectively), which are considered reliable biomarkers of AD, 11 untreated 5xFAD mice exhibited increased plasma levels of both markers. Remarkably, both the early (1 + 0) and late (0 + 1) FLAV-27 treatment regimens reversed these increases, normalizing p-tau levels to those observed in the plasma of WT controls (Figure S10F-G). Similarly, NF-L levels, which were significantly elevated in untreated 5xFAD mice, were restored to control levels in both FLAV-27 treatment groups (Figure S10H). Finally, we performed correlation analyses between all the plasma biomarkers evaluated, focusing on H3K9me2 and SMOC1, which are both regulated by G9a (Fig. 7 C). H3K9me2 showed a strong positive correlation with SMOC1 levels, while other biomarkers displayed variable trends. Notably, SMOC1 also correlated with the neuroinflammatory markers p-tau (T181) and NF-L (Fig. 7 C), highlighting the multi-targeted modulation of plasma biomarkers following G9a inhibition by FLAV-27. This evidence supports the potential of FLAV-27 as a disease-modifying therapy for AD. To assess the translational relevance of our findings, we analyzed a comprehensive panel of molecular markers in the plasma and cerebrospinal fluid (CSF) of controls with no dementia, individuals with prodromal AD, and patients with AD dementia. The plasma levels of H3K9me2 (Fig. 7 D), p-tau (T181), and TNF-α were significantly elevated in the AD patients (Figure S10I-J), while SMOC1 levels showed a trend towards an increase (Fig. 7 E). In the CSF, levels of SMOC1 (Fig. 7 F), total tau, and p-tau (T181) were significantly higher in the prodromal and AD dementia groups compared to controls (Figure S10K-L). Additionally, the CSF Aβ42/Aβ40 ratio was significantly decreased in the AD patients, driven primarily by reduced Aβ42 levels that is consistent with amyloid pathology (Figure S10M-O). Cognitive impairment, measured by the MMSE scores (Fig. 7 G), correlated negatively with plasma H3K9me2 and CSF SMOC1 levels (Fig. 7 H). Western blot analysis of post-mortem brain tissue revealed elevated SMOC1 protein levels in the AD patients compared to controls (Fig. 7 I-J). Furthermore, plasma H3K9me2 levels correlated positively with SMOC1 concentrations in the CSF, while SMOC1 also showed moderate associations with tau biomarkers and inflammatory markers (Fig. 7 H). These results support the involvement of epigenetic dysregulation and astroglial activation in human AD pathology and reinforce the potential of these markers as therapeutic targets and disease indicators. DISCUSSION In this work, we report the discovery and in-depth characterization of FLAV-27, a first-in-class, SAM-competitive, brain-penetrant, and selective inhibitor of the lysine methyltransferase G9a. Our findings position FLAV-27 as a potent epigenetic modulator with robust disease-modifying effects across multiple in vitro and in vivo models of AD, with the potential to overcome several limitations that have historically hindered the translational success of G9a inhibitors for CNS applications (Fig. 8 ). Previous G9a inhibitors 55 such as BIX-01294, UNC0638, and UNC0642 have shown limited utility in neurodegeneration due to off-target effects, the dual inhibition of GLP, and poor CNS penetration. BIX-01294 was initially reported to reduce H3K9me2 levels in cancer cells, but was associated with high cytotoxicity and a lack of in vivo viability. 56 UNC0638 and UNC0642 offered improved potency, but remained selective for GLP too and poorly penetrated the brain, thereby limiting their translational potential. 13 , 57 By contrast, FLAV-27 exhibits subnanomolar G9a inhibition, with > 30-fold selectivity for G9a over GLP and other histone methyltransferases, as well as robust brain permeability that has been confirmed both in vitro (PAMPA-BBB) and in vivo . Structurally, the SAM-competitive mechanism of FLAV-27 represents a paradigm shift from traditional substrate-competitive G9a inhibitors. The high-resolution X-ray co-crystal structure of human G9a bound to FLAV-27 (PDB ID: 9LUS) confirms a unique SAM-competitive binding mode, revealing that FLAV-27 occupies the SAM pocket of G9a. Interestingly, structural comparisons with the published G9a–SAM complex (PDB: 5TTF) show that FLAV-27 relies heavily on hydrophobic contacts, particularly between its benzyl and phenol moieties and the G9a residues Trp1050, Phe1110, Tyr1085, and Tyr1154, thereby rationalizing its superior affinity and selectivity. In fact, preliminary findings indicate that while the active sites of G9a and GLP are structurally similar, their surface charge distributions differ significantly. The SAM-binding site of G9a is predominantly hydrophobic with a minimal negative charge, whereas that of GLP is largely positively charged. This distinction may explain why the hydrophobic FLAV-27 compound preferentially binds to G9a. Moreover, the inadvertent presence of low‐occupancy SAM in our G9a crystallization likely dampened the FLAV-27 electron density, but the refined Fo–Fc maps at 1σ clearly define FLAV-27 in the SAM co-substrate pocket. Consequently, by blocking SAM binding, FLAV-27 prevents methyl transfer entirely, offering a mechanistic advantage over substrate‐competitive compounds that merely impede histone peptide docking. Thus, unlike substrate-competitive inhibitors that often affect multiple SET domain methyltransferases, 58 the occupation by FLAV-27 of the SAM pocket provides superior selectivity and reduced off-target effects. This mechanistic distinction is crucial, as cross-reactivity with other methyltransferases can have deleterious effects on chromatin integrity in neurons, where precise regulation of gene expression is essential for plasticity and survival. 59 Here, we are actively working to further elucidate the molecular determinants underlying the selectivity of FLAV-27 for G9a over GLP. Our ongoing studies focus on detailed structural and electrostatic analyses of the SAM-binding pockets in both enzymes. The exceptional safety profile of FLAV-27 (NOAEL > 1 g/kg) combined with its low effective dose (5 mg/kg/day) provides a substantial therapeutic window that enhances its clinical viability. The lack of significant off-target effects across comprehensive screening panels contrasts favorably with existing epigenetic therapeutics that often exhibit dose-limiting toxicities. This safety profile is particularly important for chronic neurodegenerative diseases requiring long-term treatment. In addition, the brain-to-plasma ratio of 3–6:1 achieved by FLAV-27 ensures adequate CNS exposure, while minimizing peripheral effects. This pharmacokinetic profile represents a significant improvement over earlier G9a inhibitors that suffer from poor BBB penetration. The achievement of sustained brain exposure with intermittent dosing schedules further enhances the clinical practicality of this approach. Epigenetic repression mediated by H3K9me2 is increasingly recognized as a driver of cognitive decline in neurodegeneration in AD models and patient samples. 19 , 48 , 60 , 61 Our data show that FLAV-27 robustly reduces H3K9me2 levels in the SAMP8 mouse brain and induces genome-wide reprogramming of heterochromatin-associated regions, particularly at the promoters of genes implicated in synaptic signaling, neurogenesis, learning and memory, and cellular differentiation. These data align with previous work showing G9a-mediated repression of plasticity‐related genes in aging and AD. 9 – 11 , 60 , 62 , 63 This is consistent with reports showing that G9a inhibition in AD models reactivates silenced neuroprotective genes, including Arc and Syt4 , which we also found to be upregulated following FLAV-27 treatment. 60 Notably, we also observed a novel reduction in H3K18me₁–₃ levels following FLAV-27 treatment, producing the first evidence that H3K18 methylation contributes to AD pathogenesis. Elevated H3K18me₁–₃ has been reported in the brains of AD patients, but its functional role is unknown. Our data imply that H3K18 methylation may cooperatively establish repressive heterochromatin with H3K9me2 to silence neuronal gene programs. The ability of FLAV-27 to reverse both the H3K9 and H3K18 marks highlights its potential for broad epigenetic reprogramming in diseased neurons. At the transcriptomic level, bulk RNA-seq in the cortex of SAMP8 mice revealed a global pattern of gene de-repression, with an upregulation of immune‐regulatory gene sets. This finding is consistent with the dual epigenetic and immunomodulatory role of FLAV-27. Single‐cell RNA-seq further refined these insights by showing that although the neuronal and glial cell proportions remained unchanged, FLAV-27 shifted gene expression within cell types. Disease ontology analysis revealed that the FLAV-27‐regulated genes are associated not only with AD, but also with schizophrenia, depression, and other mental disorders, suggesting broader utility for neuropsychiatric conditions in which G9a dysregulation has been implicated. 64 – 67 Our in vitro studies provide direct evidence of the ability of FLAV-27 to counteract AD-related cellular phenotypes. In primary neuronal and microglial cultures, FLAV-27 markedly reduced the aggregation of Aβ₁–₄₂, total tau, and phosphorylated tau (p-Tau), while restoring the neuritic integrity disrupted by these aggregates. These findings are consistent with previous work suggesting that histone methylation contributes to synaptic loss and cytoskeletal disorganization in AD neurons. 68 In addition, we evaluated its effect in the C. elegans CL2006 strain, which expresses human Aβ₁–₄₂ in its muscle cells and exhibits age-dependent paralysis. 18 FLAV-27 (1 µM) significantly reduced the H3K9me2/H3 ratio, decreased Aβ aggregation by approximately 45% (compared to ~ 26% with UNC0638), and improved locomotor behavior. Notably, FLAV-27 extended the lifespan. This is in accordance with previous observations that G9a inhibition extends the lifespan and improves stress resistance in C. elegans , 69 further supporting the idea that the epigenetic mechanisms governing proteostasis and neuronal function are conserved across phyla. SAMP8 mice, a model of late-onset AD (LOAD), and 5xFAD mice, a model of early-onset AD (EOAD), were selected for this study as it was previously reported that they show elevations in the G9a protein and its repressive histone mark H3K9me2 during disease progression. 19 , 48 , 49 , 70 Furthermore, we aimed to investigate whether selective G9a inhibition with FLAV-27 conferred disease-modifying properties due to the current lack of disease-modifying therapies for neurodegenerative diseases such as AD. For that reason, two FLAV-27 treatment regimens were tested in the 5xFAD mice: early intervention (weeks 20–24) and late intervention (weeks 24–28). Both approaches improved cognitive performance in the NORT, OLT, and social interaction paradigms, without affecting locomotor activity. Notably, early treatment tended to completely prevent cognitive deficits, while the late intervention was able to reverse established impairments. These behavioral improvements were accompanied by structural enhancements in synaptic morphology, including increased dendritic spine density and length, and by enhanced AMPAR-mediated miniature excitatory postsynaptic currents (mEPSCs), indicating a functional rescue of synaptic transmission. These findings parallel previous reports that G9a inhibition enhances synaptic function in the nucleus accumbens and hippocampus. 8 Moreover, FLAV-27 reduced the Aβ plaque burden and suppressed astrogliosis in the 5xFAD mice. FLAV-27 also ameliorated social behavioral deficits in the 5xFAD mice, as assessed by the TCT. Treated animals spent significantly more time interacting with the social stimulus compared to untreated controls, indicating a restoration of sociability, a crucial behavioral domain often impaired in AD. This rescue aligns with previous reports linking epigenetic repression to social cognition deficits, highlighting the broad functional impact of G9a inhibition. 50 , 65 , 70 Cell-type–specific marker analyses showed that microglial markers, though present at a constant level, were downregulated in FLAV-27 samples compared to SAMP8 controls, reinforcing the notion that FLAV-27 directly modulates microglial transcriptomes without altering cell numbers. To test functional consequences, we treated human iPSC-derived microglia with FLAV-27 (1 µM) and compared them to UNC0642-treated cells. Under basal conditions, FLAV-27 did not elicit IL-1β secretion, whereas UNC0642 significantly increased IL-1β levels, suggesting distinct allosteric effects on microglial activation due to SAM-competitive versus substrate-competitive binding. Importantly, FLAV-27 significantly enhanced microglial uptake of Aβ₁₋₄₂ oligomers, a critical neuroprotective mechanism in AD, whereas UNC0642 did not. These data indicate that FLAV-27 restores microglial homeostasis while enhancing phagocytosis, potentially contributing to a decreased Aβ burden in vivo . In parallel, both the bulk and single-cell transcriptomic datasets confirmed the upregulation of key synaptic genes such as Arc and Syt4 following FLAV-27 treatment, further substantiating the link between G9a inhibition and the restoration of neuronal gene expression. Collectively, these findings position H3K9me2 not only as a critical epigenetic mark mediating transcriptional repression in AD, but also as a biomarker of treatment response. They also reinforce the central role of G9a as a regulator of synaptic gene silencing and highlight the potential of epigenetic reprogramming via FLAV-27 to restore neuronal identity and function in AD. Ferroptosis has emerged as a central mechanism contributing to neuronal loss in AD. 71 – 75 SAMP8 mice displayed elevated markers of ferroptosis, including increased levels of lipid peroxidation (MDA), ROS, and Fe²⁺, and a reduced GSH:GSSG ratio. FLAV-27 reversed these alterations and upregulated Gpx4 and Fsp1 , which are critical suppressors of ferroptosis, suggesting a direct protective role through redox regulation. Additionally, due to prior studies showing abnormal mitochondrial respiration in a C. elegans model of mild pan-neuronal AD in its old stages, 76 we used this model to corroborate our findings after FLAV-27 treatment. We used the CL2006 strain, a transgenic strain in which the muscular expression of the Aβ peptide and consequent Aβ plaques lead to abnormal locomotion and paralysis. In fact, one possible cause of the muscular dysfunction could be the abnormal mitochondrial metabolism exhibited by the CL2006 strain at a younger stage (L4). However, FLAV-27 increased the oxygen consumption rate (OCR) of CL2006, probably ameliorating its locomotory behavior and well-being through aging. Regarding the proteomic study, FLAV-27 treatments modulated AD-related protein pathways in 5xFAD mice, but differed in the scope. While FLAV-27 1 + 0 treatment reduced the levels of the proteins linked to lipid metabolism (Dhcr24), synaptic plasticity (Smarcc2 and Slc12a2), and redox homeostasis (Abcd1), FLAV-27 0 + 1 treatment broadly downregulated inflammatory regulators (Ptpn6 and Igtp), proteostasis markers (Ubb and Ctsh), and transcriptional mediators (Supt4a and Ncbp2). These changes suggested that FLAV-27 normalizes microglial activation, protein degradation, and RNA processing, while restoring vesicle trafficking via Golga2 and Pak1ip1 reduction. This was also validated in the single-cell seq performed in the SAMP8 mice. Consistent with our results, the G9a inhibitor MS1262 demonstrated a similar pattern profile, 50 reversing the changes in several proteins previously reported to be upregulated in 5xFAD mice, including translation regulators (METTL3), early AD biomarkers (SMOC1), and matrisome components, while restoring PI3K/Akt/MAPK signaling, cytoskeletal phosphorylation states (DNM2 and TAGLN2), and proteostasis networks. Importantly, these proteomic brain signatures were accompanied by peripheral biomarker changes, underscoring the systemic impact of the FLAV-27 treatment. FLAV-27 reduced levels of p-tau (T181), TNF-α, and H3K9me2, three key markers linked to AD pathology, inflammation, and epigenetic repression, in the plasma of 5xFAD mice. The pattern of the changes in the Aβ 42 /Aβ 40 ratio after FLAV-27 treatment suggests that FLAV-27 exerts its effect on Aβ pathology by modulating the pathways that are active during established disease. The lack of persistence in the group receiving early treatment may reflect the dynamic reversibility of this epigenetic mechanism. Interestingly, SMOC1 has been recently identified as a biomarker for AD and its levels correlate with the Aβ plaque load. 77 – 79 More noteworthy, a preprint postulated that treatment with a G9a inhibitor can reverse SMOC1 expression and phosphorylation in the Aβ-associated matrisome module. 51 , 80 Accordingly, the 5xFAD mice had higher plasma levels of SMOC1 that were reverted by FLAV-27 treatment. To evaluate the translational relevance of our preclinical findings and support the clinical applicability of FLAV-27, we analyzed a broad panel of AD-related biomarkers in human fluids and post-mortem brain samples. Strikingly, we found that the epigenetic alterations observed in the animal models, particularly the upregulation of H3K9me2 and H3K18 methylation, were also recapitulated in the AD human brain samples. Elevated H3K9me2 levels were detected in both the plasma and brain tissue of AD individuals compared to controls with no dementia, reinforcing the notion that G9a-mediated chromatin repression is not limited to the CNS but extends to peripheral compartments. Moreover, SMOC1 was also found to be significantly elevated in the CSF and brain tissue of AD patients and showed a trend towards elevation in the plasma. Together with the consistent increase in tau levels and the expected decrease in the Aβ 42 /Aβ 40 ratio in the CSF, these findings highlight SMOC1 as a candidate biomarker of early disease progression with central and peripheral relevance. Importantly, the strong correlations observed between plasma H3K9me2 and SMOC1 levels, as well as their associations with tau pathology, inflammatory markers (TNF-α), and cognitive decline (MMSE), further support the biological linkage between epigenetic dysregulation and hallmark AD mechanisms. In fact, all of this mirrors the observations in human epigenome-wide association studies that link H3K9 methylation to cognitive impairment and neurodegeneration. 68 , 81 – 83 These translational data not only validate the relevance of our experimental models, but also underscore the utility of H3K9me2 and SMOC1 as promising peripheral biomarkers for monitoring disease progression and therapeutic response in future clinical applications of G9a-targeted interventions such as FLAV-27 treatment. Declarations All procedures involving animals, including behavioral testing and dissection and removal of brains, followed ARRIVE and the standard ethical guidelines (Council of the European Communities Directive 2010/63/EU and Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research, National Research Council 2003) and were approved by the Institutional Animal Care and Generalitat de Catalunya (#PJ_159/24, March 13, 2024). Every effort was made to minimize the number of mice used and their suffering. ACKNOWLEDGMENTS This study was supported by the Ministerio de Economía, Industria Economía, Industria y Competitividad (Agencia Estatal de Investigación, AEI) and European Union NextGenerationEU/PRTR (PID2022-139016OA-I00, PDC2022-133441-I00 ,to CGF and MP; PID2022-1380790B-I00, to C.E.), MICIU/AEI/ 10.13039/501100011033 and FEDER, UE and PDC2022-133441-I00 MICIU/AEI / 10.13039/501100011033 Europea Next GenerationEU/ PRTR), Generalitat de Catalunya (2021 SGR 00357). This study was co-financed by Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya 2023 (Product 0092; Llavor 005 and Llavor 007 to CGF). ABS acknowledges the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) for her FI-SDUR fellowship (2021FISDU 00182). NIDDK R01 230857 GRANT to SD. Financial support was provided for F.R.-B. (PREP2022-000196 Ministerio de Ciencia e Innovación). 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Barcelona","correspondingAuthor":false,"prefix":"","firstName":"Marcel.lí","middleName":"","lastName":"Carbó","suffix":""},{"id":476291653,"identity":"fcfe86ee-ea07-4321-bc51-ee3c48360452","order_by":32,"name":"Ana Guerrero","email":"","orcid":"","institution":"University of Barcelona","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"","lastName":"Guerrero","suffix":""},{"id":476291654,"identity":"a80ab2d8-0ee7-4ac7-911e-f9b3161888ab","order_by":33,"name":"Santiago Vázquez","email":"","orcid":"https://orcid.org/0000-0002-9296-6026","institution":"Universitat de Barcelona","correspondingAuthor":false,"prefix":"","firstName":"Santiago","middleName":"","lastName":"Vázquez","suffix":""},{"id":476291655,"identity":"ad1258c1-9aa6-4928-804f-d455ba3f6de3","order_by":34,"name":"Shaodong Dai","email":"","orcid":"https://orcid.org/0000-0003-4760-2773","institution":"University of Colorado Anschutz Medical Campus","correspondingAuthor":false,"prefix":"","firstName":"Shaodong","middleName":"","lastName":"Dai","suffix":""},{"id":476291656,"identity":"5906e6a2-5f56-411c-be3e-5d489f2761c8","order_by":35,"name":"Carmen Escolano","email":"","orcid":"","institution":"University of Barcelona","correspondingAuthor":false,"prefix":"","firstName":"Carmen","middleName":"","lastName":"Escolano","suffix":""},{"id":476291657,"identity":"6d440d82-724d-4821-9ded-349d98d50ea3","order_by":36,"name":"Rafael Franco","email":"","orcid":"https://orcid.org/0000-0003-2549-4919","institution":"University of Barcelona","correspondingAuthor":false,"prefix":"","firstName":"Rafael","middleName":"","lastName":"Franco","suffix":""},{"id":476291658,"identity":"477f4f87-78cf-4c79-9910-a8c1e06d7135","order_by":37,"name":"Mercè Pallàs","email":"","orcid":"https://orcid.org/0000-0003-3095-4254","institution":"University of Barcelona","correspondingAuthor":false,"prefix":"","firstName":"Mercè","middleName":"","lastName":"Pallàs","suffix":""}],"badges":[],"createdAt":"2025-06-20 15:56:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6940373/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6940373/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85455250,"identity":"3b953f3a-5f56-4283-aba4-c93a038542ea","added_by":"auto","created_at":"2025-06-26 06:12:47","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":440833,"visible":true,"origin":"","legend":"\u003cp\u003eKinetic analysis of G9a inhibition at a constant H3 peptide concentration and variable SAM concentrations (0-7 µM).\u003cstrong\u003e \u003c/strong\u003eData are represented as a Michaelis–Menten plot for (\u003cstrong\u003eA\u003c/strong\u003e) FLAV-27 and (\u003cstrong\u003eB\u003c/strong\u003e) UNC0642. Error bars represent the SEM of two replicates. \u003cstrong\u003eC. \u003c/strong\u003eCrystal structure of the G9a-FLAV-27 complex (PDB 9LUS).\u003cstrong\u003e \u003c/strong\u003eHomodimer of G9a-FLAV-27, with one subunit shown in the surface representation and the other in the cartoon representation. Superimposition of the G9a-FLAV-27 (red) (PDB 9LUS) monomer with the G9a-SET domain (yellow) monomer from PDB ID 5TTF. The inset highlights FLAV-27 occupying the SAM position in the G9a-FLAV-27 complex. \u003cstrong\u003eD. \u003c/strong\u003eDetailed interactions between the FLAV-27 inhibitor and neighboring residues. \u003cstrong\u003eE.\u003c/strong\u003e Detailed interactions between the SAM cofactor and neighboring residues, using PDB 5TTF. \u003cstrong\u003eF.\u003c/strong\u003e Comparison of FLAV-27 and SAM-binding residues. Overlay of the G9a-FLAV-27 complex with GLP-SAM (PDB ID: 5TUZ). \u003cstrong\u003eG.\u003c/strong\u003e Neighboring residues of FLAV-27 (yellow stick) are shown in gray, using the G9a-FLAV-27 complex. \u003cstrong\u003eH.\u003c/strong\u003e Neighboring residues of SAM (ruby stick) are shown in gray, using 5TUZ (PDB ID). Residues that are not involved in interactions with FLAV-27 are highlighted by the blue text. Residues that still interact with, but are changed in the FLAV complex, are highlighted by the red text.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6940373/v1/7abd84eefedf6c0202d07876.jpeg"},{"id":85455252,"identity":"ed17ab31-9140-4457-85c1-db1752472b06","added_by":"auto","created_at":"2025-06-26 06:12:47","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":419240,"visible":true,"origin":"","legend":"\u003cp\u003eChIP-seq analysis of FLAV-27 effects in cortical tissue. (\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eRepresentative images and\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003equantification levels of\u003cstrong\u003e \u003c/strong\u003eH3K9me2, H3K18me1-3, and H3K23me1-3. Values presented are the mean ± SEM (N = 2 groups: SAMP8 Control, n = 5, and SAMP8 FLAV-27 (5 mg/kg), n = 5). Student’s t-test analysis was performed: *p \u0026lt; 0.05; **p \u0026lt; 0.001. \u003cstrong\u003eC. \u003c/strong\u003eH3K9me2 enrichment heatmap of significant peaks according to macs3 in control (left) or FLAV-27-treated samples (right). \u003cstrong\u003eD. \u003c/strong\u003eAverage H3K9me2 enrichment derived from Figure 3c. \u003cstrong\u003eE.\u003c/strong\u003e H3K9me2 fold change enrichment upon FLAV-27 treatment within significant peaks. \u003cstrong\u003eF.\u003c/strong\u003e H3K9me2 enrichment heatmap in peaks with significant decrease upon FLAV-27 treatment. \u003cstrong\u003eG.\u003c/strong\u003e Average H3K9me2 enrichment derived from Figure 3f. \u003cstrong\u003eH\u003c/strong\u003e. Genome browser snapshot of genes with significant H3K9me2 changes in control (top, gray) and FLAV-27-treated (bottom, blue) samples. (\u003cstrong\u003eI\u003c/strong\u003e) Key upregulated and (\u003cstrong\u003eJ\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003edownregulated terms from the Gene Ontology and Pathway analysis using the genes close to FLAV-27-reduced H3K9me2 peaks. \u003cstrong\u003eK.\u003c/strong\u003eGenome distribution of genes from G (orange) compared with the global distribution of annotated genes (gray). \u003cstrong\u003eL\u003c/strong\u003e. Gene Ontology and Pathway analysis using genes close to FLAV-27-reduced H3K9me2 peaks. \u003cstrong\u003eM. \u003c/strong\u003eBar plot showing the top enriched biological pathways from H3K9me2 ChIP-seq analysis (FLAV-27-treated vs. control), categorized by pathway source and regulation direction. \u003cstrong\u003eN. \u003c/strong\u003eRepresentative western blot images and (\u003cstrong\u003eO\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003equantification of the H3 methylation landscape. Values presented are the mean ± SEM (N = 2 groups: no dementia, n = 5-6, and AD dementia, n = 7). Student’s t-test analysis was performed: *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6940373/v1/08d1f0224ef97f9187df7775.jpeg"},{"id":85456140,"identity":"3b5b338c-faab-4e31-a536-cc221024b6d1","added_by":"auto","created_at":"2025-06-26 06:28:47","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":386160,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of FLAV-27 (1 µM) on AD markers in primary neuronal and microglial cultures.\u003cstrong\u003e A. \u003c/strong\u003eRepresentative images of the\u003cstrong\u003e \u003c/strong\u003eeffect of FLAV-27 (1 µM) on Aß\u003csub\u003e1-42\u003c/sub\u003e aggregation (\u003cstrong\u003eB\u003c/strong\u003e) and on\u003cstrong\u003e \u003c/strong\u003etau and p-tau aggregation (\u003cstrong\u003eC\u003c/strong\u003e) in primary neuronal and microglial cultures.\u003cstrong\u003e \u003c/strong\u003eData are presented as the mean ± SEM of 5 independent experiments. One-way ANOVA or two-way ANOVA with Tukey’s post-hoc analysis was performed: *p \u0026lt; 0.05 (n ≥ 3). \u003cstrong\u003eD. \u003c/strong\u003eNeurite formation was analyzed in primary neuronal cultures treated with (\u003cstrong\u003eE\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eAβ\u003csub\u003e1-42 \u003c/sub\u003e(500 nM) or (\u003cstrong\u003eF\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003etau (1 µM) or p-tau (1 µM) for 24 h and subsequently stimulated with FLAV-27 (1 µM) or vehicle for 24 h more. Data are presented as the mean ± SEM of 5 independent experiments. One-way ANOVA or two-way ANOVA with Tukey’s post-hoc analysis was performed: *p \u0026lt; 0.05 (n ≥ 3). \u003cstrong\u003eG.\u003c/strong\u003e Treatment with FLAV-27 ameliorates the motor dysfunction of CL2006 worms. Values presented are the mean ± SEM (n = 2-3 technical replicates). At least 90-100 worms were in each group. One-way ANOVA followed by Tukey’s post-hoc analysis was performed: *p \u0026lt; 0.05; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001. \u003cstrong\u003eH\u003c/strong\u003e. Representative western blot image and (\u003cstrong\u003eI\u003c/strong\u003e) quantification of the total H3K9me/H3 ratio after the pharmacological treatments. Values presented are the mean ± SEM (n = 3 with at least 200 worms in each group). Unpaired t-test analysis was performed for N2 (WT) vs. 0 µM, 0 µM vs. FLAV-27, and UNC0638: *p \u0026lt; 0.05. \u003cstrong\u003eJ.\u003c/strong\u003e Quantification and (\u003cstrong\u003eK\u003c/strong\u003e) representative images of thioflavin S-positive particles in the head region of the CL2006 strain. Values presented are the mean ± SEM (n = 3 with 25 worms in each group). One-way ANOVA followed by Dunnett’s post-hoc analysis was performed for vehicle vs. FLAV-27: ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001. Unpaired t-test analysis was conducted for UNC0638 (0.1 µM) vs. FLAV-27 (0.1 µM): ## p \u0026lt; 0.001. \u003cstrong\u003eL.\u003c/strong\u003e Kaplan–Meier curve for the survival of CL2006 worms treated with FLAV-27. \u003cstrong\u003eM\u003c/strong\u003e. The mean lifespans of the CL2006 group treated with FLAV-27 and the control group treated with 1% DMSO. Values presented are the mean ± SEM\u0026nbsp;(n\u0026nbsp;= 3 with 50–70 worms in each group). One-way ANOVA followed by Tukey’s post-hoc analysis was performed: ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6940373/v1/6970144df465723faa44568d.jpeg"},{"id":85455254,"identity":"c8a65a15-79e6-4db0-9995-acd0e8fcf55c","added_by":"auto","created_at":"2025-06-26 06:12:47","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":336954,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of FLAV-27 on cognition and synaptic plasticity. \u003cstrong\u003eA. \u003c/strong\u003eExperimental timeline for drug administration and behavioral test. NORT evaluation by DI after (\u003cstrong\u003eB\u003c/strong\u003e) 2 h and (\u003cstrong\u003eC\u003c/strong\u003e) 24 h.\u003cstrong\u003e D. \u003c/strong\u003eOLT evaluation by DI after 24 h. Values presented are the mean ± SEM (SAMP8 Control, n = 10, and SAMP8 FLAV-27 (5 mg/kg), n = 10). Student’s t-test analysis was performed: *p \u0026lt; 0.05. \u003cstrong\u003eE.\u003c/strong\u003e Representative images of Golgi‐stained neurons and neuron spine density (scale bar = 100 µm). \u003cstrong\u003eF.\u003c/strong\u003e A number of neuronal intersections. Values presented are the mean ± SEM (SAMP8 Control, n = 50, and SAMP8 FLAV-27 (5 mg/kg), n = 50 images (from different neurons)). The Kolmogorov-Smirnov test was performed. \u003cstrong\u003eG. \u003c/strong\u003eNeuronal spine density quantification. Values presented are the mean ± SEM (SAMP8 Control, n = 60, and SAMP8 FLAV-27 (5 mg/kg), n = 60 images (from different neurons) were analyzed from 6 mice per group). The Mann-Whitney U test was conducted: **p \u0026lt; 0.01. \u003cstrong\u003eH.\u003c/strong\u003e The Aβ\u003csub\u003e42\u003c/sub\u003e/Aβ\u003csub\u003e40\u003c/sub\u003e ratio, (\u003cstrong\u003eI\u003c/strong\u003e) Aβ\u003csub\u003e40\u003c/sub\u003e, and (\u003cstrong\u003eJ\u003c/strong\u003e) Aβ\u003csub\u003e42\u003c/sub\u003e in the brain, as determined by ELISA. Values presented are the mean ± SEM (SAMP8 Control, n = 3, and SAMP8 FLAV-27 (5 mg/kg), n = 3). Student’s t-test analysis was performed: *p \u0026lt; 0.05. \u003cstrong\u003eK. \u003c/strong\u003eGene expression of \u003cem\u003eArc\u003c/em\u003e, \u003cem\u003eNT3\u003c/em\u003e, \u003cem\u003eComt\u003c/em\u003e, \u003cem\u003eSyt4\u003c/em\u003e, and (\u003cstrong\u003eL\u003c/strong\u003e) \u003cem\u003eTnf-α\u003c/em\u003e, \u003cem\u003eIl-6\u003c/em\u003e, and \u003cem\u003eIl-1b\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e. \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eM.\u003c/strong\u003e Representative traces of AMPAR-mediated miniature EPSCs recorded from hippocampal neurons from wild-type animals treated with vehicle (WT-DMSO; upper traces), knock-in mice treated with vehicle (KI-DMSO; middle traces) or knock-in mice treated with 1 μM FLAV-27 (KI-FLAV27; lower traces) for 72 hours prior to the recordings. The traces on the left show a 30-second recording period, while the traces on the right show a 2-second period. \u003cstrong\u003eN. \u003c/strong\u003eBar graph for the average values of mEPSCs for the three conditions presented previously in panel M. Columns represent the mean ± SEM. Dots in the graph represent the number of recordings (9, 12, and 13 recordings for WT, KI, and KI-FLAV27, respectively, from 3 independent cultures).\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6940373/v1/1958e46d69f29cbe837b3ed3.jpeg"},{"id":85455255,"identity":"df887bd1-7978-4e2e-886a-09b5e4a231db","added_by":"auto","created_at":"2025-06-26 06:12:47","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":594468,"visible":true,"origin":"","legend":"\u003cp\u003eFLAV-27 modulates neuronal, glial, and ferroptosis dysregulation in AD\u003cstrong\u003e. A. \u003c/strong\u003eVolcano plot of genes with a significant downregulation (blue) or upregulation (red) in FLAV-27 mice compared to control SAMP8 mice according to bulk RNA-seq. Dashed lines indicate the fold-change and FDR cut-offs used for the analysis. Numbers indicate the number of differentially expressed genes (DEGs) in each category. UMAP plots of SAMP8 control (left, \u003cstrong\u003eB\u003c/strong\u003e) and SAMP8 FLAV-27-treated mice (right, \u003cstrong\u003eC\u003c/strong\u003e) derived from single-cell RNA-seq data. Expression heatmap of gene markers for each cell type population in control (top, \u003cstrong\u003eE\u003c/strong\u003e) and FLAV-27-treated (bottom, \u003cstrong\u003eF\u003c/strong\u003e) samples. \u003cstrong\u003eG. \u003c/strong\u003eqPCR validation of genes with significant changes in different single-cell clusters: \u003cem\u003eElavl4\u003c/em\u003e, \u003cem\u003eSyt7\u003c/em\u003e, \u003cem\u003eFtl-1\u003c/em\u003e, and \u003cem\u003eFth-1\u003c/em\u003e, respectively. Values presented are the mean ± SEM (N = 2 groups: SAMP8 Control, n = 5, and SAMP8 FLAV-27 (5 mg/kg), n = 5). Student’s t-test analysis was performed: *p \u0026lt; 0.05; **p \u0026lt; 0.001. \u003cstrong\u003eH.\u003c/strong\u003e \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eBasal and LPS/IFN-γ-induced pro-inflammatory release of IL-1β was assessed by ELISA in the culture medium of iPSC-derived microglia treated with the G9a inhibitors FLAV-27 and UNC0642 at 1 μΜ for 24 h. \u003cstrong\u003eI.\u003c/strong\u003e The ability of human iPSC-derived microglia to phagocytose Aβ\u003csub\u003e1-42 \u003c/sub\u003eoligomers\u003cstrong\u003e \u003c/strong\u003eupon G9a inhibition with FLAV-27 or UNC0642 at 1 μΜ for 24 h was assessed by FACS. CytoD, an inhibitor of actin-mediated phagocytosis, was used as a negative control for Aβ\u003csub\u003e1-42 \u003c/sub\u003euptake. Values presented are the mean ± SEM. Statistical significance was assessed with one-way or two-way ANOVA with Tukey’s post-hoc analysis: *p \u0026lt; 0.05 (n ≥ 3). \u003cstrong\u003eJ. \u003c/strong\u003eGene expression of \u003cem\u003eGpx4\u003c/em\u003e and\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eFsp1\u003c/em\u003e. \u003cstrong\u003eK.\u003c/strong\u003e Levels of the GSG:GSSG ratio. Levels of (\u003cstrong\u003eL\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eROS, (\u003cstrong\u003eM\u003c/strong\u003e) Fe\u003csup\u003e2+\u003c/sup\u003e\u003cstrong\u003e, \u003c/strong\u003eand (\u003cstrong\u003eN\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eMDA in SAMP8 control and FLAV-27-treated (5 mg/kg) samples\u003cstrong\u003e.\u0026nbsp; \u003c/strong\u003eValues presented are the mean ± SEM (N = 2 groups: SAMP8 Control, n = 5-6, and SAMP8 FLAV-27 (5 mg/kg), n = 5-4). Student’s t-test analysis was performed: *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001. \u003cstrong\u003eO. \u003c/strong\u003eExperimental timeline for drug administration in \u003cem\u003eC. elegans\u003c/em\u003e.\u003cstrong\u003e \u003c/strong\u003eOCR values for (\u003cstrong\u003eP\u003c/strong\u003e) basal and (\u003cstrong\u003eQ\u003c/strong\u003e) mitochondrial respiration of WT and CL2006 control groups. Student’s t-test analysis with Welch’s correction was performed: *p \u0026lt; 0.05.\u0026nbsp; Percentage change of (\u003cstrong\u003eR\u003c/strong\u003e) basal and (\u003cstrong\u003eS\u003c/strong\u003e) mitochondrial OCR of WT and CL2006 worms after FLAV-27 exposure for 37 h. These values were normalized with the respective control condition. Values presented are the mean ± SEM. Each dot indicates values of technical replicates. Levels of (\u003cstrong\u003eT\u003c/strong\u003e) the GSH:GSSH ratio, (\u003cstrong\u003eU\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eROS, (\u003cstrong\u003eV\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eMDA, and (\u003cstrong\u003eW\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eFe\u003csup\u003e2+\u003c/sup\u003e in the brains of AD patients compared to controls with no dementia. Values presented are the mean ± SEM (N = 2 groups: no dementia, n = 3-4, and AD dementia, n = 4-5). Student’s t-test analysis was conducted: *p \u0026lt; 0.05; **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6940373/v1/dc9e0c2367b22089a6b5e1c7.jpeg"},{"id":85455257,"identity":"bbbf4ca2-58c2-4cd4-ba61-31d4ddaf74a0","added_by":"auto","created_at":"2025-06-26 06:12:47","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":852371,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of FLAV-27 on AD hallmarks in 5xFAD mice. \u003cstrong\u003eA. \u003c/strong\u003eExperimental timeline for drug administration and behavioral test. NORT:\u003cstrong\u003e \u003c/strong\u003eEvaluation of the working memory by DI after (\u003cstrong\u003eB\u003c/strong\u003e) 2 h and\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eC\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003e24 h. \u003cstrong\u003eD.\u003c/strong\u003e OF: Total distance. \u003cstrong\u003eE.\u003c/strong\u003e The number of rearings recorded during the OF test. \u003cstrong\u003eF. \u003c/strong\u003eOLT: Evaluation of the spatial memory by DI after changing the location of one object. \u003cstrong\u003eG. \u003c/strong\u003eSniffing time in the three-chamber test. Values are the mean ± SEM (N = 4 groups: WT Control, n = 8, 5xFAD Control, n = 7, 5xFAD FLAV-27 1+0 (5 mg/kg), n = 8, and 5xFAD FLAV-27 0+1 (5 mg/kg), n = 7). One-way ANOVA followed by Tukey’s post-hoc analysis was performed: *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001. Quantitative analysis of thioflavin-S staining and the average size of the Aβ plaques in\u003cstrong\u003e \u003c/strong\u003ethe (\u003cstrong\u003eH\u003c/strong\u003e) hippocampus and (\u003cstrong\u003eI\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003ecortex. \u003cstrong\u003eJ. \u003c/strong\u003eRepresentative images of Aβ plaques labeled with thioflavin-S. \u003cstrong\u003eK. \u003c/strong\u003eRepresentative images of Golgi‐stained neurons and the spine density of the different experimental conditions (scale bar = 100 µm). \u003cstrong\u003eL.\u003c/strong\u003e Number of neuronal intersections. Values presented are the mean ± SEM (N = 4 groups: WT Control, n = 50 neurons, 5xFAD Control, n = 50 neurons, , 5xFAD FLAV-27 1+0 (5 mg/kg), n = 50 neurons, and 5xFAD FLAV-27 0+1 (5 mg/kg), n = neurons). The Mann-Whitney U test was performed: ***p \u0026lt; 0.001.\u003cstrong\u003e M. \u003c/strong\u003eSpine density quantification of neurons. Values presented are the mean ± SEM (N = 4 groups: WT Control, n = 90-100, 5xFAD Control, n = 90-100, 5xFAD FLAV-27 1+0 (5 mg/kg), n = 90-100 dendrites, and 5xFAD FLAV-27 0+1 (5 mg/kg), n = 90-100 images (from different neurons)). The Mann-Whitney U test was conducted: *p \u0026lt; 0.05; **p \u0026lt; 0.001; ****p \u0026lt; 0.0001. \u003cstrong\u003eN. \u003c/strong\u003eRepresentative images of GFAP (red) and Hoechst (blue) positive staining and \u003cstrong\u003eO.\u003c/strong\u003e quantification of the whole hippocampus. \u003cstrong\u003eP. \u0026nbsp;\u003c/strong\u003eQuantification of GFAP levels is shown specifically in the CA3 area. The mean intensity of GFAP staining is hereby indicated. Values presented are the mean ± SEM (N = 5 groups: WT Control, n = 4, 5xFAD Control, n = 4, n = 4, 5xFAD FLAV-27 1+0 (5 mg/kg), n = 4, and 5xFAD FLAV-27 0+1 (5 mg/kg), n = 4). One-way ANOVA followed by Tukey’s post-hoc analysis was conducted: *p \u0026lt; 0.05; **p \u0026lt; 0.01. \u003cstrong\u003eQ.\u003c/strong\u003e Heatmap of the top differentially expressed proteins across all groups, clustered by the Euclidean distance. Volcano plots depicting differential protein expression in 5XFAD mice with respect to (\u003cstrong\u003eR\u003c/strong\u003e) early and (\u003cstrong\u003eS\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003elate treatment groups. \u003cstrong\u003eT. \u003c/strong\u003eProtein-protein interaction networks of differentially regulated proteins in 5xFAD mice compared to WT, showing a network of downregulated proteins in 5xFAD mice. The map displays distinct functional groups involved in neuronal and synaptic signaling, cellular metabolism, proteostasis, as well as membrane trafficking and signal transduction. Proteins with a minimum interaction score of 0.5 were included, and clustering was performed using the MCL algorithm with an inflation parameter of 2.3. Proteins significantly reversed by treatment interventions are highlighted by the frames. \u003cstrong\u003eU.\u003c/strong\u003e A network of upregulated proteins in 5xFAD mice. Distinct clusters center on processes such as synaptic pruning, phosphatidylinositol signaling, B cell signaling, and cytoskeletal organization. Here, proteins were included at a minimum interaction score of 0.65, and MCL clustering was conducted with an inflation parameter of 1.7. Proteins significantly reversed by treatment are also indicated by the frames. These data suggest that increases and decreases in protein levels contribute to disease-related alterations in metabolic pathways, vesicle trafficking, immune function, and neuronal signaling. Clustering was performed using the MCL algorithm.\u003csup\u003e32\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6940373/v1/ae33d9b9387e2b721932c69d.jpeg"},{"id":85455258,"identity":"4d5cc7b6-7618-400a-81c5-f26f6882890c","added_by":"auto","created_at":"2025-06-26 06:12:47","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":231821,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFluid biomarkers after FLAV-27 treatment in 5xFAD mice.\u003c/strong\u003e Plasma levels of (\u003cstrong\u003eA\u003c/strong\u003e) H3K9me2 and (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eSMOC1. Values presented are the mean ± SEM (N = 4 groups: WT Control, n = 8, 5xFAD Control, n = 7, 5xFAD FLAV-27 1+0 (5 mg/kg), n = 8, and 5xFAD FLAV-27 0+1 (5 mg/kg), n = 7). One-way ANOVA followed by Tukey’s post-hoc analysis was performed. In some cases, Student’s t-test was also performed: *\u003csup\u003e/#\u003c/sup\u003ep \u0026lt; 0.05; **p \u0026lt; 0.01. \u003cstrong\u003eC. \u003c/strong\u003eCorrelation heatmap between all the plasma biomarkers for all the experimental groups. The numbers represent Spearman’s correlation coefficients, indicating the strength and direction of the relationships between the biomarkers. Blue indicates positive correlations and orange indicates negative correlations. Plasma levels of (\u003cstrong\u003eD\u003c/strong\u003e) H3K9me2 and (\u003cstrong\u003eE\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eSMOC1, as well as (\u003cstrong\u003eF\u003c/strong\u003e) CSF levels of\u003cstrong\u003e \u003c/strong\u003eSMOC1 in human samples. \u003cstrong\u003eG. \u003c/strong\u003eMMSE scores. Values presented are the mean ± SEM (N = 3 groups: No dementia, n = 13, AD Prodromal, n = 14, and AD dementia, n = 12). One-way ANOVA followed by Tukey’s post-hoc analysis was performed. In some cases, Student’s t-test was also performed: *p \u0026lt; 0.05; **p. \u0026lt;0.01; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001. \u003cstrong\u003eH. \u003c/strong\u003eCorrelation heatmap between all the plasma and CSF biomarkers, and the MMSE scores for all the experimental groups. The numbers represent Spearman’s correlation coefficients, indicating the strength and direction of the relationships between the biomarkers. Red indicates\u003cstrong\u003e \u003c/strong\u003epositive correlations and gray indicates negative correlations. \u003cstrong\u003eI. \u003c/strong\u003eRepresentative western blot images and (\u003cstrong\u003eJ\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003equantification of SMOC1 levels. Values presented are the mean ± SEM (N = 2 groups: No dementia, n = 3-4, and AD dementia, n = 4-5). Student’s t-test analysis was conducted: *p \u0026lt; 0.05; **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6940373/v1/a5b5a08e90ca08a293d6fd5a.jpeg"},{"id":85455454,"identity":"fae97047-08ec-4588-8f21-f1a5e93614dd","added_by":"auto","created_at":"2025-06-26 06:20:47","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":294026,"visible":true,"origin":"","legend":"\u003cp\u003eSummary of the neuroprotective and disease-modifying effects of FLAV-27 across experimental models, and the validation of different AD biomarker in AD human patients.\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6940373/v1/1c0131fce658b9d95e716aa6.jpeg"},{"id":85684297,"identity":"c63c2b51-d905-4818-b820-df2ffb940358","added_by":"auto","created_at":"2025-06-30 15:43:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4767543,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6940373/v1/8c120bb5-0c1e-47a0-aa76-14367a83fb31.pdf"},{"id":85455261,"identity":"09d6bf0e-a27e-449e-9ef4-03dae03bc91c","added_by":"auto","created_at":"2025-06-26 06:12:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5788476,"visible":true,"origin":"","legend":"supplementary tables","description":"","filename":"SupplementaryinformationFigureandTables050625.docx","url":"https://assets-eu.researchsquare.com/files/rs-6940373/v1/5c5f2d066d272bb3637d56ff.docx"},{"id":85455273,"identity":"1ae2cdb6-e48c-4117-b1a5-16d1f5e7d3d8","added_by":"auto","created_at":"2025-06-26 06:12:47","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13592352,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"SupplementaryinformationSyntheticcharacterizationandstructureactivityrelationshipsdetails050625.docx","url":"https://assets-eu.researchsquare.com/files/rs-6940373/v1/2abe474af231e7fa6cd4c8c6.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"First-in-Class SAM-Competitive G9a Inhibitor FLAV-27 as a Disease-Modifying Therapy for Alzheimer’s Disease","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAlzheimer\u0026rsquo;s disease (AD) is one of the most pressing and unresolved medical challenges of our time. Collectively affecting hundreds of millions of individuals worldwide, this neurodegenerative disorder is characterized by progressive neuronal loss and cognitive decline, without any effective disease-modifying treatments.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Despite intensive research, clinical progress has been limited by the multifactorial nature of the neurodegeneration, an incomplete understanding of its molecular underpinnings, and the formidable barrier posed by the blood\u0026ndash;brain barrier (BBB) that restricts CNS drug delivery.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eEmerging evidence implicates epigenetic dysregulation as a central contributor to the pathogenesis of neurodegenerative diseases. Unlike irreversible genetic mutations, epigenetic marks such as histone methylation are dynamic and potentially reversible, making them attractive therapeutic targets.\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e In particular, two histone methyltransferases (HMTs), GLP (EHMT2) and G9a (EHMT2), have attracted increasing attention due to their role in catalyzing the dimethylation of histone H3 at lysine 9 (H3K9me2), a repressive mark associated with transcriptional silencing. G9a/GLP-mediated epigenetic repression has been shown to influence critical processes such as neuronal development, synaptic plasticity, and memory consolidation.\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIntriguingly, an aberrant upregulation of G9a activity has been linked to increased oxidative stress (OS), neuroinflammation, and neuronal dysfunction, which are hallmarks of AD and other neurodegenerative conditions. However, translating G9a inhibition into a viable therapeutic strategy has proven to be difficult. Most known G9a inhibitors, including BIX-01294, UNC0638, and A-366, suffer from poor selectivity, high cytotoxicity, and inadequate BBB permeability, which are limitations that are less critical in oncology but represent major obstacles for CNS applications.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Consequently, the therapeutic potential of G9a inhibition in neurodegeneration remains largely untapped.\u003c/p\u003e \u003cp\u003eHere, we report the discovery and characterization of FLAV-27, a brain-penetrant, subnanomolar inhibitor of G9a with exceptional selectivity for G9a over the closely related GLP and other methyltransferases. Unlike previously reported G9a inhibitors, FLAV-27 exhibits favorable CNS drug-like properties, including excellent BBB permeability and a strong safety profile. Structural studies revealed a unique SAM-competitive binding mode, as demonstrated by the first X-ray co-crystal structure of FLAV-27 bound to human G9a. To evaluate its therapeutic potential, we employed a comprehensive, cross-species strategy incorporating primary neuronal and microglial cultures, the SAMP8 mouse model of AD, and \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e (\u003cem\u003eC. elegans\u003c/em\u003e) models of neurodegeneration. The results showed that FLAV-27 reduced H3K9me2 levels, attenuated neuroinflammation, mitigated pathological protein aggregation, and restored cognitive and motor functions. In SAMP8 mice, FLAV-27 treatment led to marked improvements in memory performance, the reactivation of neuroprotective gene expression, and the suppression of the pro-inflammatory cytokine IL-1β. These effects were supported by converging evidence from behavioral, molecular, and epigenetic profiling. In \u003cem\u003eC. elegans\u003c/em\u003e, FLAV-27 decreased Aβ aggregation, enhanced mitochondrial respiration, and extended the lifespan. Building on these promising findings, we further assessed the disease-modifying potential of FLAV-27 in the 5xFAD transgenic mouse model of early-onset AD, observing that FLAV-27 reversed the established cognitive and social deficits, reduced plaque burden, normalized markers of ferroptosis, and reprogrammed pathological proteomic signatures. Importantly, to assess clinical relevance, we analyzed human post-mortem brain tissue, cerebrospinal fluid (CSF), and plasma from AD patients. We found that the key epigenetic and molecular markers altered in animal models, namely H3K9me2, H3K18 methylation, and SMOC1, were significantly elevated in human AD samples. Furthermore, SMOC1 and H3K9me2 plasma levels correlated with tau pathology, neuroinflammatory markers, and cognitive decline, supporting their potential use as peripheral biomarkers of disease progression and therapeutic response. Together, our results position FLAV-27 as a first-in-class selective G9a inhibitor with disease-modifying potential for treating AD and related neurodegenerative disorders, highlighting the translational value of integrated epigenetics- and biomarker-based therapeutic strategies.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eBuilding on prior efforts to identify novel epigenetic modulators, we undertook a structure-based design strategy to discover new G9a inhibitors. Interestingly, a promising chemotype emerged, featuring a central 1,4-oxazepane scaffold flanked by a piperidine ring and an aryl moiety. This scaffold served as the foundation for a medicinal chemistry campaign to optimize potency, selectivity, and brain penetration (see SI for details on synthesis, characterization, and structure-activity relationships, Tables S1-S5, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-E). In the initial evaluations, all compounds were analyzed as racemic mixtures.\u003c/p\u003e \u003cp\u003eIn the initial phase, modifications were introduced to the aryl ring attached to the piperidine. A wide range of substituents at the meta, para, and ortho positions were tested (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The majority of the analogs displayed weak or no activity (IC₅₀ \u0026gt; 1 \u0026micro;M), but several compounds, such as 2 (m-NH₂), 4 (m-CO₂CH₃), 10 (p-OH), 12 (p-Cl), and 17 (o-OH), showed inhibition in the low nanomolar range. These results pointed to the critical role of the \u003cem\u003em\u003c/em\u003e-hydroxyl group in achieving potent inhibition.\u003c/p\u003e \u003cp\u003eSubsequently, the \u003cem\u003em\u003c/em\u003e-hydroxyphenyl group was replaced by various heterocycles (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), but none of the resulting analogs retained nanomolar activity. Similarly, ring contraction of the piperidine to pyrrolidine abolished activity entirely, likely due to conformational changes and the generation of a new stereocenter.\u003c/p\u003e \u003cp\u003eFurther efforts focused on the oxazepane core. Replacement with a morpholine ring (Table S3) yielded analogs such as 32 and 35 with reduced but measurable activity. Methylation of the nitrogen atom within the oxazepane ring (Table S4) significantly diminished potency, as did modifications of the benzylic moiety. Among the latter, only compound 47 (p-chlorobenzyl) showed activity close to that of the reference compounds (IC₅₀ = 1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 nM; Table S5).\u003c/p\u003e \u003cp\u003eUltimately, the most promising compound emerged as a result of this iterative process. Compound 1, herein referred to as FLAV-27, featuring an \u003cem\u003em\u003c/em\u003e-hydroxyphenyl ring, a piperidine linker, a 1,4-oxazepane core, and a benzylic substituent, exhibited the highest potency (IC₅₀ = 0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001 nM) and excellent selectivity over GLP (3.2% inhibition at 1 \u0026micro;M). FLAV-27 also outperformed standard G9a inhibitors such as UNC0638 (IC₅₀ = 11.1\u0026thinsp;\u0026plusmn;\u0026thinsp;8 nM) and UNC0642 (IC₅₀ = 6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7 nM), being more selective for G9a. It should be noted that neither UNC0642 nor UNC0638 are selective as it has been proven that they inhibit both G9a and GLP.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Moreover, an enantiomeric separation using semi-preparative chiral HPLC of FLAV-27 was performed, although no significant differences in ADME properties were observed (see SI for details on synthesis, characterization, and structure-activity relationships).\u003c/p\u003e \u003cp\u003eA further relevant finding was the impressive selectivity profile. FLAV-27 demonstrated high selectivity for G9a, exhibiting\u0026thinsp;\u0026lt;\u0026thinsp;15% inhibition at 1 \u0026micro;M across a panel of 32 HMTs, including H3K9 (SUV39H1, SUV39H2, and SUV420H1), H3K27 (EZH2), H3K4 (SETD7 and MLL), H3K79 (DOT1L), and H4K20 (SETD8), as well as various protein arginine methyltransferases (PRMTs) (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF). In addition, FLAV-27 showed no inhibitory activity at 10 \u0026micro;M against a panel of 50 off-target kinases, supporting its clean selectivity profile (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG). This result is highly relevant as other compounds that are considered selective inhibitors of methyl transferases, such as UNC0638 and UNC0642, inhibit both G9a and GLP.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eX-ray crystallography examining the binding of FLAV-27 at the human G9a SAM-site\u003c/h2\u003e \u003cp\u003eG9a inhibitors can interfere with either the lysine-binding site or the binding site of S-adenosylmethionine (SAM), the methyl donor. Inhibitors targeting the latter are referred to as SAM-competitive. Enzyme kinetics conducted at a fixed concentration of the lysine-containing H3 peptide and increasing levels of SAM (0\u0026ndash;7 \u0026micro;M) revealed a significant reduction of the maximum velocity (V\u003csub\u003emax\u003c/sub\u003e) by FLAV-27 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Consistent with its low IC\u003csub\u003e50\u003c/sub\u003e, the decrease in V\u003csub\u003emax\u003c/sub\u003e was already significant even at the lowest FLAV-27 concentration tested (1 nM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). UNC0642, a non-selective G9a inhibitor, did not reduce the V\u003csub\u003emax\u003c/sub\u003e of the enzyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), suggesting that its interaction with G9a differs mechanistically from that of FLAV-17. This implies that UNC0642 and FLAV-17 bind to distinct sites on the enzyme. Moreover, co-treatment with FLAV-27 and UNC0642 did not result in a synergistic inhibition of G9a activity (Figure S6C\u0026ndash;D), further supporting the hypothesis of non-overlapping binding sites. To elucidate the precise binding mode of FLAV-27, we employed a structural biology approach and successfully resolved the crystal structure of the human G9a enzyme in complex with FLAV-27. Provided that FLAV-27 binds to the SAM-binding site, it would confer unprecedented selectivity and position FLAV-27 as the first G9a inhibitor to directly block co-substrate binding, thereby inhibiting both methyl transfer and overall enzymatic activity.\u003c/p\u003e \u003cp\u003eProtein\u0026ndash;inhibitor crystals were obtained under the conditions detailed in the methods (see SI for more details). Subsequent X-ray crystallographic analyses revealed a homodimeric architecture, with the inhibitor occupying the binding pocket of each protomer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-E, Table S6, Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The active site in the structure reported in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC is similar to the structure reported for the heterodimer formed by the suppressor of variegation 3\u0026thinsp;\u0026minus;\u0026thinsp;9, enhancer of zeste, and trithorax (SET) domain of G9a (residues 913 to 1193 of the human sequence) and the SET domain of GLP (residues 982 to 1266 of the human sequence) (PDB 5TTF).\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn the reported G9a SET\u0026ndash;SAM complex structure (PDB: 5TTF), the position of the SAM co-substrate is defined in Xiong et al. (2017).\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Structural comparison between the G9a\u0026ndash;FLAV-27 complex and the 5TTF crystal structure revealed that FLAV-27 occupies the same binding pocket as SAM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The binding mode was, however, different as FLAV-27 binding is primarily driven by hydrophobic interactions (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD-E), in contrast to SAM, which establishes a more extensive hydrogen bonding network (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Notably, the nitrogen atom of the piperidine ring in FLAV-27 forms hydrogen bonds with the hydroxyl group of Tyr1154 and the backbone carbonyl of Ser1084 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). These interactions are absent in the SAM-bound structure and may contribute to the enhanced stability of FLAV-27 binding.\u003c/p\u003e \u003cp\u003eAdditionally, the hydroxyl group of the phenol ring in FLAV-27 forms a hydrogen bond with the side chain hydroxyl of Tyr1085, an interaction that is also observed in the SAM complex. The phenol ring of FLAV-27 is further stabilized by hydrophobic contacts with Trp1050 and Phe1110 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), which are interactions that are not present in the G9a SET\u0026ndash;SAM structure. These contacts may underlie the stronger binding of FLAV-27. Finally, the benzyl ring of FLAV-27 is deeply buried within a hydrophobic pocket in G9a, forming significant hydrophobic interactions. This increased hydrophobic contribution in the G9a\u0026ndash;FLAV-27 complex likely explains its enhanced binding affinity and may underlie its greater inhibitory potency.\u003c/p\u003e \u003cp\u003eIn fact, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-H and Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eF-J explain the difference between G9a and GLP. The active sites of the G9a and GLP proteins look very similar, but the surface charge distribution is different. The surface charge distribution (SCD) of G9a clearly suggests that the SAM-binding site is hydrophobic with little negative charge distribution, while the GLP SCD for SAM binding is mostly positively charged. This could be a possible reason for why the hydrophobic FLAV-27 compound might favor binding to G9a instead of GLP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eassessment of FLAV-27 efficacy\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn our previous experiments with the SAMP8 senescence model, we detected epigenetic alterations related to the methylation of histones. To better characterize the epigenetic landscape and simultaneously evaluate the \u003cem\u003ein vivo\u003c/em\u003e efficacy of FLAV-27, we first assessed global histone methylation profiles in cortical tissue. As expected, SAMP8 mice treated with FLAV-27 exhibited markedly reduced levels of H3K9me2 in the brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B), confirming effective inhibition of the primary enzymatic activity of G9a. Given that G9a has also been implicated in catalyzing methylation at additional histone sites, we extended our analysis to other epigenetic marks. FLAV-27 treatment selectively reversed the aberrant levels of H3K18me1/2/3, modifications that have been previously linked to G9a function,\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e without having detectable effects on H3K23me1/2/3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003eTo gain a genome-wide perspective on these epigenetic changes, ChIP-seq analysis for H3K9me2 was performed in the cortical tissue of control SAMP8 mice and SAMP8 mice treated with FLAV-27. This approach enabled us to map the epigenetic changes induced by FLAV-27 and to identify the genomic regions where the G9a-regulated repressive histone modification H3K9me2 is dynamically altered in the context of aging. Our results revealed a clear global decrease in H3K9me2 levels in FLAV-27-treated samples, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC. Furthermore, across different false discovery rate (FDR) thresholds during peak calling, we observed that FLAV-27-treated samples consistently presented fewer H3K9me2 peaks (Figure S3A), with the remaining peaks showing significantly lower H3K9me2 enrichment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D). While the majority of the peaks exhibited reduced methylation after FLAV-27 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), we focused specifically on the subset of peaks that showed the strongest H3K9me2 decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-G). To investigate the functional impact of these changes, we identified the genes whose promoters were closest to these downregulated H3K9me2 peaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). We then performed gene set enrichment and ontology analyses on this gene set. Validating our approach, these genes showed overlap with known repressive histone modifications (H3K9me3 and H3K27me3) from the ENCODE datasets (Figure S3B), supporting the notion that FLAV-27 broadly affects heterochromatin organization. Strikingly, there was a notable enrichment of GO terms associated with synaptic structure and function after FLAV-27 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Pathways linked to synaptic plasticity and structural synapse development, such as \"synapse assembly\u0026rdquo;, \"regulation of synapse structure or activity\", and \"positive regulation of synapse assembly\u0026rdquo;, were significantly upregulated in the FLAV-27-treated SAMP8 mice, according to the GO enrichment analysis. These pathways, which regulate dendritic growth, receptor trafficking, and synaptic potentiation, involve important genes like \u003cem\u003eGria1\u003c/em\u003e, \u003cem\u003eShank3\u003c/em\u003e, \u003cem\u003eSnap25\u003c/em\u003e, \u003cem\u003eSlitrk5/6\u003c/em\u003e, and \u003cem\u003eDlgap1\u003c/em\u003e. An enrichment was also observed of pre-synapse and post-synapse organization terms highlighting a coordinated reconstruction of both ends of the synaptic cleft that is necessary for effective neurotransmission. The presence of other GO terms like \"cognition\" and \"regulation of synaptic plasticity\" that directly align with higher-order neurological processes in the FLAV-27-treated group suggested possible behavioral and memory-level rescue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Conversely, maladaptive neuroplasticity and the suppression of inhibitory synaptic signaling were found in the GO pathways downregulated in the FLAV-27-treated group. The terms \"inhibitory synapse assembly\", \"GABAergic synaptic transmission\", and \"GABA signaling\", which involve genes like \u003cem\u003eGabra1\u003c/em\u003e, \u003cem\u003eGad1\u003c/em\u003e, \u003cem\u003eGabrg2\u003c/em\u003e, and \u003cem\u003eSlc6a1\u003c/em\u003e, were significantly enriched. This indicates that FLAV-27 improves synaptic plasticity and the excitatory/inhibitory rebalance by suppressing dysfunctional inhibitory circuits. Indirect epigenetic silencing of neuroinflammatory pathways by FLAV-27 is suggested by the repression of several genes associated with inflammation and oxidative stress. The suppression of redox-sensitive genes indicates possible dampening of pro-degenerative oxidative pathways, even though ferroptosis itself was not enriched. The downregulation of the terms associated with inhibitory and inflammatory signals represents a neuroprotective response (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003eFurthermore, many sequences near the hypomethylated regions are transcribed into non-coding RNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK), suggesting that FLAV-27 may impact the expression of regulatory RNA species whose functions are still largely unknown. Strikingly, the genes associated with regions of H3K9me2 loss were enriched in functions related to serotonin signaling, the regulation of nervous system processes, anxiety, and behavioral responses to fear (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL-M), indicating that the central impairments previously reported in this senescence-accelerated SAMP8 model correlate with epigenetic traits. Complementary to these findings, western blot analysis of histone modifications in human post-mortem brain tissue was performed. Increased levels of H3K9me2 and H3K18 mono-, di-, and tri-methylation were observed in the AD brains, with no significant changes detected in H3K23 methylation states (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN-O).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eModulation of Aβ₁₋₄₂, tau, and p-tau aggregation and neuritic integrity in primary neuronal cultures\u003c/h3\u003e\n\u003cp\u003eAD is characterized by the accumulation of toxic protein aggregates, including Aβ and hyperphosphorylated tau (p-tau), which disrupt neuronal function and contribute to synaptic degeneration and cognitive decline. To initially explore the potential neuroprotective properties of FLAV-27, \u003cem\u003ein vitro\u003c/em\u003e experiments were conducted using mixed neuronal and microglial primary cultures treated with Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e (500 nM), tau (1 \u0026micro;M), or p-tau (1 \u0026micro;M) protein aggregates for 48 hours, before exposure to FLAV-27 (1 \u0026micro;M) for 24 h. Results demonstrated a significant reduction in Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e, tau, and p-tau aggregation after FLAV-27 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). Additionally, neurite patterning was analyzed by immunocytochemistry in primary cortical neuronal cultures under the same treatment conditions. The data revealed a marked loss of neurite formation after exposure to Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e, tau, or p-tau aggregates, which was largely reversed by FLAV-27 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F).\u003c/p\u003e\n\u003ch3\u003eReversion of cognitive impairments in a worm and a mouse model\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eReversion of cognitive impairments in a worm and a mouse model\u003c/div\u003e \u003cp\u003eWe investigated whether the selective inhibition of G9a would be efficacious in restoring central impairments by using FLAV-27 and non-mammalian and mammalian models of AD that are widely accepted as tools to test the efficacy of potential anti-AD drugs.\u003c/p\u003e \u003cp\u003eFirst, FLAV-27 was tested on the CL2006 transgenic strain of \u003cem\u003eC. elegans\u003c/em\u003e, which develops age-related paralysis upon expression of human Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e in the muscle cells.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e FLAV-27 treatment reduced locomotor impairment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), indicating its potential to alleviate Aβ-associated phenotypes. Notably, CL2006 worms exhibit elevated H3K9me2 levels and increased expression of \u003cem\u003eset-25\u003c/em\u003e, the ortholog of human G9a/EHMT2, linking epigenetic dysregulation to Aβ pathology.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Treatment with FLAV-27 (1 \u0026micro;M) significantly reduced the H3K9me2/H3 ratio compared to vehicle, while the reference inhibitor UNC0638 showed only a non-significant trend towards reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-I). Moreover, FLAV-27 significantly reduced Aβ aggregation by approximately 45% relative to the vehicle group, surpassing the effect of UNC0638 (~\u0026thinsp;26% reduction) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ-K). To evaluate the potential impact on organismal longevity, we conducted survival analyses in CL2006 worms. Although no significant differences in the overall lifespan were observed, FLAV-27 extended the mean lifespan by up to 22% in the CL2006 strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL-M).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFLAV-27 was also tested in the senescence-accelerated SAMP8 model, which is considered a model of late-onset AD in which non-selective methyltransferase inhibitors have shown efficacy.\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e These experiments were essential to refine and standardize drug administration protocols, ensuring optimal delivery and reproducibility. Drug administration commenced at 4 months of age, as this time point corresponds to the early symptomatic phase of age-related cognitive decline in the SAMP8 mouse model.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e FLAV-27 was administered orally at a dose of 5 mg/kg body weight once daily for one month (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To assess the effects of FLAV-27 on working memory, the novel object recognition test (NORT) was performed. During the familiarization phase (Figure S3C), FLAV-27 had no effect on the total exploration time. Importantly, FLAV-27 treatment resulted in a significant improvement in both short-term (measured at 2 hours post-training) and long-term (measured at 24 hours post-training) object recognition memory, as evidenced by the NORT performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). Similarly, exploration during the habituation phase remained unchanged by FLAV-27 treatment in the object location test (OLT) (Figure S3D). Additionally, a significant enhancement in the discrimination index (DI) was observed during the OLT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTo gain a deeper insight into the mechanisms by which selective G9a inhibition might enhance cognitive function in the SAMP8 model, cortical brain sections from vehicle-treated and FLAV-27-treated animals were processed using the Golgi staining method. This technique allowed for a detailed morphological analysis of individual neurons, focusing on two key indicators of synaptic plasticity: dendritic spine density and neuronal branching complexity. Dendritic complexity was quantified using the Sholl analysis, a well-established neuroanatomical method that involves centering concentric circles on the neuronal soma and counting the number of dendritic intersections at increasing radial distances. This approach provides a robust measure of dendritic arborization, which is closely linked to the capacity for synaptic integration of a neuron (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-F). Remarkably, animals treated with FLAV-27 exhibited a significant increase in both dendritic spine density and the number of neuronal intersections compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-G). These findings support the hypothesis, previously proposed by the \u003cem\u003ein vitro\u003c/em\u003e studies, that selective G9a inhibition enhances neuronal plasticity. Importantly, the observed improvements in the dendritic architecture correlated with the reversal of cognitive deficits in the SAMP8 model, reinforcing the therapeutic potential of G9a inhibitors for age-related neurodegenerative disorders.\u003c/p\u003e \u003cp\u003eAβ levels are associated with behavioral abnormalities and cognitive decline; thus, we evaluated the Aβ\u003csub\u003e42\u003c/sub\u003e/Aβ\u003csub\u003e40\u003c/sub\u003e ratio in the brain. After FLAV-27 treatment, brain levels of the Aβ\u003csub\u003e42\u003c/sub\u003e/Aβ\u003csub\u003e40\u003c/sub\u003e ratio was reduced in the SAMP8 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Levels of Aβ\u003csub\u003e42\u003c/sub\u003e were also reduced after FLAV-27 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI), while Aβ\u003csub\u003e40\u003c/sub\u003e levels remained unaltered (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003eFLAV-27 treatment also induced significant changes in the expression of genes associated with synaptic plasticity and neuroinflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK-L). Specifically, FLAV-27 administration increased the expression of \u003cem\u003eSyt4\u003c/em\u003e and \u003cem\u003eArc\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK), two genes that are critically involved in synaptic function, neurotransmitter release, and activity-dependent neuronal remodeling.\u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e The increase in these markers in the hippocampus suggests a potential enhancement of synaptic integrity and cognitive resilience. In parallel, FLAV-27 treatment led to a marked reduction in the expression of \u003cem\u003eIl-6\u003c/em\u003e and \u003cem\u003eTnf-α\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL), which are key pro-inflammatory cytokines known to be elevated in neurodegenerative conditions and implicated in AD progression. These findings suggest that FLAV-27 supports synaptic gene programs and exerts anti-inflammatory effects in the aging AD brain.\u003c/p\u003e \u003cp\u003eFurthermore, to explore the effects of G9a inhibition on synaptic transmission, we measured AMPAR-mediated spontaneous miniature excitatory postsynaptic currents (mEPSCs) in primary cortical neurons from knock-in (KI) mice after exposure to FLAV-27 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM-N). To isolate AMPAR-mEPSCs, D-(-)-2-amino-5-phosphonopentanoic acid and picrotoxin were added to the perfusion to block NMDA and GABA\u003csub\u003eA\u003c/sub\u003e receptors, respectively. FLAV-27 treatment for 24 hours significantly increased mEPSC amplitudes, suggesting an enhancement in postsynaptic AMPAR expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM). By contrast, the mEPSC frequency remained unchanged, indicating no presynaptic alterations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM). These findings indicate that FLAV-27 promotes neuronal plasticity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eN).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eReversal of neuronal, glial, and ferroptosis dysregulation in AD\u003c/h3\u003e\n\u003cp\u003eTo further characterize the molecular effects of FLAV-27 in the SAMP8 AD mouse model, we began by performing bulk RNA sequencing on samples of hippocampal tissue from vehicle-treated and FLAV-27\u0026ndash;treated mice. Differential gene expression (DEG) analysis revealed a pattern of de-repression consistent with G9a inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Gene Ontology (GO) enrichment analysis of the upregulated genes indicated a strong association with immune-related pathways (Figure S3E). Additionally, we applied the RNA-Age Calculator tool\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e to estimate the molecular age of the treated and control animals based on transcriptomic data. Although differences were not statistically significant due to sample size limitations, FLAV-27\u0026ndash;treated mice exhibited a trend towards a reduced molecular age relative to controls (Figure S3F).\u003c/p\u003e \u003cp\u003eTo further dissect the cell-type-specific mechanisms underlying these transcriptomic changes, we employed single-cell RNA sequencing (scRNA-seq). Unlike bulk RNA-seq, which captures average gene expression across heterogeneous tissue, scRNA-seq enables the examination of transcriptional dynamics at the individual cell level. This approach allowed us to characterize cellular heterogeneity and identify distinct populations that are particularly responsive to FLAV-27 treatment. Through this analysis, we aimed to pinpoint key cell types and molecular pathways affected by selective G9a inhibition, thereby advancing our understanding of the therapeutic potential of FLAV-27 in AD.\u003c/p\u003e \u003cp\u003eGene expression analyses revealed important transcriptional changes in response to FLAV-27 treatment. We successfully sequenced 6,316 cells for SAMP8 controls, with a median of 1,215 UMIs per cell, and 11,919 cells for FLAV-27-treated mice, with a median of 1,050 UMIs per cell. Subsequent clustering analysis based on transcriptional profiles identified distinct cell types in both the control and FLAV-27 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). Notably, while analyzing cell type composition, we observed a two-fold decrease in microglia, a known disease driver, upon FLAV-27 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Notably, neurons in FLAV-27-treated mice displayed a transcriptional profile consistent with enhanced synaptic function, which aligns with our previous findings indicating that FLAV-27 supports neuronal activity. Interestingly, in the control samples, cluster 1 exhibited signatures of microglial and astrocytic activation, reflecting the characteristic pro-inflammatory environment seen in AD. By contrast, cluster 1 in FLAV-27-treated samples showed a gene expression profile associated with pathways related to synaptic transmission and nervous system development (Figure S3G-H). In fact, the most prominent upregulated pathways were those that regulated vesicle-mediated transport in the synapse, synapse assembly, post-synapse organization, synapse structure or activity, and signal release from the synapse. Furthermore, GO terms associated with the modulation of neuroinflammatory responses, such as gliogenesis, regulation of cytokine production, and the response to interleukin-1, were enriched, with an upregulation of genes like \u003cem\u003eSerpine1\u003c/em\u003e, \u003cem\u003eNfkbia\u003c/em\u003e, and \u003cem\u003eCcl3\u003c/em\u003e. Changes in the genes related to the redox balance and iron-related stress responses indicated that FLAV-27 could indirectly inhibit ferroptosis, contributing to its neuroprotective action, even though ferroptosis-related GO terms were not directly enriched. Together, these findings highlight how FLAV-27 triggers a transcriptional program that supports synaptic integrity, cognitive function, and inflammatory control, while potentially reducing the onset of neurodegenerative diseases. The potential of FLAV-27 to preserve or restore excitatory neurotransmission and memory function in an aging brain is strongly supported by the inclusion of genes related to glutamatergic synapses (Figure S3I-J).\u003c/p\u003e \u003cp\u003eAccordingly, analysis of differentially expressed genes (DEGs) revealed that FLAV-27-treated samples uniquely expressed genes implicated in glutamatergic and serotonergic synapses, as well as long-term potentiation, supporting the notion that FLAV-27 enhances processes critical for learning and memory (Figure S3K). GO enrichment analysis indicated that FLAV-27-induced genes were associated with neuronal development, regulation of neuronal differentiation, and neuronal migration (Figure S3L), consistent with our previous observations of FLAV-27 playing a role in promoting dendritic growth and improving memory.\u003c/p\u003e \u003cp\u003eTo refine our understanding of cell-type-specific transcriptional responses, we analyzed the top expressed genes within each identified cell population (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F, Figure S3G-H). While the overall abundance of microglial markers was similar between the groups (as expected in the single-cell analyses), the expression levels of these markers were reduced in FLAV-27-treated samples compared to controls, indicating that FLAV-27 modulates microglial gene expression without altering the population size.\u003c/p\u003e \u003cp\u003eTo validate these transcriptomic findings, we selected several DEGs from key cell types, particularly neurons, that are relevant to AD pathology and evaluated their expression following FLAV-27 treatment. All the selected genes exhibited increased expression upon FLAV-27 administration, confirming the robustness of our scRNA-seq results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, Figure S3M).\u003c/p\u003e \u003cp\u003eGiven the observed modulation of microglial gene expression, we further investigated the functional impact of FLAV-27 on human iPSC-derived microglia. Cells treated with FLAV-27 (1 \u0026micro;M) were assessed for cytokine secretion and phagocytic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Under basal conditions, neither FLAV-27 nor the reference G9a inhibitor UNC0642 significantly altered cytokine secretion across most targets. Interestingly, UNC0642 induced a significant increase in IL-1β secretion, an effect not observed with FLAV-27 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH), suggesting distinct mechanisms of action that could lead to different microglial responses. Notably, since Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e uptake by microglia is a key event in AD pathology, we evaluated this process using flow cytometry. FLAV-27 significantly enhanced the uptake of Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e oligomers by iPSC-derived microglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). This effect was not observed with UNC0642 pre-treatment, further supporting the unique potential of FLAV-27 to modulate microglial function and its promise as a therapeutic candidate. To further assess the anti-inflammatory effects of FLAV-27, we tested its impact on glial cells exposed to LPS. FLAV-27 pre-treatment preserved viability in LPS-challenged HEK-293T cells (Figure S7A) and significantly reduced nitrite production in primary astrocyte and microglia cultures (9.65 \u0026micro;M and 7.15 \u0026micro;M, respectively), outperforming UNC0642 (Figure S7B-C). These results reinforce the anti-inflammatory and neuroprotective potential of FLAV-27.\u003c/p\u003e \u003cp\u003eFinally, among the DEGs validated in our scRNA-seq dataset, some of them were associated with ferroptosis pathways, prompting us to explore this regulated form of iron-dependent cell death that has been increasingly implicated in AD. Supporting its relevance, ferroptosis markers were significantly elevated in SAMP8 mice compared to the senescence-resistant SAMR1 strain (Figure S7D-H), indicating enhanced susceptibility to ferroptosis in this AD model. In line with this, FLAV-27 treatment in SAMP8 mice led to a significant upregulation of \u003cem\u003eGpx4\u003c/em\u003e and \u003cem\u003eFsp1\u003c/em\u003e, two key ferroptosis suppressor genes acting through glutathione-dependent and -independent mechanisms, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). Additionally, FLAV-27 restored the redox balance, as evidenced by a higher GSH:GSSG ratio and reduced levels of ROS, Fe\u0026sup2;⁺, and MDA when compared to vehicle-treated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK-N).\u003c/p\u003e \u003cp\u003eTo evaluate the impact of FLAV-27 on mitochondrial function and oxidative stress, we turned to the \u003cem\u003eC. elegans\u003c/em\u003e CL2006 model, which enables high-resolution metabolic profiling \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eO, Figure S7I-L). Seahorse assays revealed that FLAV-27 significantly improved mitochondrial respiration, increasing basal and mitochondrial oxygen consumption rates by up to 36% in CL2006 worms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eP-S). FLAV-27 also conferred protection against oxidative stress induced by tert-butyl hydroperoxide, with survival rates approaching those of vitamin C\u0026ndash;treated controls (Figure S7M). Furthermore, expression of the oxidative stress\u0026ndash;responsive gene \u003cem\u003esod-1\u003c/em\u003e, which is elevated in the AD model, was normalized by FLAV-27 treatment (Figure S7N), suggesting improved redox homeostasis.\u003c/p\u003e \u003cp\u003eFinally, to assess translational relevance, we examined post-mortem brain tissue from AD patients. While \u003cem\u003eGPX4\u003c/em\u003e and \u003cem\u003eFSP1\u003c/em\u003e transcript levels did not differ significantly from controls (Figure S7O), markers of oxidative stress and ferroptosis vulnerabilities were evident in the tissue from AD patients, including a reduced GSH:GSSG ratio and increased levels of ROS, Fe\u0026sup2;⁺, and MDA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eT-W).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFLAV-27 as a promising disease-modifying therapy for AD\u003c/h2\u003e \u003cp\u003eTo assess whether selectivity in the inhibition of G9a could lead to novel properties in the way to combat AD, we aimed to test the effects of FLAV-27 on both cognition and the disease course. The effects of FLAV-27 were evaluated in the 5xFAD transgenic mouse model, a well-established model of early-onset AD. Two treatment regimens were implemented: (i) an early intervention schedule (treatment 1\u0026thinsp;+\u0026thinsp;0; weeks 20\u0026ndash;24) designed to mimic preventive therapy and (ii) a late intervention schedule (treatment 0\u0026thinsp;+\u0026thinsp;1; weeks 24\u0026ndash;28) simulating treatment during an established disease state. The design in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA allowed us to assess both the symptomatic effects on cognition (using standard behavioral assays) and the potential of FLAV-27 as a disease-modifying agent in a transgenic mouse model of early-onset hereditary AD. The NORT was used to assess cognition in these animals. The results revealed that FLAV-27 treatment significantly improved both short-term and long-term memory (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C, Figure S8A). Both FLAV-27 regimens provided cognitive benefits. Despite a lack of significance, the earlier treatment showed a trend towards preventing cognitive deficits. Importantly, FLAV-27 did not affect locomotor activity, as the total distance traveled in the open field (OF) test remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, Figure S8B-H). The amount of rearing behavior decreased only in the animals treated with FLAV-27 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), while the amount of grooming did not change after treatment, with significant differences only observed between the two control groups (WT vs. 5xFAD) (Figure S8I). To examine spatial memory, we performed the OLT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, Figure S8J), which showed a significant enhancement in the discrimination index following all pharmacological interventions. Notably, both FLAV-27 treatment regimens resulted in higher discrimination index values than those observed in the WT controls, suggesting a robust cognitive-enhancing effect of FLAV-27. Furthermore, sociability was assessed using the three-chamber test (TCT) by recording the time spent sniffing (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, Figure S8K-L). The WT control group and both 5xFAD groups treated with FLAV-27 exhibited greater sniffing time, suggesting a social improvement compared to the 5xFAD control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Histopathological analysis of Aβ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH-J) showed that FLAV-27 significantly reduced Aβ plaque numbers in both the hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH) and cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). In addition, FLAV-27 treatment significantly increased dendritic spine length (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK, M) and, recapitulating the effect observed in SAMP8 mice, FLAV-27 significantly increased spine density (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL, N).\u003c/p\u003e \u003cp\u003eAstrogliosis increases progressively in 5xFAD mice as they age, aligning with the onset of cognitive impairments.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Accordingly, GFAP immunolabeling was conducted to ascertain the effect of the different treatments on the course of astrogliosis. This evaluation was performed using an immunofluorescence assay against GFAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eO). The results confirmed a significant increase in GFAP immunostaining in 5XFAD mice compared to WT mice in the entire hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eP, Figure S8M). This increase was also observed in specific regions, including the dentate gyrus (DG), CA1, and CA3 regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eO, Figure S8N-P). Quantification of the GFAP levels in the whole hippocampus revealed a decrease after both FLAV-27 treatment regimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eP). Interestingly, while the beneficial effects were negligible in the DG and CA1 regions (Figure S8N-P), both the early and late FLAV-27 treatments demonstrated a significant reduction in GFAP immunoreactivity in the hippocampal CA3 region (see Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eO).\u003c/p\u003e \u003cp\u003eFLAV-27 restored the expression of the immediate-early gene \u003cem\u003eArc\u003c/em\u003e, a key regulator of synaptic plasticity, particularly in the 1\u0026thinsp;+\u0026thinsp;0 group (Figure S8Q), indicating enhanced neuronal activity and cognitive function. Moreover, FLAV-27 significantly reduced the expression of the pro-inflammatory cytokine \u003cem\u003eIl-6\u003c/em\u003e (Figure S8R), while upregulating \u003cem\u003eFtl-1\u003c/em\u003e (Figure S8S), suggesting a reduction in neuroinflammatory signaling and a potential activation of ferroptosis-related protective mechanisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo validate the molecular alterations underlying key pathological events in AD, such as synaptic plasticity, neuroinflammation, and ferroptosis, comprehensive proteomic analyses were performed following FLAV-27 treatment. WT mice were clearly separated from the 5xFAD mice in a principal component analysis (PCA), with treated groups clustering in intermediate positions, suggesting partial reversal of disease-associated proteomic changes by the treatments (Figure S9A). The heatmap confirmed distinct expression patterns among the groups, with a characteristic proteomic signature in 5xFAD mice and notable differences between the treatment conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eQ). Furthermore, global enrichment analysis revealed that several biological processes were significantly altered when all the groups were considered together, indicating broad functional differences driven by disease state and modulated by treatment (Figure S9B).\u003c/p\u003e \u003cp\u003eNext, we compared the 5xFAD and WT proteomes. As shown in Figure S9C, ~\u0026thinsp;40% of the differentially expressed proteins were nuclear or plasma membrane proteins, while 6.5% were mitochondrial, underscoring the potential contribution of mitochondrial dysfunction to AD pathogenesis. Pathway analysis revealed a reduction in Hippo signaling (Figure S9D), which is involved in cell proliferation and apoptosis regulation,\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and an upregulation of glycosphingolipid biosynthesis and glycosaminoglycan degradation (Figure S9E), both of which may disrupt membrane composition and promote inflammation and aggregation in AD.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Finally, to assess treatment impact, we compared each group to untreated 5xFAD mice. All treatments significantly altered the proteomic profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eR-S, Figure S9F), indicating disease-modifying potential.\u003c/p\u003e \u003cp\u003eA detailed analysis of the 5xFAD proteome compared to the WT proteome revealed widespread alterations in the pathways related to synaptic function, energy metabolism, and protein homeostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Figure S9G-J). Notably, 5xFAD mice showed a downregulation of the proteins involved in mitochondrial function, ATP synthesis, ubiquitination, and RNA processing, alongside an upregulation of the proteins linked to the innate immunity, synaptic pruning, and inflammatory responses. Additional changes were observed in membrane trafficking, cytoskeletal organization, and proteasome function, indicating broader disturbances in neuronal structure and intracellular signaling. Metabolic imbalances, including disruptions in sphingolipid metabolism, glycolysis, lipid transport, and mitochondrial translation, were also evident, suggesting deficits in energy production and the biosynthetic capacity.\u003c/p\u003e \u003cp\u003eIn the context of the molecular alterations observed in 5xFAD mice, the tested treatments modulated protein expression in key disease-related pathways. The FLAV-27 early treatment (1\u0026thinsp;+\u0026thinsp;0) upregulated Grid2, Ist1, and Gng3, suggesting restoration of synaptic signaling and vesicle trafficking mechanisms that are both impaired in 5xFAD mice.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e The late treatment (0\u0026thinsp;+\u0026thinsp;1) additionally increased the expression of Pigt, Cpped1, Cox14, Atp5mpl, Nf2, Ints8, Mkln1, Ube2v1, Smpd3, and Ndufa3, pointing to improvements in mitochondrial bioenergetics (e.g., Cox14, Atp5mpl, and Ndufa3), protein homeostasis and ubiquitination (e.g., Ube2v1), and RNA metabolism (e.g., Ints8), which are all commonly dysregulated in AD.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e In addition to upregulating beneficial proteins, the FLAV-27 treatments reversed the increases in several proteins previously found to be upregulated in 5xFAD mice, thereby targeting pathways implicated in AD pathology. The early treatment (1\u0026thinsp;+\u0026thinsp;0) reduced the levels of Dhcr24, Smarcc2, Slc12a2, Ints1, Josd2, and Abcd1, which are associated with lipid metabolism, redox homeostasis, synaptic gene expression, and ion transport.\u003c/p\u003e \u003cp\u003eThe late treatment (0\u0026thinsp;+\u0026thinsp;1) led to a broader downregulation of disease-elevated proteins, including Ptpn6, Golga2, Pik3ca, Igtp, Ubb, Ctsh, Supt4a, Dhcr24, Ncbp2, Pak1ip1, B3galnt1, and Ptcd1, suggesting a widespread therapeutic impact. The reduced expression of inflammatory regulators (Ptpn6 and Igtp) aligns with the prior findings in the single-cell RNA-seq analysis of attenuated microglial activation. The downregulation of proteostasis-related proteins (Ubb and Ctsh) suggests an improved protein degradation capacity,\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e while decreased Golga2 and Pak1ip1 may indicate recovery of vesicle trafficking and cytoskeletal structure, which are both essential for proper APP processing and synaptic function.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e Furthermore, the downregulation of Pik3ca, a PI3K/Akt signaling component, may reflect rebalanced cell survival and autophagy pathways, while the decreased levels of Supt4a and Ncbp2 (involved in RNA processing) may indicate a partial reversal of transcriptional dysregulation. A lower Dhcr24 expression points to corrected cholesterol biosynthesis and OS regulation,\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e while B3galnt1 downregulation may modulate glycosylation processes that influence protein aggregation and synaptic signaling.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e Together, these changes suggest that FLAV-27 exerts multi-target neuroprotective effects, modulating inflammation, metabolism, vesicle trafficking, and neuronal architecture in a manner consistent with a disease-modifying therapeutic profile.\u003c/p\u003e \u003cp\u003e \u003cb\u003eModulation of the plasma biomarkers of AD and of plasma epigenetic-, synaptic plasticity-, and neuroinflammatory-related parameters\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe next aimed to correlate the effects of FLAV-27 with the levels of biomarkers associated with AD pathology that are commonly measured in clinical settings and are detectable in the plasma of 5xFAD mice.\u003csup\u003e\u003cspan additionalcitationids=\"CR41 CR42 CR43 CR44 CR45 CR46\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e All the data presented below were obtained from plasma samples collected from different experimental groups.\u003c/p\u003e \u003cp\u003eGiven that H3K9me2 levels are known to be elevated in the brains of 5xFAD mice and given its role in transcriptional repression,\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e we conducted a preliminary experiment to determine whether H3K9me2 is detectable in the plasma and whether FLAV-27 alters its levels. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, untreated 5xFAD mice displayed higher plasma levels of H3K9me2 compared to control animals. Notably, FLAV-27 treatment resulted in a significant reduction of H3K9me2 levels, consistent with its anticipated activity as a lysine methyltransferase. Next, we assessed the plasma levels of SMOC1, an Aβ plaque-associated synaptic protein identified as a biomarker of early-stage AD.\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e Plasma concentrations of SMOC1 were significantly elevated in untreated 5xFAD mice compared to controls. Importantly, treatment with FLAV-27 reduced these elevated levels, restoring them to values comparable to those observed in control animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe levels of GFAP, a marker of astrocytic activation and neuroinflammation, as well as TNF-α, a proinflammatory cytokine, were also assessed, given that both are elevated in the plasma of AD patients.\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e As expected, the plasma levels of both inflammatory markers were higher in the 5xFAD mice compared to wild-type controls (Figure S10A-B). Notably, plasma TNF-α levels were only normalized following the early (1\u0026thinsp;+\u0026thinsp;0) FLAV-27 treatment regimen (Figure S10B), while a reduction in GFAP plasma levels was observed exclusively with the late (0\u0026thinsp;+\u0026thinsp;1) treatment (Figure S10A).\u003c/p\u003e \u003cp\u003eWe then aimed to correlate the potential benefits of the early and late FLAV-27 treatments with the plasma biomarkers associated with AD. Specifically, we focused on Aβ peptides, p-tau, and neurofilament light chain (NF-L), which are key indicators of disease pathology and progression. These correlations are critical to determine whether FLAV-27 acts as a disease-modifying therapy rather than offering merely symptomatic relief. To this end, we measured the plasma levels of Aβ peptides, two p-tau forms recognized as clinical biomarkers of AD, and NF-L, a well-established marker of neuroaxonal damage in neurological disorders.\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e Notably, the early (1\u0026thinsp;+\u0026thinsp;0) treatment regimen showed no significant changes in Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e or Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e levels, nor in the Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e/Aβ\u003csub\u003e1\u0026minus;40\u003c/sub\u003e ratio, suggesting that the preventive benefits of FLAV-27 do not stem from reducing the amyloid burden (Figure S10C-E). Regarding p-Tau181 and p-Tau217 (phosphorylated tau forms identified by antibodies specific to tau phosphorylated at Thr181 and Thr217, respectively), which are considered reliable biomarkers of AD,\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e untreated 5xFAD mice exhibited increased plasma levels of both markers. Remarkably, both the early (1\u0026thinsp;+\u0026thinsp;0) and late (0\u0026thinsp;+\u0026thinsp;1) FLAV-27 treatment regimens reversed these increases, normalizing p-tau levels to those observed in the plasma of WT controls (Figure S10F-G). Similarly, NF-L levels, which were significantly elevated in untreated 5xFAD mice, were restored to control levels in both FLAV-27 treatment groups (Figure S10H).\u003c/p\u003e \u003cp\u003eFinally, we performed correlation analyses between all the plasma biomarkers evaluated, focusing on H3K9me2 and SMOC1, which are both regulated by G9a (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). H3K9me2 showed a strong positive correlation with SMOC1 levels, while other biomarkers displayed variable trends. Notably, SMOC1 also correlated with the neuroinflammatory markers p-tau (T181) and NF-L (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), highlighting the multi-targeted modulation of plasma biomarkers following G9a inhibition by FLAV-27. This evidence supports the potential of FLAV-27 as a disease-modifying therapy for AD.\u003c/p\u003e \u003cp\u003eTo assess the translational relevance of our findings, we analyzed a comprehensive panel of molecular markers in the plasma and cerebrospinal fluid (CSF) of controls with no dementia, individuals with prodromal AD, and patients with AD dementia. The plasma levels of H3K9me2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), p-tau (T181), and TNF-α were significantly elevated in the AD patients (Figure S10I-J), while SMOC1 levels showed a trend towards an increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). In the CSF, levels of SMOC1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF), total tau, and p-tau (T181) were significantly higher in the prodromal and AD dementia groups compared to controls (Figure S10K-L). Additionally, the CSF Aβ42/Aβ40 ratio was significantly decreased in the AD patients, driven primarily by reduced Aβ42 levels that is consistent with amyloid pathology (Figure S10M-O). Cognitive impairment, measured by the MMSE scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG), correlated negatively with plasma H3K9me2 and CSF SMOC1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). Western blot analysis of post-mortem brain tissue revealed elevated SMOC1 protein levels in the AD patients compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI-J). Furthermore, plasma H3K9me2 levels correlated positively with SMOC1 concentrations in the CSF, while SMOC1 also showed moderate associations with tau biomarkers and inflammatory markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). These results support the involvement of epigenetic dysregulation and astroglial activation in human AD pathology and reinforce the potential of these markers as therapeutic targets and disease indicators.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this work, we report the discovery and in-depth characterization of FLAV-27, a first-in-class, SAM-competitive, brain-penetrant, and selective inhibitor of the lysine methyltransferase G9a. Our findings position FLAV-27 as a potent epigenetic modulator with robust disease-modifying effects across multiple \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models of AD, with the potential to overcome several limitations that have historically hindered the translational success of G9a inhibitors for CNS applications (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrevious G9a inhibitors\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e such as BIX-01294, UNC0638, and UNC0642 have shown limited utility in neurodegeneration due to off-target effects, the dual inhibition of GLP, and poor CNS penetration. BIX-01294 was initially reported to reduce H3K9me2 levels in cancer cells, but was associated with high cytotoxicity and a lack of \u003cem\u003ein vivo\u003c/em\u003e viability.\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e UNC0638 and UNC0642 offered improved potency, but remained selective for GLP too and poorly penetrated the brain, thereby limiting their translational potential.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e By contrast, FLAV-27 exhibits subnanomolar G9a inhibition, with \u0026gt;\u0026thinsp;30-fold selectivity for G9a over GLP and other histone methyltransferases, as well as robust brain permeability that has been confirmed both \u003cem\u003ein vitro\u003c/em\u003e (PAMPA-BBB) and \u003cem\u003ein vivo\u003c/em\u003e. Structurally, the SAM-competitive mechanism of FLAV-27 represents a paradigm shift from traditional substrate-competitive G9a inhibitors. The high-resolution X-ray co-crystal structure of human G9a bound to FLAV-27 (PDB ID: 9LUS) confirms a unique SAM-competitive binding mode, revealing that FLAV-27 occupies the SAM pocket of G9a. Interestingly, structural comparisons with the published G9a\u0026ndash;SAM complex (PDB: 5TTF) show that FLAV-27 relies heavily on hydrophobic contacts, particularly between its benzyl and phenol moieties and the G9a residues Trp1050, Phe1110, Tyr1085, and Tyr1154, thereby rationalizing its superior affinity and selectivity. In fact, preliminary findings indicate that while the active sites of G9a and GLP are structurally similar, their surface charge distributions differ significantly. The SAM-binding site of G9a is predominantly hydrophobic with a minimal negative charge, whereas that of GLP is largely positively charged. This distinction may explain why the hydrophobic FLAV-27 compound preferentially binds to G9a. Moreover, the inadvertent presence of low‐occupancy SAM in our G9a crystallization likely dampened the FLAV-27 electron density, but the refined Fo\u0026ndash;Fc maps at 1σ clearly define FLAV-27 in the SAM co-substrate pocket. Consequently, by blocking SAM binding, FLAV-27 prevents methyl transfer entirely, offering a mechanistic advantage over substrate‐competitive compounds that merely impede histone peptide docking. Thus, unlike substrate-competitive inhibitors that often affect multiple SET domain methyltransferases,\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e the occupation by FLAV-27 of the SAM pocket provides superior selectivity and reduced off-target effects. This mechanistic distinction is crucial, as cross-reactivity with other methyltransferases can have deleterious effects on chromatin integrity in neurons, where precise regulation of gene expression is essential for plasticity and survival.\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eHere, we are actively working to further elucidate the molecular determinants underlying the selectivity of FLAV-27 for G9a over GLP. Our ongoing studies focus on detailed structural and electrostatic analyses of the SAM-binding pockets in both enzymes.\u003c/p\u003e \u003cp\u003eThe exceptional safety profile of FLAV-27 (NOAEL\u0026thinsp;\u0026gt;\u0026thinsp;1 g/kg) combined with its low effective dose (5 mg/kg/day) provides a substantial therapeutic window that enhances its clinical viability. The lack of significant off-target effects across comprehensive screening panels contrasts favorably with existing epigenetic therapeutics that often exhibit dose-limiting toxicities. This safety profile is particularly important for chronic neurodegenerative diseases requiring long-term treatment. In addition, the brain-to-plasma ratio of 3\u0026ndash;6:1 achieved by FLAV-27 ensures adequate CNS exposure, while minimizing peripheral effects. This pharmacokinetic profile represents a significant improvement over earlier G9a inhibitors that suffer from poor BBB penetration. The achievement of sustained brain exposure with intermittent dosing schedules further enhances the clinical practicality of this approach.\u003c/p\u003e \u003cp\u003eEpigenetic repression mediated by H3K9me2 is increasingly recognized as a driver of cognitive decline in neurodegeneration in AD models and patient samples.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e Our data show that FLAV-27 robustly reduces H3K9me2 levels in the SAMP8 mouse brain and induces genome-wide reprogramming of heterochromatin-associated regions, particularly at the promoters of genes implicated in synaptic signaling, neurogenesis, learning and memory, and cellular differentiation. These data align with previous work showing G9a-mediated repression of plasticity‐related genes in aging and AD.\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e This is consistent with reports showing that G9a inhibition in AD models reactivates silenced neuroprotective genes, including \u003cem\u003eArc\u003c/em\u003e and \u003cem\u003eSyt4\u003c/em\u003e, which we also found to be upregulated following FLAV-27 treatment.\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e Notably, we also observed a novel reduction in H3K18me₁\u0026ndash;₃ levels following FLAV-27 treatment, producing the first evidence that H3K18 methylation contributes to AD pathogenesis. Elevated H3K18me₁\u0026ndash;₃ has been reported in the brains of AD patients, but its functional role is unknown. Our data imply that H3K18 methylation may cooperatively establish repressive heterochromatin with H3K9me2 to silence neuronal gene programs. The ability of FLAV-27 to reverse both the H3K9 and H3K18 marks highlights its potential for broad epigenetic reprogramming in diseased neurons. At the transcriptomic level, bulk RNA-seq in the cortex of SAMP8 mice revealed a global pattern of gene de-repression, with an upregulation of immune‐regulatory gene sets. This finding is consistent with the dual epigenetic and immunomodulatory role of FLAV-27. Single‐cell RNA-seq further refined these insights by showing that although the neuronal and glial cell proportions remained unchanged, FLAV-27 shifted gene expression within cell types. Disease ontology analysis revealed that the FLAV-27‐regulated genes are associated not only with AD, but also with schizophrenia, depression, and other mental disorders, suggesting broader utility for neuropsychiatric conditions in which G9a dysregulation has been implicated.\u003csup\u003e\u003cspan additionalcitationids=\"CR65 CR66\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOur \u003cem\u003ein vitro\u003c/em\u003e studies provide direct evidence of the ability of FLAV-27 to counteract AD-related cellular phenotypes. In primary neuronal and microglial cultures, FLAV-27 markedly reduced the aggregation of Aβ₁\u0026ndash;₄₂, total tau, and phosphorylated tau (p-Tau), while restoring the neuritic integrity disrupted by these aggregates. These findings are consistent with previous work suggesting that histone methylation contributes to synaptic loss and cytoskeletal disorganization in AD neurons.\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e In addition, we evaluated its effect in the \u003cem\u003eC. elegans\u003c/em\u003e CL2006 strain, which expresses human Aβ₁\u0026ndash;₄₂ in its muscle cells and exhibits age-dependent paralysis.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e FLAV-27 (1 \u0026micro;M) significantly reduced the H3K9me2/H3 ratio, decreased Aβ aggregation by approximately 45% (compared to ~\u0026thinsp;26% with UNC0638), and improved locomotor behavior. Notably, FLAV-27 extended the lifespan. This is in accordance with previous observations that G9a inhibition extends the lifespan and improves stress resistance in \u003cem\u003eC. elegans\u003c/em\u003e,\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e further supporting the idea that the epigenetic mechanisms governing proteostasis and neuronal function are conserved across phyla.\u003c/p\u003e \u003cp\u003eSAMP8 mice, a model of late-onset AD (LOAD), and 5xFAD mice, a model of early-onset AD (EOAD), were selected for this study as it was previously reported that they show elevations in the G9a protein and its repressive histone mark H3K9me2 during disease progression.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e Furthermore, we aimed to investigate whether selective G9a inhibition with FLAV-27 conferred disease-modifying properties due to the current lack of disease-modifying therapies for neurodegenerative diseases such as AD. For that reason, two FLAV-27 treatment regimens were tested in the 5xFAD mice: early intervention (weeks 20\u0026ndash;24) and late intervention (weeks 24\u0026ndash;28). Both approaches improved cognitive performance in the NORT, OLT, and social interaction paradigms, without affecting locomotor activity. Notably, early treatment tended to completely prevent cognitive deficits, while the late intervention was able to reverse established impairments. These behavioral improvements were accompanied by structural enhancements in synaptic morphology, including increased dendritic spine density and length, and by enhanced AMPAR-mediated miniature excitatory postsynaptic currents (mEPSCs), indicating a functional rescue of synaptic transmission. These findings parallel previous reports that G9a inhibition enhances synaptic function in the nucleus accumbens and hippocampus.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Moreover, FLAV-27 reduced the Aβ plaque burden and suppressed astrogliosis in the 5xFAD mice. FLAV-27 also ameliorated social behavioral deficits in the 5xFAD mice, as assessed by the TCT. Treated animals spent significantly more time interacting with the social stimulus compared to untreated controls, indicating a restoration of sociability, a crucial behavioral domain often impaired in AD. This rescue aligns with previous reports linking epigenetic repression to social cognition deficits, highlighting the broad functional impact of G9a inhibition.\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eCell-type\u0026ndash;specific marker analyses showed that microglial markers, though present at a constant level, were downregulated in FLAV-27 samples compared to SAMP8 controls, reinforcing the notion that FLAV-27 directly modulates microglial transcriptomes without altering cell numbers. To test functional consequences, we treated human iPSC-derived microglia with FLAV-27 (1 \u0026micro;M) and compared them to UNC0642-treated cells. Under basal conditions, FLAV-27 did not elicit IL-1β secretion, whereas UNC0642 significantly increased IL-1β levels, suggesting distinct allosteric effects on microglial activation due to SAM-competitive versus substrate-competitive binding. Importantly, FLAV-27 significantly enhanced microglial uptake of Aβ₁₋₄₂ oligomers, a critical neuroprotective mechanism in AD, whereas UNC0642 did not. These data indicate that FLAV-27 restores microglial homeostasis while enhancing phagocytosis, potentially contributing to a decreased Aβ burden \u003cem\u003ein vivo\u003c/em\u003e. In parallel, both the bulk and single-cell transcriptomic datasets confirmed the upregulation of key synaptic genes such as \u003cem\u003eArc\u003c/em\u003e and \u003cem\u003eSyt4\u003c/em\u003e following FLAV-27 treatment, further substantiating the link between G9a inhibition and the restoration of neuronal gene expression. Collectively, these findings position H3K9me2 not only as a critical epigenetic mark mediating transcriptional repression in AD, but also as a biomarker of treatment response. They also reinforce the central role of G9a as a regulator of synaptic gene silencing and highlight the potential of epigenetic reprogramming via FLAV-27 to restore neuronal identity and function in AD.\u003c/p\u003e \u003cp\u003eFerroptosis has emerged as a central mechanism contributing to neuronal loss in AD.\u003csup\u003e\u003cspan additionalcitationids=\"CR72 CR73 CR74\" citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e SAMP8 mice displayed elevated markers of ferroptosis, including increased levels of lipid peroxidation (MDA), ROS, and Fe\u0026sup2;⁺, and a reduced GSH:GSSG ratio. FLAV-27 reversed these alterations and upregulated \u003cem\u003eGpx4\u003c/em\u003e and \u003cem\u003eFsp1\u003c/em\u003e, which are critical suppressors of ferroptosis, suggesting a direct protective role through redox regulation. Additionally, due to prior studies showing abnormal mitochondrial respiration in a \u003cem\u003eC. elegans\u003c/em\u003e model of mild pan-neuronal AD in its old stages,\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e we used this model to corroborate our findings after FLAV-27 treatment. We used the CL2006 strain, a transgenic strain in which the muscular expression of the Aβ peptide and consequent Aβ plaques lead to abnormal locomotion and paralysis. In fact, one possible cause of the muscular dysfunction could be the abnormal mitochondrial metabolism exhibited by the CL2006 strain at a younger stage (L4). However, FLAV-27 increased the oxygen consumption rate (OCR) of CL2006, probably ameliorating its locomotory behavior and well-being through aging.\u003c/p\u003e \u003cp\u003eRegarding the proteomic study, FLAV-27 treatments modulated AD-related protein pathways in 5xFAD mice, but differed in the scope. While FLAV-27 1\u0026thinsp;+\u0026thinsp;0 treatment reduced the levels of the proteins linked to lipid metabolism (Dhcr24), synaptic plasticity (Smarcc2 and Slc12a2), and redox homeostasis (Abcd1), FLAV-27 0\u0026thinsp;+\u0026thinsp;1 treatment broadly downregulated inflammatory regulators (Ptpn6 and Igtp), proteostasis markers (Ubb and Ctsh), and transcriptional mediators (Supt4a and Ncbp2). These changes suggested that FLAV-27 normalizes microglial activation, protein degradation, and RNA processing, while restoring vesicle trafficking via Golga2 and Pak1ip1 reduction. This was also validated in the single-cell seq performed in the SAMP8 mice. Consistent with our results, the G9a inhibitor MS1262 demonstrated a similar pattern profile,\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e reversing the changes in several proteins previously reported to be upregulated in 5xFAD mice, including translation regulators (METTL3), early AD biomarkers (SMOC1), and matrisome components, while restoring PI3K/Akt/MAPK signaling, cytoskeletal phosphorylation states (DNM2 and TAGLN2), and proteostasis networks.\u003c/p\u003e \u003cp\u003eImportantly, these proteomic brain signatures were accompanied by peripheral biomarker changes, underscoring the systemic impact of the FLAV-27 treatment. FLAV-27 reduced levels of p-tau (T181), TNF-α, and H3K9me2, three key markers linked to AD pathology, inflammation, and epigenetic repression, in the plasma of 5xFAD mice. The pattern of the changes in the Aβ\u003csub\u003e42\u003c/sub\u003e/Aβ\u003csub\u003e40\u003c/sub\u003e ratio after FLAV-27 treatment suggests that FLAV-27 exerts its effect on Aβ pathology by modulating the pathways that are active during established disease. The lack of persistence in the group receiving early treatment may reflect the dynamic reversibility of this epigenetic mechanism. Interestingly, SMOC1 has been recently identified as a biomarker for AD and its levels correlate with the Aβ plaque load.\u003csup\u003e\u003cspan additionalcitationids=\"CR78\" citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e More noteworthy, a preprint postulated that treatment with a G9a inhibitor can reverse SMOC1 expression and phosphorylation in the Aβ-associated matrisome module.\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e Accordingly, the 5xFAD mice had higher plasma levels of SMOC1 that were reverted by FLAV-27 treatment.\u003c/p\u003e \u003cp\u003eTo evaluate the translational relevance of our preclinical findings and support the clinical applicability of FLAV-27, we analyzed a broad panel of AD-related biomarkers in human fluids and post-mortem brain samples. Strikingly, we found that the epigenetic alterations observed in the animal models, particularly the upregulation of H3K9me2 and H3K18 methylation, were also recapitulated in the AD human brain samples. Elevated H3K9me2 levels were detected in both the plasma and brain tissue of AD individuals compared to controls with no dementia, reinforcing the notion that G9a-mediated chromatin repression is not limited to the CNS but extends to peripheral compartments. Moreover, SMOC1 was also found to be significantly elevated in the CSF and brain tissue of AD patients and showed a trend towards elevation in the plasma.\u003c/p\u003e \u003cp\u003eTogether with the consistent increase in tau levels and the expected decrease in the Aβ\u003csub\u003e42\u003c/sub\u003e/Aβ\u003csub\u003e40\u003c/sub\u003e ratio in the CSF, these findings highlight SMOC1 as a candidate biomarker of early disease progression with central and peripheral relevance. Importantly, the strong correlations observed between plasma H3K9me2 and SMOC1 levels, as well as their associations with tau pathology, inflammatory markers (TNF-α), and cognitive decline (MMSE), further support the biological linkage between epigenetic dysregulation and hallmark AD mechanisms. In fact, all of this mirrors the observations in human epigenome-wide association studies that link H3K9 methylation to cognitive impairment and neurodegeneration.\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e,\u003cspan additionalcitationids=\"CR82\" citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e These translational data not only validate the relevance of our experimental models, but also underscore the utility of H3K9me2 and SMOC1 as promising peripheral biomarkers for monitoring disease progression and therapeutic response in future clinical applications of G9a-targeted interventions such as FLAV-27 treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAll procedures involving animals, including behavioral testing and dissection and removal of brains, followed ARRIVE and the standard ethical guidelines (Council of the European Communities Directive 2010/63/EU and Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research, National Research Council 2003) and were approved by the Institutional Animal Care and Generalitat de Catalunya (#PJ_159/24, March 13, 2024). Every effort was made to minimize the number of mice used and their suffering.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGMENTS\u003c/h2\u003e \u003cp\u003eThis study was supported by the Ministerio de Econom\u0026iacute;a, Industria Econom\u0026iacute;a, Industria y Competitividad (Agencia Estatal de Investigaci\u0026oacute;n, AEI) and European Union NextGenerationEU/PRTR (PID2022-139016OA-I00, PDC2022-133441-I00 ,to CGF and MP; PID2022-1380790B-I00, to C.E.), MICIU/AEI/\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.13039/501100011033\u003c/span\u003e\u003cspan address=\"10.13039/501100011033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e and FEDER, UE and PDC2022-133441-I00 MICIU/AEI /\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.13039/501100011033\u003c/span\u003e\u003cspan address=\"10.13039/501100011033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e Europea Next GenerationEU/ PRTR), Generalitat de Catalunya (2021 SGR 00357). This study was co-financed by Secretaria d\u0026rsquo;Universitats i Recerca del Departament d\u0026rsquo;Empresa i Coneixement de la Generalitat de Catalunya 2023 (Product 0092; Llavor 005 and Llavor 007 to CGF). ABS acknowledges the Ag\u0026egrave;ncia de Gesti\u0026oacute; d'Ajuts Universitaris i de Recerca (AGAUR) for her FI-SDUR fellowship (2021FISDU 00182). NIDDK R01 230857 GRANT to SD. Financial support was provided for F.R.-B. (PREP2022-000196 Ministerio de Ciencia e Innovaci\u0026oacute;n).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCummings J et al (2023) Alzheimer\u0026rsquo;s disease drug development pipeline: 2023. Alzheimer\u0026rsquo;s Dementia: Translational Res Clin Interventions 9:e12385\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcFarthing K et al (2024) Parkinson\u0026rsquo;s Disease Drug Therapies in the Clinical Trial Pipeline: 2024 Update. 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Nat Commun 12:3517\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer’s disease, epigenetics, cognition, neuroprotection, SAM-competitive inhibitor","lastPublishedDoi":"10.21203/rs.3.rs-6940373/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6940373/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlzheimer\u0026rsquo;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, 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 \u003cem\u003ein vitro\u003c/em\u003e. In \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e, 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.\u003c/p\u003e","manuscriptTitle":"First-in-Class SAM-Competitive G9a Inhibitor FLAV-27 as a Disease-Modifying Therapy for Alzheimer’s Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-26 06:12:42","doi":"10.21203/rs.3.rs-6940373/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"515387ad-b2eb-4d5f-82bc-5abd7949046b","owner":[],"postedDate":"June 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50576997,"name":"Biological sciences/Drug discovery/Medicinal chemistry/Drug discovery and development"},{"id":50576998,"name":"Biological sciences/Neuroscience/Epigenetics in the nervous system/Epigenetics and behaviour"}],"tags":[],"updatedAt":"2025-06-30T15:35:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-26 06:12:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6940373","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6940373","identity":"rs-6940373","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}