The dual G9a inhibitor and histamine H3 receptor antagonist A-366 improves repetitive and social behaviors and attenuates neuroinflammation in BTBR T + tf/J mice

Autism spectrum disorder (ASD) is a prevalent neurodevelopmental condition characterized by repetitive behaviors, impaired sociability, and persistent neuroinflammation. While both epigenetic dysregulation and histaminergic imbalance contribute to ASD pathology, no therapy targets both aspects simultaneously. This study investigated A-366, a selective histone H3K9 methyltransferase G9a inhibitor with additional histamine H3 receptor (H3R) antagonist properties, in BTBR T + tf/J mice (BTBR mice). Sub-chronic administration of A-366 (0.5, 1, or 2 mg/kg, i.p.) dose-dependently reduced repetitive behaviors in marble burying, nestlet shredding, and self-grooming tests, and improved spatial working memory in spontaneous alternation in spontaneous alternation (p < 0.01−0.001 overall). A-366 also restored sociability and social novelty preference (p < 0.001 overall), with the highest dose (2 mg/kg) fully normalizing performance to wild-type levels. At the molecular level, A-366 significantly lowered pro-inflammatory cytokines in cerebellar and hippocampal tissues (p < 0.001 for TNF-α and IL-6; p < 0.05–0.001 for IL-1β). Compared with the reference H3R antagonist Pitolisant, A-366 produced broader behavioral and anti-inflammatory improvements. A-366 also produced broader behavioral and anti-inflammatory improvements and outperformed the dopaminergic–serotonergic modulator, Aripiprazole, at its effective dose. Partial reversal of these effects by coadministration with the selective H3R agonist RAMH indicated that histamine H3R antagonism contributes to its mechanism of action, while G9a inhibition remained unaffected. These findings suggest that A-366 exerts dual therapeutic action by improving ASD-like repetitive and social behaviors and attenuating neuroinflammation, providing a promising scaffold for next-generation multi-target therapeutics.