Mygalin reduces inflammation by targeting TLR3 signaling pathway in macrophages

Toll-like receptors (TLRs) are central regulators of innate immunity, enabling macrophages to detect pathological changes through both cell surface and intracellular compartments. Several molecules have been investigated for their ability to modulate TLR-mediated immune responses. Mygalin, a synthetic acylpolyamine analogue of spermidine, has been reported to modulate macrophage activation via extracellular TLR2 and TLR4 pathways. Here, we investigated the immunomodulatory effects of Mygalin on RAW 264.7 macrophages activated with Poly I:C (TLR3 agonist), LPS (TLR4 agonist), and IFN-γ using in vitro assays and in silico molecular docking. Mygalin treatment significantly attenuated the production of inflammatory mediators, reduced IRF3 phosphorylation without affecting STAT1, and selectively downregulated CD40 expression, while CD86, and F4/80 levels remained. No significant cytotoxicity was detected in any of the analyzed groups. Docking analyses revealed that Mygalin interacts with three distinct binding sites on TLR3, with the highest affinity for site 3, and engages specific residues of the IFN-γ receptor. Molecular dynamics simulations confirmed the stable association of Mygalin with TLR3 and the IFN-γ receptor. These findings suggest that Mygalin may represent a promising immunomodulatory candidate for controlling inflammatory conditions associated with dysregulated TLR signaling.