Structural and molecular characterization of a small-molecule TNF-α-TNFR1 inhibitor modulating cell death signaling

Tumor necrosis factor alpha (TNF-α) is a central mediator of inflammation and autoimmunity, where dysregulated activation of apoptotic and necroptotic pathways drives progressive tissue damage. Although monoclonal antibody therapies against TNF-α have provided clinical benefit, limitations related to parenteral delivery, immunogenicity, and incomplete suppression of downstream signaling underscore the need for alternative therapeutic approaches. Despite the pivotal role of TNF-α in chronic inflammation, no small-molecule inhibitor has yet reached clinical approval, with only a few candidates currently under clinical investigation. Here, we report the identification and mechanistic characterization of TI-16, a novel small-molecule inhibitor of TNF-α-TNFR1 interaction, discovered through structure-based virtual screening and validated by biophysical and cellular assays. TI-16 effectively protected fibroblasts from TNF-α-induced apoptosis and necroptosis and reduced the release of proinflammatory cytokines. Mechanistic analyses using immunoblotting, surface plasmon resonance, and molecular dynamics simulations demonstrated that TI-16 selectively disrupts tumor necrosis factor-alpha/tumor necrosis factor receptor 1 (TNF-α/TNFR1) interaction without altering TNF-α trimerization. By simultaneously suppressing apoptotic and necroptotic signaling, TI-16 overcomes key limitations of current biologics and represents a promising lead for the development of a novel class of orally available TNF-α-targeted therapeutics.