In silico discovery and mechanistic profiling of STING agonists engaging the transmembrane domain.

Stimulator of interferon genes (STING) is an ER resident cytosolic pattern recognition receptor involved in innate immune signaling and is a promising therapeutic target in immuno-oncology and vaccine adjuvant design. While canonical STING agonists typically activate the receptor via direct engagement with the cytosolic cyclic dinucleotide (CDN)-binding domain (CBD), recent high-resolution structural studies have uncovered a distinct allosteric binding site within the transmembrane domain (TMD). Here, we report the identification and characterization of a novel STING agonist, compound 7k, which uniquely engages the TMD rather than the cytosolic domain. Through comparative molecular docking and binding site validation, the TMD of STING was computationally identified as the preferential site of engagement, diverging from the classical CBD. This mode of activation is functionally significant, as it leads to a demonstrably distinct set of downstream molecular phenotypes. Furthermore, our study led to the discovery of structurally related series of potent, small-molecule human STING activators with potential utility as immunomodulatory therapeutics. A lead compound, 7k, emerged with potent STING-dependent activity in vitro and displayed adjuvant efficacy in vivo, as shown by enhanced antigen-specific IgG production and Th1/Th2 cytokine responses in a genetically humanized STING mouse model. These findings support the TMD as a druggable allosteric site and highlight 7k as a promising candidate for next-generation STING-targeted immunotherapeutics.