Discovery of pyrazolo[1,5-a]pyrimidine derivatives targeting TLR4-TLR4∗ homodimerization via AI-powered next-generation screening.

TLR4 signaling is instrumental in orchestrating multiple aspects of innate immunity. Developing small molecule inhibitors targeting the TLR4 pathway holds potential therapeutic promise for TLR4-related disorders. Herein, an artificial intelligence (AI)-powered next-generation screening approach, employing HelixVS and HelixDock, was utilized to focus on the TLR4-TLR4∗ (a second copy of TLR4) homodimerization surface, leading to the identification of a potent pyrazolo[1,5-a]pyrimidine derivative, designated as compound 1. An extensive structure-activity relationship (SAR) exploration culminated in the discovery of the lead compound TH023, which effectively blocked the LPS-stimulated NF-κB activation and nitric oxide overproduction in HEK-Blue hTLR4 and RAW264.7 cells, with IC values of 0.354 and 1.61 μM, respectively. Molecular dynamic (MD) simulations indicated that TH023 stabilized TLR4-MD-2 and disrupted its association with TLR4∗. Moreover, TH023 alleviated the lung injury and decreased pro-inflammatory cytokine levels in LPS-induced septic mice. These findings not only illuminated the strategic advantage of HelixDock in advancing the frontiers of AI-driven drug discovery, but also provided valuable structural insights for the rational design of TLR4-TLR4∗ protein-protein interaction (PPI) inhibitors based on the pyrazolo[1,5-a]pyrimidine scaffold. Overall, this study validated a new strategy for TLR4 signaling regulation by targeting its dimerization, thereby underscoring the therapeutic promise of TH023 in treating TLR4-mediated inflammatory diseases.