Abstract 474: A comprehensive LinkLight platform for GPCR mechanistic profiling to support cancer drug discovery.

Abstract Background: G protein-coupled receptors (GPCRs) are increasingly recognized as important targets in cancer therapy due to their roles in tumor growth, angiogenesis, and metastasis. LinkLight technology is a protein-protein interaction (PPI) detection platform that monitors β-arrestin recruitment to activated G protein-coupled receptors (GPCRs), complementing second messenger-based assays for ligand activity characterization. Here, we use LinkLight stable cell lines co-expressing TEV-protease-tagged GPCRs and permuted luciferase (pLuc)-tagged β-arrestin to study β-arrestin recruitment, cAMP signal, and calcium-influx within the same cellular context. Methods: Cell-based functional assays were conducted in both agonist and antagonist modes across three GPCR LinkLight stable cell lines representing distinct G-protein couplings (Gs, Gi, and Gq). β-Arrestin-1/2 recruitment was quantified using the LinkLight PPI luminescence readout, while G-protein-mediated signaling was measured via cAMP or calcium-influx fluorescence, depending on receptor class. Results: Ligands produced consistent, mechanism-appropriate responses across β-arrestin and second-messenger pathways. For Gq-coupled ADRA1A, the agonist Cirazoline induced robust β-arrestin-1/2 recruitment (EC50 = 2.51×10-8 M) and calcium influx (EC50 = 8.2×10-9 M), while the antagonist Prazosin inhibited both (β-arrestin EC50 = 2.51×10-8 M; calcium EC50 = 2.06×10-7 M). For Gi-coupled ADRA2A, the agonist Brimonidine stimulated β-arrestin-1/2 recruitment (EC50 = 2.64×10-8 M) and suppressed cAMP (EC50 = 8.88×10-10 M), whereas the antagonist Yohimbine blocked β-arrestin recruitment (EC50 = 1.78×10-8 M) and reversed cAMP inhibition (EC50 = 2.69×10-7 M). For Gs-coupled ADRB2, the agonist Fenoterol induced β-arrestin-2 recruitment (EC50 = 8.83×10-10 M) and cAMP signaling (EC50 = 1.52×10-9 M), while the antagonist Yohimbine (S)-Propranolol inhibited both (β-arrestin EC50 = 3.98×10-8 M; cAMP EC50 = 3.6×10-8 M). Across all receptors, β-arrestin recruitment aligned with G-protein-specific signaling in response to the same ligand, demonstrating the robustness of the comprehensive GPCR portfolio. Summary: By enabling simultaneous assessment of β-arrestin recruitment and G-protein signaling in a unified cellular context, LinkLight technology offers a comprehensive, mechanistically informative platform for GPCR pharmacology. Integrated with G-protein activation data, LinkLight technology also supports high throughput profiling of ligand efficacy, bias, and receptor regulation for GPCR drug discovery against cancer. Citation Format: Yong Wan, Alisha D. Ianni, Haiching Ma, Jianghong Wu. A comprehensive LinkLight platform for GPCR mechanistic profiling to support cancer drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 474.