ライブ細胞ルミネッセンスベースのGαq-PLC-β相互作用アッセイ：セロトニン5-HT2A受容体への応用開発

G protein-coupled receptors (GPCRs) remain among the most important drug targets. To characterize their ligands' activity, a range of in vitro assays is available, all with their inherent advantages and limitations. In the Gα_q signaling pathway, the interaction between Gα_q and phospholipase C (PLC-)β is a receptor-proximal event, and its monitoring does not require modification of the GPCR. With this in mind, we set out to develop a luminescence-based assay gauging this interaction. The resulting assay format encompassed functional complementation of a split nanoluciferase following the interaction of SmBiT, N-terminally fused to PLC-β, with LgBiT, intramolecularly integrated within Gα_q. When applied to assess activation of the Gα_q-coupled serotonin 2A receptor (5-HT_2AR), an excellent assay performance was demonstrated, as evidenced by an auspicious Z'-factor of 0.75 and by confirming the specificity for both the GPCR and the Gα_q signaling pathway. The optimized Gα_q-PLC-β assay was successfully applied on a diverse panel of 5-HT_2AR ligands and the resulting output was compared with that of a more upstream miniGα_q recruitment assay, and more downstream IP₁ accumulation and Ca²⁺ mobilization assays. Overall, the Gα_q-PLC-β assay yielded efficacy, potency and relative activity data that correlated very well with those obtained with the IP₁ accumulation and miniGα_q recruitment assays, although with the former two assays consistently higher potencies and a smaller range of efficacies were observed. Comparison with the Ca²⁺ mobilization assay did not yield such good correlation, presumably because of kinetic implications. Last, the Gα_q-PLC-β interaction assay allowed to detect signals emerging from endogenously expressed GPCRs, indicative of its broad applicability.