Monitoring G-Protein Coupled Receptors using a Bioluminescent, Homogenous, and High Throughput Formatted Assay for cAMP, cGMP, and IP₃

Abstract ID 91255 Poster Board 144 Second messengers play a major role in mediating the effect of modulators of G Protein Coupled Receptors (GPCR) in a wide range of activities in cell signaling and human health. There are more than 800 GPCRs representing one of the major cell surface receptors families and encoding 3-4% of the human genome. The three most targeted G proteins for tracking GPCR activation are Gas, Gai, and Gaq, resulting in activation of adenylate cyclase (Gas), inhibition of adenylate cyclase (Gai) and activation of phospholipase b (Gaq), respectively. As researchers continue to study and target these pathways, it will be critical to develop assays that accurately determine the levels of second messenger molecules using one platform. Here we present sensitive and specific bioluminescent detection systems to monitor the levels of cAMP, cGMP, and IP₃. Taking advantage of splitting an engineered luciferase from deep sea shrimp (Nanoluc) which has small molecular size (19 kD) and 100-fold more luminescence than firefly luciferase, we were able to use protein complementation platform to develop assays for the second messengers cAMP, cGMP, and IP₃. The enzyme is split into small BiT (Sm BiT,11 amino acids, 1.3kD) and large BiT (Lg BiT, 1.7 kDa), and by linking Sm BiT to the second messenger of interest via a linker (Sensor) and Lg BiT linked to antibodies selective for the target (cAMP, cGMP, and IP3), the interaction of the small molecule with the antibody, brings the Sm BiT to the Lg BiT leading to complementation of Nanoluc and generating luminescence upon addition of the substrate. We demonstrate the utility of these systems in both enzymatic assays and cell-based assays. We have tested these assays using cells that express the appropriate GPCR for generating or inhibiting cAMP production and those that result in IP₃ generation. We have successfully demonstrated the detection of the second messengers using stably transfected receptors as well as endogenous receptors. We also tested these assays for determining the potency of selective phosphodiesterase inhibitors for cGMP-PDE and cAMP-PDE and these assays yielded IC₅₀ values that are very similar to those reported in the literature. These assays are easy to perform, homogeneous, highly sensitive, minimal, or no false hits, and compatible with high throughput formats. It is anticipated that these assays will be of significant value to aid in further understanding the role of second messenger signaling in physiology and support further drug discovery efforts towards the treatment of human disease.