GPCR activation-based in vitro luminescent assays for high-throughput drug screening and extracted GPCR characterization

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PROPOSAL SUMMARY G-protein Coupled Receptors (GPCR) are transmembrane signaling proteins found abundantly throughout the body. GPCRs are responsible for regulating many physiological processes including pain modulation and drug addiction. The mu-opioid receptor (MOR) is one of the most significant GPCRs since it is implicated in the opioid epidemic as the primary target for therapeutic analgesics. High-throughput screening (HTS) methods for detecting GPCR activity are highly desirable for discovering alternative, nonaddictive agonists. All the existing screening platforms are based on maintaining live-cell cultures due to the lack of methods for characterizing GPCR activity in-solution. Here I propose the design of the first in-solution HTS bioluminescent assay for detecting GPCR activity which will greatly streamline the process of GPCR ligand screening. The new assay features conformation specific binders, including nanobodies (Nb) and peptidomimetics, which will bind to the activated conformation of the GPCR, both in-solution and in live-cell cultures. Components of a split bioluminescent enzyme, Nanoluciferase (split NanoLuc), will be attached to the GPCR and the conformation specific binder. When the agonist-dependent interaction occurs, the NanoLuc will reconstitute and produce a quantifiable bioluminescent signal in the presence of furimazine. I have already validated the feasibility of this in-solution bioluminescent assay using beta2-adrenergic receptor (β2AR) and its conformation-specific binders, a G-protein mimic Nanobody and a novel peptidomimetic. The assay will be applied for screening MOR agonists and antagonists using its conformation-specific nanobody binders, Nb39 and Nb44. Additionally, I propose to adapt the in-solution bioluminescent assay for broader applications using G-protein peptidomimetics. The platform will be used to screen agonists and antagonists for a wide variety of GPCRs, extending the screening capabilities to include additional GPCRs implicated in drug abuse. Lastly, this assay will provide a new approach to validate the structural integrity of GPCRs isolated from membranes using nanodiscs. Overall, the screening assay proposed here could have far-reaching therapeutic impacts resulting from the discovery of non-addictive pain medications or agonists to suppress opioid-induced addiction.