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

> **NIH NIH F31** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2022 · $39,350

## Abstract

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.

## Key facts

- **NIH application ID:** 10462863
- **Project number:** 1F31DA056192-01
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Ruby May Miller
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $39,350
- **Award type:** 1
- **Project period:** 2022-05-30 → 2025-05-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10462863

## Citation

> US National Institutes of Health, RePORTER application 10462863, GPCR activation-based in vitro luminescent assays for high-throughput drug screening and extracted GPCR characterization (1F31DA056192-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10462863. Licensed CC0.

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