# A robust assay for detecting agonist bias in living cells.

> **NIH NIH R44** · MONTANA MOLECULAR, LLC · 2020 · $600,181

## Abstract

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Project Summary
The objective of this SBIR Phase II project is to optimize and validate a prototype assay
for ligand bias to be used in screening drugs that target seven transmembrane G-
protein coupled receptors (GPCRs). The assay is based on genetically-encoded
fluorescent biosensors that report signaling with a change in fluorescence intensity.
Aim 1 of this project involves engineering and testing to increase the change in
fluorescence intensity of biosensors for β arrestin1 and β arrestin2. This involves using
an established workflow and screening process to iteratively improve linkers in between
a very bright green fluorescent protein and β-arrestin. This proprietary process consists
of randomly mutagenizing key amino acids to create low diversity libraries of candidate
biosensors that are screened for improved fluorescence properties.
Aim 2 involves optimizing assay parameters and validating the multiplex assay
comprised of the optimized green fluorescent biosensor for β arrestin, co-expressed
with existing red fluorescent biosensors for diacylglycerol (DAG) or cyclic AMP. The red
sensor indicates G-protein mediated activity while the green sensor indicates arrestin
activity. The assay will be validated with a select panel of GPCRs and small molecule
and peptide agonists.
GPCRs, are one of the largest family of drug targets. Their activity is mediated by both
G proteins and β-arrestins which activate a network of distinct signaling pathways.
Depending upon the GPCR and cell type, biased agonism can be therapeutically
beneficial or produce unwanted or harmful side effects (Violin and Lefkowitz, 2007).
Quantifying bias in GPCR signaling could help to identify new therapeutic drugs that
avoid adverse effects. Detecting agonist bias early in the screening process, in cell
types that are relevant to disease, has the potential to reduce the risk and cost of drug
discovery.

## Key facts

- **NIH application ID:** 9964847
- **Project number:** 5R44GM125390-03
- **Recipient organization:** MONTANA MOLECULAR, LLC
- **Principal Investigator:** THOMAS E HUGHES
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $600,181
- **Award type:** 5
- **Project period:** 2017-08-21 → 2022-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9964847

## Citation

> US National Institutes of Health, RePORTER application 9964847, A robust assay for detecting agonist bias in living cells. (5R44GM125390-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9964847. Licensed CC0.

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