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A robust assay for detecting agonist bias in living cells.

Award Number: R43GM125390
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 08/21/2017
PERIOD OF PERFORMANCE END DATE: 08/20/2018

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A robust assay for detecting agonist bias in living cells. - PROJECT SUMMARY The goal this Phase 1 SBIR project is to develop an agonist bias assay for screening drugs that target seven transmembrane G-protein coupled receptors (GPCRs). Phase 1 will establish the feasibility of a robust assay platform to detect when particular GPCR agonists activate both ß-arrestin and G-protein pathways. This assay will detect responses in living cells on automated fluorescence plate readers, and will be compatible with screening in any cell type including neurons, pancreatic islets, cardiomyocytes, transformed cell lines and iPSC-derived cells. 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 treatments 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. The following specific aims will establish the feasibility of this assay: Aim 1: Develop dimerization-dependent fluorescent proteins to report ß-arrestin protein interactions involved in signaling. The dimerization-dependent fluorescent protein (ddFP) sensors will serve as a rapid prototype to be used as a standard for verifying sensors described in Aim 2. The ddFP sensors will be a functional readout for the protein interactions characteristic of ß-arrestin signaling. The ddFP sensors will be built using standard molecular cloning in the matter of a few weeks and will be detectable using epi- fluorescence microscopy. These prototype sensors are not amenable for detection on automated plate readers but will be useful for lower throughput, nonquantitative applications. Aim 2: Develop ß-arrestin signaling sensors that are based on one, bright fluorescent protein that have such large signals that they are detectable on an automated fluorescence plate reader. Building on the ß-arrestin protein interactions in aim one, we will harness the large scale movements of ß-arrestin associated proteins to create a bright green sensor with circularly permuted mNeonGreen. Libraries of candidate sensors will be built and screened using an iterative, directed evolution approach. Aim 3: Test a fluorescence plate reader assay that simultaneously detects G-protein and ß-arrestin signaling typical of biased agonism. ß2-adrenergic receptor activation by agonists that signal through either or both the Gs pathway and the ß-arrestin pathway (Drake et al., 2008) will be used to test the assay on a standard fluorescence plate reader. Responses from prototype sensors for ß-arrestin signaling, developed in aims one and two, and a previously developed red cAMP sensor (Tewson et al., 2016) will be compared to determine levels of agonist bias from known ß2-adrenergic agonists. www.montanamolecular.com


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2019 ( Subtotal = $0 )
2019	2017	MONTANA MOLECULAR LLC	366 GALLATIN PARK DR STE A	BOZEMAN	MT	59715	GALLATIN	USA	Biomedical Research and Research Training	000	1	11/17/2018	NEW	$0
														Subtotal = $0

Issue Date FY: 2017 ( Subtotal = $193,000 )
2017	2017	MONTANA MOLECULAR LLC	347 S FERGUSON AVE	BOZEMAN	MT	59718	GALLATIN	USA	Biomedical Research and Research Training	000	1	8/15/2017	NEW	$193,000
														Subtotal = $193,000

Grand Total All Awards = $193,000

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A robust assay for detecting agonist bias in living cells.

Award Number: R43GM125390

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 08/21/2017

PERIOD OF PERFORMANCE END DATE: 08/20/2018

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