R43CA295295

Fluorescent biosensors for measuring the signaling kinetics of GPCR ligands that activate G12/13 pathways.

- Abstract

G-protein coupled receptors are outstanding drug targets.

When the receptor is activated, it signals through heterotrimeric G proteins, which in turn activate various downstream effector proteins.

The process of discovering new drugs that activate particular G-protein coupled receptors depends on having functional assays that provide insights into which compounds activate the receptor, which block it, and how long the receptor is active.

Today there are good assays for the receptors that activate the GS, GI, and GQ proteins.

There are remarkably few for the G12/13 family, which is particularly important because this pathway is involved in cancer.

The receptors that signal through the G12/13 proteins are involved in cell adhesion, cell invasion, or cell motility, the hallmarks of cancer behavior.

Indeed the constitutively active G12 protein can act as an oncogene.

Given the biological importance of the receptors that signal through the G12/13 pathway, reliable assays for the activation of this pathway are needed.

The first aim of this work is to determine the feasibility of creating a live cell, fluorescent assay for G12/13 pathway activation that 1) can be used on standard plate readers, 2) provides kinetic data about when the pathway is activated, and for how long, 3) involves no additional enzyme substrates that can be depleted, and 4) can be used on a variety of cell types ranging from standard fibroblasts to cancer cell lines.

Cross talk often occurs between different G-protein signaling pathways.

Some receptors activate multiple different G proteins, and it is often the case that a receptor will activate both the GQ and G12/13 pathways.

The second goal of this project is to determine the feasibility of creating CRISPR/Cas9 tools with the specific guide RNAs to selectively knock out different G proteins.

These tools would be capable of removing the function of a G protein in a matter of days such that receptor driven signaling could be compared in parallel wells of identical cells missing different signaling pathways.

The final goal of the project is to combine the RhoA sensor, CRISPR tools, and biosensors for DAG, Ca2+, cAMP, β-arrestin to completely characterize the PAR1 receptor and potential agonist bias at this receptor in breast cancer cell lines.

Montana Molecular

TO IMPROVE SCREENING AND EARLY DETECTION STRATEGIES AND TO DEVELOP ACCURATE DIAGNOSTIC TECHNIQUES AND METHODS FOR PREDICTING THE COURSE OF DISEASE IN CANCER PATIENTS. SCREENING AND EARLY DETECTION RESEARCH INCLUDES DEVELOPMENT OF STRATEGIES TO DECREASE CANCER MORTALITY BY FINDING TUMORS EARLY WHEN THEY ARE MORE AMENABLE TO TREATMENT. DIAGNOSIS RESEARCH FOCUSES ON METHODS TO DETERMINE THE PRESENCE OF A SPECIFIC TYPE OF CANCER, TO PREDICT ITS COURSE AND RESPONSE TO THERAPY, BOTH A PARTICULAR THERAPY OR A CLASS OF AGENTS, AND TO MONITOR THE EFFECT OF THE THERAPY AND THE APPEARANCE OF DISEASE RECURRENCE. THESE METHODS INCLUDE DIAGNOSTIC IMAGING AND DIRECT ANALYSES OF SPECIMENS FROM TUMOR OR OTHER TISSUES. SUPPORT IS ALSO PROVIDED FOR ESTABLISHING AND MAINTAINING RESOURCES OF HUMAN TISSUE TO FACILITATE RESEARCH. SMALL BUSINESS INNOVATION RESEARCH (SBIR) PROGRAM: TO EXPAND AND IMPROVE THE SBIR PROGRAM, TO INCREASE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, TO INCREASE SMALL BUSINESS PARTICIPATION IN FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION. SMALL BUSINESS TECHNOLOGY TRANSFER (STTR) PROGRAM: TO STIMULATE AND FOSTER SCIENTIFIC AND TECHNOLOGICAL INNOVATION THROUGH COOPERATIVE RESEARCH AND DEVELOPMENT CARRIED OUT BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO FOSTER TECHNOLOGY TRANSFER BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO INCREASE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION.

93.396 - Cancer Biology Research

National Cancer Institute (NCI) [HHS - NIH]

Montana United States

State-Wide

PHS 2024-2 Omnibus Solicitation of the NIH, CDC and FDA for Small Business Innovation Research Grant Applications (Parent SBIR [R43/R44] Clinical Trial Not Allowed) (PA-24-245)