G protein-coupled receptors (GPCRs) represent one of the highest priority targets for the pharmaceutical industryand roughly one-third of all approved drugs target on specific GPCRs, which corresponds to global sales of overUSD 180 billion annually. As one of the largest gene families in humans with varied physiology functions, GPCRshave also been implicated in the pathophysiology of numerous diseases such as cancer, diabetes insipidus,heart disease, and numerous metabolic disorders. Understanding the characteristics of GPCRs remains acentral theme in biology and various commercial assays are available to characterize ligand binding affinity andthe identification of agonists and antagonists for members of this receptor family. Despite their utility, theseassays have a number of limitations that preclude their widespread use. These include the need for expensive,artificial GPCR cell lines; limited analytical output; indirect measures of GPCR properties; and the potential forphotobleaching and photodamage during data collection. Surface-enhanced Raman spectroscopy (SERS) is anextremely sensitive and highly specific means to analyze surface molecules in the cellular and subcellulardomains. The proposed Phase I research plan focuses on the development of SERS-based probes for thecharacterization of two GPCRs that are members of the predominant class of these receptors, the Class A GPCRfamily. This approach has noted advantages over existing technology including greater analytical output, directmeasures of the GPCR rather than downstream targets, and the ability to apply this technology to any nativecells with their normal complement of cellular proteins. The long-term goal of this research is to develop a lowcost, versatile and robust platform for the characterization of GPCRs that is a significant advancement over thoseassays currently available. To meet this goal, we have the following specific objectives."¢ Development of GPCR SERS probes (Specific Aim 1). The initial goal (Obj. 1.1) in this part of the proposed plan is to identify suitable antibodies for eventual construction of SERS probes using immuno histochemistry and surface plasma resonance for GPR84 and GPR120 in cell lines expressing these receptors. Objective 1.2 will use the identified antibodies to synthesize, construct and characterize SERS probes specific for GPR84 andGPR120."¢ Development of the SERS-based GPCR activation assay and validation with existing technology(Specific Aim 2). The SERS probes will be used in Objective 2.1 to develop the point-by-point and spectralmapping modules to provide the kinetic analyses, dose-dependence, relative expression area, and activationprobabilities for these two GPCRs. Data generated in this objective will be compared to the exiting technologyby running commercially available assays (PathHunter® Ã-arrestin assays; Eurofins) on the same two cell lines(Objective 2.2).
Public Health Relevance Statement: PROJECT NARRATIVE The importance of understanding the properties of G protein-coupled receptors (GPCRs) is underscored by the fact that these proteins are the most common druggable target in the pharmaceutical industry with a global sales volume of over USD 180 billion annually. Existing technology for assaying GPCR function has several limitations including the need for expensive, engineered cell lines, limited information on receptor characteristics, and indirect measurement properties. This proposal is focused on the development and implementation of novel, highly sensitive SERS-based probes for exploring GPCRs that obviate these limitations and allow the investigation of GPCRs in any native cell type at the single cell or population levels, and offer greater analytical power than approaches currently available.
Project Terms: Antibodies ; Monoclonal Antibodies ; Clinical Treatment Moab ; mAbs ; Biological Assay ; Assay ; Bioassay ; Biologic Assays ; Biology ; Malignant Neoplasms ; Cancers ; Malignant Tumor ; malignancy ; neoplasm/cancer ; Cell Line ; CellLine ; Strains Cell Lines ; cultured cell line ; Cell Survival ; Cell Viability ; Cells ; Cell Body ; Complement ; Complement Proteins ; Data Analyses ; Data Analysis ; data interpretation ; Data Collection ; Diabetes Insipidus ; Disadvantaged ; Disease ; Disorder ; Drug Industry ; Pharmaceutic Industry ; Pharmaceutical Industry ; Endocrine System Diseases ; Endocrine Diseases ; Endocrine Diseases and Manifestations ; endocrine disorder ; Engineering ; Family ; Feasibility Studies ; Florida ; Fluorescence ; Goals ; Heart Diseases ; Cardiac Diseases ; Cardiac Disorders ; heart disorder ; Human ; Modern Man ; Immunochemistry ; Immunohistochemistry ; Immunohistochemistry Cell/Tissue ; Immunohistochemistry Staining Method ; Kinetics ; Ligands ; luminescence ; Membrane Proteins ; Membrane Protein Gene ; Membrane-Associated Proteins ; Surface Proteins ; Metabolic Diseases ; Metabolic Disorder ; Thesaurismosis ; metabolism disorder ; Mission ; Oligonucleotides ; Oligo ; oligos ; Pathology ; Peptides ; Physiology ; Plasma ; Blood Plasma ; Plasma Serum ; Reticuloendothelial System, Serum, Plasma ; Play ; Probability ; Proteins ; Publications ; Scientific Publication ; Cell Surface Receptors ; Research ; Role ; social role ; Running ; Sales ; Sensitivity and Specificity ; Computer software ; Software ; Raman Spectrum Analysis ; IR/UV/Raman Spectroscopy ; Raman Spectroscopy ; Raman spectrometry ; Technology ; Time ; Universities ; Promega ; Pro-Mega ; Measures ; Family member ; GTP-Binding Protein alpha Subunits, Gs ; G(s), alpha Subunit ; G(s), α Subunit ; G(s)alpha ; G(s)α ; GTP-Binding Protein α Subunits, Gs ; Gs alpha Family G-Protein ; Gsα ; Gαs ; Regulatory Ns Protein ; Stimulatory Gs G-Protein ; alpha Subunit Stimulatory GTP-Binding Protein ; alpha-Gs ; α-Gs ; enhancing factor ; beta-arrestin ; arrestin B ; β-arrestin ; base ; Area ; Surface ; Phase ; Physiological ; Physiologic ; Medical ; Measurement ; Selection Criteria ; Agonist ; Functional disorder ; Dysfunction ; Physiopathology ; pathophysiology ; tool ; instrument ; Life ; Investigation ; Dependence ; cell type ; System ; extracellular ; experience ; Performance ; Receptor Protein ; receptor ; success ; novel ; member ; G Protein-Complex Receptor ; G Protein-Coupled Receptor Genes ; GPCR ; G-Protein-Coupled Receptors ; Cell surface ; Devices ; Modeling ; Property ; response ; drug development ; receptor function ; Photobleaching ; Molecular Interaction ; Binding ; Pharmaceutical Agent ; Pharmaceuticals ; Pharmacological Substance ; Pharmacologic Substance ; Preparedness ; Readiness ; ARNT ; Aryl Hydrocarbon Receptor Nuclear Translocator ; Dioxin Receptor, Nuclear Translocator ; HIF-1 Beta Gene ; HIF-1Beta Gene ; HIF-1beta ; HIF1-Beta Gene ; HIF1B ; HIF1B Gene ; HIF1Beta ; HIF1beta Gene ; Hypoxia-Inducible Factor 1 Beta Gene ; Hypoxia-Inducible Factor 1, Beta subunit ; Nuclear Translocator Gene Dioxin Receptor ; TANGO ; ARNT gene ; Dose ; Affinity ; Aptamer Technology ; Data ; Detection ; Receptor Activation ; Emerging Technologies ; Emergent Technologies ; Gene Family ; Ligand Binding ; Validation ; Characteristics ; Molecular ; Process ; Development ; developmental ; Output ; cost ; Population ; data acquisition ; Cell model ; Cellular model ; commercialization ; overexpression ; overexpress ; Drug Targeting ; GPR84 gene ; GPR84 ; druggable target ; detection method ; detection procedure ; detection technique ;
