A New Versatile, SERS-based GPCR assay

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PROJECT SUMMARY G protein-coupled receptors (GPCRs) represent one of the highest priority targets for the pharmaceutical industry and roughly one-third of all approved drugs target on specific GPCRs, which corresponds to global sales of over USD 180 billion annually. As one of the largest gene families in humans with varied physiology functions, GPCRs have 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 a central theme in biology and various commercial assays are available to characterize ligand binding affinity and the identification of agonists and antagonists for members of this receptor family. Despite their utility, these assays 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 for photobleaching and photodamage during data collection. Surface-enhanced Raman spectroscopy (SERS) is an extremely sensitive and highly specific means to analyze surface molecules in the cellular and subcellular domains. The proposed Phase I research plan focuses on the development of SERS-based probes for the characterization of two GPCRs that are members of the predominant class of these receptors, the Class A GPCR family. This approach has noted advantages over existing technology including greater analytical output, direct measures of the GPCR rather than downstream targets, and the ability to apply this technology to any native cells with their normal complement of cellular proteins. The long-term goal of this research is to develop a low cost, versatile and robust platform for the characterization of GPCRs that is a significant advancement over those assays 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 immunohistochemistry 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 and GPR120. • 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 spectral mapping modules to provide the kinetic analyses, dose-dependence, relative expression area, and activation probabilities for these two GPCRs. Data generated in this objective will be compared to the exiting technology by running commercially available assays (PathHunter® ß-arrestin assays; Eurofins) on the same two cell lines (Objective 2.2).