SBIR-STTR Award

G protein-coupled receptors (GPCRs) are major drug targets for a wide spectrum of diseases including neurological diseases such as migraine, anxiety, psychosis, and schizophrenia Novel robust assay technologies are continuously demanded for GPCR targets to increase odds for leads generation in a large-scale compound screening. Activation of GPCRs results in changes in intracellular calcium and cAMP About 70 percent of nonchemosensory GPCRs mediate their signals through up- or down-regulation of intracellular Camp via Gs and Gi proteins respectively We propose to develop a high-throughput technology for real time assay of Gs protein-coupled receptors (Gs-GPCRs) In this application, cyclic nucleotide-gated (CNG) channel variants are specifically used as a physiological sensor for cAMP, coupling a change in intracellular cAMP concentration to changes in cytosolic calcium concentration and membrane potential Our results demonstrate that the cAMP change induced by activation of adenylyl cyclase and GPCRs can be sensitively monitored in the living mammalian cells expressing the CNG channel variants by using calcium and voltage indicators Stable cells with the sensitive CNG channel mutants have been developed to enable the assay in 96-well microplates This convenient assay allows for the kinetic detection of transient cAMP signals with a high signal-tonoise ratio CNG channels may also provide a means to detect local cAMP concentrations in proximity to adenylylcyclase, allowing for a sensitive detection of GPCR activity Towards next stage of commercialization, we propose to further improve and adapt this robust GPCR assay to standard high-throughput screening platforms including FLIPR.

Thesaurus Terms:
G protein, bioassay, cell surface receptor, high throughput technology, receptor expression, technology /technique development adenylate cyclase, calcium, cyclic AMP, fluorescence, membrane channel

All human cells are regulated by extracellular signals that are transmitted through cell surface receptors. The vast majority of these receptors constitute a superfamily of G-protein coupled receptors (GPCRs), Drugs that act on GPCRs are widely used in therapies against a large variety of human diseases, including neurological diseases such as Parkinson's disease. GPCRs are the single largest target of current drug discovery programs yet, of the approximately 200 GPCR with known functions, only a very small fraction of them are currently utilized in drug discovery due to technology limitations, leaving many of them as putative new targets for the development of new therapeutics. The majority of them signal through second messenger cAMP. Atto Bioscience has developed a novel cAMP assay for a real-time detection of cAMP in living cells using a proprietary cyclic nucleotide-gated (CNG) channel mutant. With the support of a Phase 1 grant from NINDS, we created stable cell lines and optimized assay conditions with both Ca 2+and membrane potential dyes to detect activity of the CNG channels. The CNG-cAMP technology was adapted to multiple assay platforms including FLIPR and bench-top fluorescence plate readers. This is the only commercially available high throughput screen (HTS) assay system capable of detecting receptor activations with temporal resolution. The technology has been licensed for drug discovery to pharmaceutical companies, validating both the technology and its potential market. Our goal is to develop full lines of products to enable commercialization of CNG-cAMP biosensor technology. It includes the development of additional Gs- and Gi-coupled receptor cell lines, the development of CNG cell lines in different cell types, and the development of dye reagents for the technology. We believe that this cAMP biosensor technology will not only support drug discovery, but also allow researchers to examine receptor activation and signal transduction in a new way previously not possible. The specific aims are: (1) generate, evaluate and produce another 20 Gs-coupled receptor cell lines for drug discovery and receptor biological studies; (2) develop and produce 40 Gi-coupled receptor ceil lines; (3) introduce and evaluate CNG sensor into 10 different cell lines and develop them into marketable products; (4) design, synthesize, and evaluate new membrane potential dyes, and produce sufficient reagents for 30,000,000 tests; (5) identify and test new quenchers, and develop 2 reagent kits for CNG-cAMP assay; (6) develop assay for GPCR arrays.

Thesaurus Terms:
G protein, bioassay, biological transport, cell line, cell surface receptor, cyclic AMP, fluorescent dye /probe, high throughput technology, membrane channel, technology /technique development drug discovery /isolation, receptor expression biotechnology, time resolved data, transfection /expression vector