EFC Assay to Measure Growth Factor Receptors

Discoverx Corporation, Fremont CA

Investigators

Abstract

DESCRIPTION (provided by applicant): Growth factors are a family of polypeptides necessary for the development of all cells in the body. They regulate cell proliferation and abnormal growth factor activity is involved in cancer and other cell proliferative disorders. Because of their medical uses in treating disease, growth factors have become important targets for drug development in the pharmaceutical industry. Growth factors induce their actions by binding to receptor tyrosine kinases (RTK). The binding induces a cascade of phosphorylation events leading to changes in gene expression and also causes receptor internalization and subsequent degradation resulting in desensitization of growth factor signaling. Recent studies have shown that RTK internalization involves the association of the activated receptor with beta arrestin (betaarr) which serves as an adaptor by attaching RTKs to clathrin in vesicles to promote receptor internalization. Technologies that could measure the association of betaarr with RTKs could be developed into assays to discover small molecule drugs to prolong the actions of growth factors by blocking interaction of ?arr with RTK or facilitate RTK internalization to attenuate growth factor actions. Such drugs could supplement the growing family of protein therapeutics used as growth factor agonists or antagonists. In this Phase I SBIR, DiscoveRx proposes to develop its proprietary enzyme fragment complementation (EFC) technology to monitor RTK internalization. The technology is based on a sensitive beta-galactosidase (beta-gal) complementation assay and utilizes two genetically engineered fragments of E. coli beta-gal. The larger fragment, Enzyme Acceptor (EA), contains a deletion near the amino terminus, while the smaller fragment, ProLabel, contains the amino-terminal sequence missing from EA. Alone, EA is inactive, but in vitro it can spontaneously recombine with ProLabel to form an active enzyme that can catalyze the formation of a fluorescent or chemiluminescent product that can be detected photometrically as a visually amplified response. Using this technology, we will develop an assay to measure betaarr association with RTKs. We will also develop a parallel EFC assay to measure the internalization of RTKs. Combined, these two technologies will be further developed in Phase II SBIR studies to provide novel high throughput approaches to pharmaceutical and biotechnology companies to discover unique small molecule drugs to modulate growth factor signaling.