EFC Assay to Measure Growth Factor Receptors

描述（由申请人提供）：生长因子是体内所有细胞发育所必需的多肽家族。 它们调节细胞增殖，并且异常生长因子活性参与癌症和其它细胞增殖性病症。由于它们在治疗疾病中的医学用途，生长因子已成为制药工业中药物开发的重要目标。 生长因子通过与受体酪氨酸激酶（RTK）结合来诱导其作用。结合诱导级联的磷酸化事件，导致基因表达的变化，也导致受体内化和随后的降解，导致生长因子信号转导的脱敏。最近的研究表明，RTK内化涉及活化受体与β抑制蛋白（β抑制蛋白）的缔合，β抑制蛋白通过将RTK连接到囊泡中的网格蛋白以促进受体内化而充当衔接子。技术，可以测量与RTKs的β-内酰胺酶协会可以发展成检测发现小分子药物，以延长行动的生长因子通过阻断相互作用？或促进RTK内化以减弱生长因子的作用。这类药物可以补充作为生长因子激动剂或拮抗剂的蛋白质治疗剂的不断增长的家族。 在第一阶段SBIR中，DiscoveRx建议开发其专有的酶片段互补（EFC）技术来监测RTK内化。该技术是基于一种敏感的β-半乳糖苷酶（β-gal）互补测定，并利用两个基因工程片段的E。coli beta-gal.较大的片段，酶受体（EA），含有氨基末端附近的缺失，而较小的片段，ProLabel，含有EA缺失的氨基末端序列。单独使用时，EA是无活性的，但在体外，它可以自发地与ProLabel重组，形成一种活性酶，该酶可以催化荧光或荧光发光产物的形成，该产物可以通过光度法检测为视觉放大反应。 使用这种技术，我们将开发一种测定方法来测量β-内酰胺酶与RTK的结合。我们还将开发一种平行的EFC试验来测量RTK的内化。结合起来，这两种技术将在第二阶段SBIR研究中进一步开发，为制药和生物技术公司提供新的高通量方法，以发现独特的小分子药物来调节生长因子信号传导。

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.