EFC Assay to Measure Growth Factor Receptors

描述(申请人提供)：生长因子是一种多肽家族，对体内所有细胞的发育都是必需的。它们调节细胞增殖和异常生长因子活性，与癌症和其他细胞增殖性疾病有关。由于其在治疗疾病方面的医疗用途，生长因子已成为制药行业药物开发的重要靶点。 生长因子通过与受体酪氨酸激酶(RTK)结合而发挥作用。这种结合诱导了一系列的磷酸化事件，导致基因表达的变化，还导致受体内化和随后的降解，从而导致生长因子信号的脱敏。最近的研究表明，RTK内化涉及激活的受体与β-arrestin(Betaarr)的结合，β-arrestin通过将RTK连接到囊泡中的网状蛋白来促进受体内化，从而作为适配器。可以测量β-ARR与RTK相互作用的技术可以发展为发现小分子药物，通过阻断？ARR与RTK的相互作用来延长生长因子的作用，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疗法的不断壮大的家族。 在这个第一阶段的SBIR中，DiscoveRx公司建议开发其专有的酶片段互补(EFC)技术来监控RTK的内化。这项技术基于一种敏感的β-半乳糖苷酶(β-GAL)互补分析，并利用了两个经过基因工程的大肠杆菌β-半乳糖苷酶片段。较大的片段酶受体(EA)在氨基末端附近有缺失，而较小的片段ProLabel含有EA缺失的氨基末端序列。单独而言，EA是无效的，但在体外，它可以自发地与ProLabel重新结合，形成一种活性酶，可以催化形成荧光或化学发光产物，可以通过光度法检测到作为视觉放大反应的产物。 利用这项技术，我们将开发一种检测Betaar与RTK关联的方法。我们还将开发一种平行的EFC试验来衡量RTK的内化。合并后，这两项技术将在第二阶段SBIR研究中得到进一步开发，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。

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.