Solid-state analysis of kinase activity in cell extracts

细胞提取物中激酶活性的固态分析

• 批准号: 7102233
• 负责人: Sean P Palecek
• 金额: $ 25.02万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7102233
• 关键词: acrylamides; antineoplastics; bioassay; chronic myelogenous leukemia; copolymer; crosslink; diagnosis design /evaluation; diagnostic tests; drug resistance; enzyme activity; enzyme inhibitors; enzyme substrate; flow cytometry; human tissue; immunologic assay /test; kinase inhibitor; microcapsule; microfluidics; neoplasm /cancer pharmacology; peptides; pharmacogenetics; phosphorylation; protein tyrosine kinase; tissue /cell preparation

DESCRIPTION (provided by applicant): The goal of the work described in this proposal is to construct a quantitative and clinically useful assay to measure activity of multiple tyrosine kinases from cellular extracts of chronic myelogenous leukemia (CML) and acute myelogenous leukemia (AML) patients. Most CML cases are associated with a chromosomal translocation resulting in the consitutively active BCR-ABL tyrosine kinase while a large number of AML patients overexpress or have activating mutations in the FLT3 tyrosine kinase. Imatinib mesylate, an inhibitor of BCR-ABL, demonstrates tremendous efficacy in inactivating BCR-ABL in vivo and achieving remission in CML patients. However, certain BCR-ABL mutations are imatinib-resistant. Therefore, an in vitro measurement to assess the level of BCR-ABL activity and its dose-dependent inhibition upon treatment with imatinib would provide valuable information for tailoring individual CML patient treatment programs. Similarly, FLT3 kinase inhibitors are in clinical trials and resistant mutants are likewise expected to exist. BCR-ABL and FLT3 substrates will be synthesized and labeled with an acrylic moiety. Then these substrates will be copolymerized with acrylamide and crosslinker in an emulsion polymerization to generate microspheres via two-phase microfluidic flow. The microspheres will be exposed to extracts from cell lines expressing BCR-ABL and FLT3 and to white blood cell extracts derived from CML patients. Quantitative levels of substrate phosphorylation will be detected by anti-phosphotyrosine antibody labeling and flow cytometry. In addition, a method using thiophosphorylation and subsequent labeling of thiophosphates will be developed as an alternative to antibody labeling. Sensitivity and specificity of detection via antiphosphotyrosine antibodies and thiophosphorylation will be compared. The assay developed in this study will be compared to standard in vitro kinase assays as well as to methods currently used to diagnose CML, with respect to sensitivity and specificity. In this study our specific aims are: 1. Develop acrylamide-peptide copolymer microspheres for measuring Bcr-Abl activity in cell extracts 2. Quantify Bcr-Abl inhibition by imatinib mesylate in cell extracts and tissue samples from CML patients 3. Assess activity of multiple kinases using an acrylamide-peptide copolymer microsphere assay Relevance: The work described in this proposal will develop an assay to directly diagnose elevated protein tyrosine kinase activity related to chronic myeloid leukemia and predict how an individual patient will respond to drug therapy. This assay will likely be more rapid and less expensive than current methods, and have the advantage of predicting response to drug treatment.

描述（由申请人提供）：本提案中所述工作的目标是构建一种定量和临床有用的测定方法，以测量慢性髓性白血病（CML）和急性髓性白血病（AML）患者细胞提取物中多种酪氨酸激酶的活性。大多数CML病例与染色体易位相关，导致BCR-ABL酪氨酸激酶的结构活性，而大量AML患者过度表达FLT 3酪氨酸激酶或在FLT 3酪氨酸激酶中存在激活突变。甲磺酸伊马替尼是BCR-ABL的抑制剂，在体内灭活BCR-ABL并在CML患者中实现缓解方面表现出巨大的疗效。然而，某些BCR-ABL突变对伊马替尼具有耐药性。因此，评估伊马替尼治疗后BCR-ABL活性水平及其剂量依赖性抑制的体外测量将为定制个体CML患者治疗方案提供有价值的信息。同样，FLT 3激酶抑制剂也在临床试验中，预计同样存在耐药突变体。将合成BCR-ABL和FLT 3底物，并用丙烯酸部分标记。然后，这些基板将共聚与丙烯酰胺和交联剂在乳液聚合，以生成微球通过两相微流控流动。将微球暴露于表达BCR-ABL和FLT 3的细胞系提取物以及CML患者的白色血细胞提取物。将通过抗磷酸酪氨酸抗体标记和流式细胞术检测底物磷酸化的定量水平。此外，将开发使用硫代磷酸化和随后标记硫代磷酸盐的方法作为抗体标记的替代方法。将比较通过抗磷酸酪氨酸抗体和硫代磷酸化检测的灵敏度和特异性。本研究中开发的检测方法将与标准体外激酶检测方法以及目前用于诊断CML的方法在灵敏度和特异性方面进行比较。本研究的具体目标是：1.开发丙烯酰胺-肽共聚物微球用于测量细胞提取物中的Bcr-Abl活性2.在CML患者的细胞提取物和组织样品中定量甲磺酸伊马替尼对Bcr-Abl的抑制3.使用丙烯酰胺-肽共聚物微球测定法评估多种激酶的活性相关性：本提案中描述的工作将开发一种测定法，以直接诊断与慢性髓性白血病相关的蛋白酪氨酸激酶活性升高，并预测个体患者对药物治疗的反应。这种检测方法可能比目前的方法更快速，更便宜，并且具有预测对药物治疗反应的优势。