Peptide Reporters for Estimation of BCR-Abl Kinase Activity in ALL Patient Cells

用于评估所有患者细胞中 BCR-Abl 激酶活性的肽报告基因

• 批准号: 8911135
• 负责人: Imola G. Zigoneanu
• 金额: $ 2万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2015-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8911135
• 关键词: Acute Lymphocytic Leukemia; Address; Aftercare; Amino Acids; Biochemical; Biological Assay; Blood capillaries; Bone Marrow Cells; Bone Marrow Transplantation; Capillary Electrophoresis; Cells; Cellular Assay; Clinic; Clinical; Coupled; Custom; Data; Development; Diagnosis; Drug Targeting; Drug resistance; Electroporation; Enzymes; Evaluation; Exhibits; Face; Genetic; Goals; Grant; Half-Life; Heterogeneity; High Pressure Liquid Chromatography; In Vitro; Individual; Kinetics; Label; Lead; Malignant Neoplasms; Measurement; Measures; Methods; Microinjections; Molecular; Monitor; Oncogenes; Oncogenic; Outcome Study; Patients; Peptides; Ph+ ALL; Pharmaceutical Preparations; Phase; Philadelphia Chromosome; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Primary Neoplasm; Proteins; Protocols documentation; Reporter; Reporting; Research; Resistance; Sampling; Signal Transduction Pathway; Solid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; System; Technology; Testing; Treatment Efficacy; Variant; Work; base; cancer recurrence; cancer therapy; capillary; chemotherapy; clinically relevant; design; effective therapy; improved; individualized medicine; instrument; instrumentation; kinase inhibitor; myristoylation; neoplastic cell; novel; public health relevance; response; targeted treatment; therapeutic target; therapy resistant; training project; tumor

DESCRIPTION (provided by applicant): Molecularly targeted therapies against kinases are changing the face of cancer treatment; however, there are no assays currently in clinical use that directly monitor the inhibition of these drugs in patient cells. Moreover, cellular heterogeneity both at the genetic and biochemical level underlies resistance to these therapies, while bulk cellular assays do not accurately predict or monitor therapeutic efficacy. A biochemical assay for monitoring BCR-Abl kinase activity in single cells has been developed. The assay quantitatively measures the enzymatic activity of BCR-Abl kinase in patients with Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). The goal of this grant is to optimize the assay to test the hypothesis that the enzymatic activity of BCR-Abl exhibits a great degree of variability amongst individual cells of Ph+ ALL patients allowing prediction of response or resistance to targeted therapies. The proposed research takes into consideration the design, synthesis and evaluation of fluorescently labeled peptide substrates as a novel method of quantitatively estimate BCR-Abl kinase activity in patient samples. Aim 1 is focused on reporter synthesis by standard FMOC solid phase peptide protocol and characterization by MALDI and HPLC. Two design strategies are selected to generate reporters with increased intracellular resistance to degradation: 1) a beta-hairpin peptide attached through a linker to N-terminus of the substrate, and 2) strategic incorporation of un-natural amino acids into the substrate. In Aim 2, the substrates are evaluated in vitro for determination of kinetic parameters KM and Vmax, and also in cell lysates and intact BCR-Abl+ cells for quantification of phosphorylation and determination of substrate half-life. Capillary electrophoreses is used for quantification with an automated system for cell lysate studies and a custom-built instrument for single cell analyses. Ultimately, the substrates will be selected based on phosphorylation and resistance to degradation. Aim 3 will quantify BCR-Abl kinase activity in Ph+ ALL patient samples at the single-cell level using the lead substrates developed in Aim 2. Microinjection is utilized to deliver the substrates into cells but other methods ranging from electroporation to myristoylation will be tested. These data will be used to gain a greater understanding on kinase phosphorylation in tumor cells, to predict the efficacy of drug-targeted therapy for ALL patients and to facilitate development of individualized therapies.

描述（由申请人提供）：针对激酶的分子靶向疗法正在改变癌症治疗的面貌;然而，目前临床上没有直接监测这些药物在患者细胞中的抑制作用的测定法。此外，在遗传和生物化学水平上的细胞异质性是对这些疗法的抗性的基础，而大量细胞测定不能准确地预测或监测治疗功效。已开发了一种用于监测单细胞中BCR-Abl激酶活性的生化测定法。该检测定量测量费城染色体阳性（Ph+）急性淋巴细胞白血病（ALL）患者BCR-Abl激酶的酶活性。该研究的目的是优化检测方法，以检验BCR-Abl的酶活性在Ph+ ALL患者的单个细胞中表现出很大程度的变异性，从而预测对靶向治疗的应答或耐药性。该研究考虑了荧光标记肽底物的设计、合成和评价，作为定量估计患者样本中BCR-Abl激酶活性的新方法。目的1：采用标准FMOC固相肽合成报告基因，并通过MALDI和HPLC进行表征。选择两种设计策略来产生具有增加的细胞内降解抗性的报告物：1）通过接头连接至底物的N末端的β-发夹肽，和2）将非天然氨基酸策略性地掺入底物中。在目的2中，在体外评价底物以测定动力学参数KM和Vmax，还在细胞裂解物和完整BCR-Abl+细胞中评价底物以定量磷酸化和测定底物半衰期。毛细管电泳用于定量，自动化系统用于细胞裂解物研究，定制仪器用于单细胞分析。最终，将根据磷酸化和抗降解性选择底物。目标3将使用目标2中开发的先导底物在单细胞水平上定量Ph+ ALL患者样本中的BCR-Abl激酶活性。利用显微注射将底物递送到细胞中，但将测试从电穿孔到肉豆蔻酰化的其他方法。这些数据将用于更好地了解肿瘤细胞中的激酶磷酸化，预测药物靶向治疗对ALL患者的疗效，并促进个体化治疗的发展。