Development of an ICMT Supported Membrane Sensor

ICMT 支持的薄膜传感器的开发

• 批准号: 7037706
• 负责人: DAVID H THOMPSON
• 金额: $ 37.93万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7037706
• 关键词: Saccharomyces cerevisiae; antineoplastics; artificial membranes; atomic force microscopy; carboxyl group; chimeric proteins; cysteine; drug discovery /isolation; fluorescence polarization; fluorescence recovery after photobleaching; guanine nucleotide binding protein; hemagglutinin; high throughput technology; immunofluorescence technique; membrane lipids; membrane model; membrane proteins; membrane reconstitution /synthesis; method development; methylation; methyltransferase; neoplasm /cancer chemotherapy; oncogenes; polyethylene glycols; protein structure function

DESCRIPTION (provided by applicant): The long term goal of this project is the development of multi-element membrane-based sensor arrays on a single chip for high-throughput, parallel sensing of therapeutic agent candidates acting on specific membrane protein targets. Successful development of this sensing technology can lead to accelerated drug discovery targeted to membrane proteins involved in a variety of diseases. We will develop a stabilized asymmetric membrane structure containing isoprenylcysteine carboxylmethyltransferase (ICMT) in this project as a potential new tool for drug discovery in cancer chemotherapy. ICMT is a membrane protein in the endoplasmic reticulum responsible for the carboxylmethylation of -CaaX motif proteins, including the Ras signal transduction proteins. This membrane sensor architecture will enable the detection of Icmt-mediated methylation of the model substrate N-acetylfarnesylcysteine as a change in fluorescence emission due to the coupled cleavage of a disulfide-linked molecular beacon. Sensors developed from these asymmetric structures will provide a direct indication of a drug candidate's ability to inhibit methylation catalyzed by Icmt. This approach will serve as a powerful tool for screening drug libraries for lead compounds that are likely to inhibit the methylation of cellular oncogenic Ras proteins. Discovery and development of these compounds are important because inhibition of Ras carboxylmethylation promotes not only the mislocalization of the Ras proteins, but also inhibits the ability of Ras to transform cells. ICMT is an excellent model system for development of this membrane-based sensor because many well-characterized substrates exist to provide data validation. These substrates will be used as tools to develop a high-throughput screening approach that may lead to improved chemotherapeutic agents for refractory tumors. Subsequent phases of the project will address the design, fabrication, characterization, and validation of multi-element sensor arrays on an optically transparent substrate. A multidisciplinary team approach will be used, combining expertise in biochemistry, materials synthesis and characterization, analytical chemistry, and theory to achieve the target supported membrane device. Future extension of this detector array concept could have far reaching potential for accelerating the discovery of new therapeutic agents targeted to many other classes of membrane-associated proteins.

描述(由申请人提供)：该项目的长期目标是在单个芯片上开发基于多元素膜的传感器阵列，用于高通量、并行地检测作用于特定膜蛋白靶标的候选治疗剂。这种传感技术的成功开发可以加速针对多种疾病中涉及的膜蛋白的药物发现。在本项目中，我们将开发一种稳定的含有异丙基半胱氨酸羧甲基转移酶(ICMT)的不对称膜结构，作为一种潜在的癌症化疗药物发现的新工具。ICMT是内质网中的一种膜蛋白，负责-CAAX基序蛋白的羧甲基化，包括RAS信号转导蛋白。这种膜传感器体系结构将能够检测ICMT介导的模型底物N-乙酰法尼基半胱氨酸的甲基化，这是由于二硫键连接的分子信标的耦合裂解导致的荧光发射的变化。从这些不对称结构开发的传感器将直接指示候选药物抑制ICMT催化的甲基化的能力。这种方法将成为筛选药物库中可能抑制细胞致癌RAS蛋白甲基化的先导化合物的有力工具。这些化合物的发现和发展具有重要意义，因为抑制RAS羧甲基化不仅会促进RAS蛋白的错误定位，而且还会抑制RAS转化细胞的能力。ICMT是开发这种基于膜的传感器的一个很好的模型系统，因为存在许多表征良好的衬底来提供数据验证。这些底物将被用作开发高通量筛选方法的工具，这种方法可能会导致对难治性肿瘤的化疗药物的改进。该项目的后续阶段将涉及在光学透明衬底上的多元素传感器阵列的设计、制造、表征和验证。将使用多学科团队的方法，结合生物化学、材料合成和表征、分析化学和理论方面的专业知识来实现目标支撑膜装置。这种探测器阵列概念的未来扩展可能会对加速发现针对许多其他类别的膜相关蛋白的新的治疗剂具有深远的潜力。