Development of an ICMT Supported Membrane Sensor

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多元件膜基传感器阵列，用于高通量、平行感测作用于特定膜蛋白靶点的治疗剂候选物。这种传感技术的成功开发可以加速针对各种疾病中涉及的膜蛋白的药物发现。我们将开发一种稳定的不对称膜结构，包含异戊二烯半胱氨酸羧基甲基转移酶（ICMT）在这个项目中作为一个潜在的新工具，用于药物发现在癌症化疗。ICMT是内质网中的膜蛋白，负责-CaaX基序蛋白（包括Ras信号转导蛋白）的羧基甲基化。这种膜传感器结构将使Icmt介导的甲基化的模型底物N-乙酰法尼基半胱氨酸的检测作为荧光发射的变化，由于二硫键连接的分子信标的耦合裂解。从这些不对称结构开发的传感器将提供候选药物抑制ICMT催化的甲基化的能力的直接指示。这种方法将作为一个强大的工具，筛选药物库的铅化合物，可能会抑制细胞致癌Ras蛋白的甲基化。这些化合物的发现和开发是重要的，因为Ras羧甲基化的抑制不仅促进Ras蛋白的错误定位，而且抑制Ras转化细胞的能力。ICMT是开发这种基于膜的传感器的优秀模型系统，因为存在许多表征良好的底物来提供数据验证。这些底物将被用作开发高通量筛选方法的工具，该方法可能导致用于难治性肿瘤的改进的化疗剂。该项目的后续阶段将解决在光学透明基板上的多元件传感器阵列的设计、制造、表征和验证。将使用多学科团队方法，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，以实现目标支持的膜装置。这种检测器阵列概念的未来扩展可能具有深远的潜力，可以加速发现针对许多其他类型的膜相关蛋白的新治疗剂。

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.