Development of ICMT Supported Membrane Sensor

The long term goal of this project is the development of multi-element membrane-based sensor arrays on asingle chip for high-throughput, parallel sensing of therapeutic agent candidates acting on specificmembrane protein targets. Successful development of this sensing technology can lead to accelerated drugdiscovery targeted to membrane proteins involved in a variety of diseases. We will develop a stabilizedasymmetric membrane structure containing isoprenylcysteine carboxylmethyltransferase (ICMT) in thisproject as a potential new tool for drug discovery in cancer chemotherapy. ICMT is a membrane protein inthe endoplasmic reticulum responsible for the carboxylmethylation of -CaaX motif proteins, including the Rassignal transduction proteins. This membrane sensor architecture will enable the detection of Icmt-mediatedmethylation of the model substrate N-acetylfarnesylcysteine as a change in fluorescence emission due to thecoupled cleavage of a disulfide-linked molecular beacon. Sensors developed from these asymmetricstructures 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 toinhibit the methylation of cellular oncogenic Ras proteins. Discovery and development of these compoundsare important because inhibition of Ras carboxylmethylation promotes not only the mislocalization of the Rasproteins, but also inhibits the ability of Ras to transform cells. ICMT is an excellent model system fordevelopment of this membrane-based sensor because many well-characterized substrates exist to providedata validation. These substrates will be used as tools to develop a high-throughput screening approachthat may lead to improved chemotherapeutic agents for refractory tumors. Subsequent phases of the projectwill address the design, fabrication, characterization, and validation of multi-element sensor arrays on anoptically transparent substrate. A multidisciplinary team approach will be used, combining expertise inbiochemistry, materials synthesis and characterization, analytical chemistry, and theory to achieve the targetsupported membrane device. Future extension of this detector array concept could have far reachingpotential for accelerating the discovery of new therapeutic agents targeted to many other classes ofmembrane-associated proteins.