HIGH CONTENT SCREENING FOR EPITHELIAL BIOLOGY

DESCRIPTION (provided by applicant): BioTechPlex proposes to develop a high content, high throughput, cell-based screening system for drug discovery using epithelial cells. This system will enable the dynamic responses of multiple cellular fluorophore-repo rtable molecular species to be simultaneously measured in a 96 well plate. The dynamic responses will be measured in a time frame consistent with the temporal nature of the respective intracellular kinetic processes. The activation or inhibition of a targe t site will be determined with high specificity following the characterization and classification of the dynamic fingerprint from three fluorescent probes. In Phase I, BioTechPlex developed the optics and the firmware of the Multi-Fluorophore Acousto Opt ic Plate Reader (MAPR(tm)) system. MAPR simultaneously measures three fluorescent signals from 96 cell samples with a dwell time of 10 ms per well. In Phase II, BioTechPlex will further develop MAPR, add bioinformatics data base, Al decision making and val idate the entire system making. This will be achieved by accomplishing the following 3 objectives. 1) Add fluid handling and environmental conditioning to MAPR by incorporating robotic plate and liquid handling as well as temperature and atmospheric regula tion. 2) Develop Target Identification Algorithms that will classify, characterize and file the unique identifiers of the dynamics of three fluorophores as well as extract this information for target identification and decision marking. 3) Validate the com plete MAPR system using normal, pathologic and engineered respiratory epithelial cell lines prior to and following perturbation of muscarinic, adrenergic and IPS receptor activated signal transduction pathways which predictably perturb intracellular ions a nd thus plasma membrane potential. This enabling technology will provide the pharmaceutical industry a novel, multi-probe cell-based platform for high content screening and high-throughput drug screening for drug discovery, validation, and cytotoxicity. Th is system addresses the needs of FDA (funded by FDA). In addition, the project directly responds to the NIH Roadmap Initiatives released in 2004. This project also directly responds to the 2005 SBIR general solicitations by NCRR for the development of high throughput screening technologies as well as by NIDDK for general interest in epithelial biology.