Research Project
2423693
In the drug discovery process, there is increased need for greater throughput screening for lead compounds at reduced cost. Concomitant with this is a greater use of living cells in screening assays to yield more accurate information on biological efficacy. We propose a new technology that builds on the success of firefly luciferase as a rapid and quantitative indicator of cellular physiology in screening operations. The new technology is based on the combined use of luciferase capable of emitting green and red light, respectively. By using two colors of light simultaneously, the overall information throughput is increased, and new assay strategies are enable such as quantitation by ratiometric and relational measurements. These assay strategies could increase the throughput in the primary screens, allow greater miniaturization of the screening assays, and reduce the number of required assays in second screens. Development of enzymes capable of emitting green and red light has been completed, as well as demonstration of their co-expression in living cells. Remaining technical objectives include characterization of enzyme performance as reporters in mammalian cells, development of assay chemistries optimized for laboratory automation, and methods development for high throughput quantitation of cell physiology. PROPOSED COMMERCIAL APPLICATION There is a strong need in the drug discovery process for a sensitive and rapid screening system for synthetic libraries of candidate molecules. We forecast a market for our live cell high throughput screening system of at least 10 million dollars annually in five years. Such a system would be amenable to laboratory automation and a variety of high throughput assay formats.
