Research Project
7650748
[unreadable] DESCRIPTION (provided by applicant): The broad, long-term objective of this research is to develop a sensitive high throughput method for determining the substrate specificity for proteases involved in human diseases and cellular signaling events that are disease related. Accurately determining the specificity and/or biological substrates for proteases involved in various diseases should then allow for the identification of lead compounds for drug design applications and elucidation of biological signaling pathways for the treatment of these diseases. This long term goal will be accomplished by first developing a method to rapidly express, purify, and assay a large number of "library" proteins that contain both a fluorescent resonance energy transfer (FRET) donor and acceptor with a linking region that contains different combinations of amino acids. Proteins from this FRET library will then be used as substrates to determine the substrate specificity for "model" proteases that will serve as test enzymes to optimize and test the sensitivity and throughput capacity of this method. This will be accomplished in three steps; 1) Constructing and evaluating a Fluorescence Resonance Energy Transfer (FRET) library, 2) Initiating and screening the FRET library using a protease with known specificity to determine statistical parameters, and 3) Initiating and screening the FRET library with two proteases of unknown specificity to fully test the methods. The relevance of this project to public health can be understood by examining the methods by which lead compounds to treat diseases are identified today and the number of proteases involved in disease states. The method described in this proposal would allow lead compounds to be more rapidly identified and provide much more accurate methods of analysis due to the sensitivity and accuracy of this assay method. The end result would be more throughput of lead compounds for drug design, which would eventually result in an overall increase in human health. [unreadable] [unreadable] [unreadable]
