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Microsphere based protease assays and high throughput screening of bacterial toxin proteases


Please use this identifier to cite or link to this item: http://hdl.handle.net/1928/10270

Microsphere based protease assays and high throughput screening of bacterial toxin proteases

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Title: Microsphere based protease assays and high throughput screening of bacterial toxin proteases
Author: Saunders, Matthew
Advisor(s): Edwards, Bruce
Graves, Steven
Committee Member(s): Edwards, Bruce
Graves, Steven
McGuire, Paul
Sklar, Larry
Wilson, Michael
Department: University of New Mexico. Biomedical Sciences Graduate Program
Subject: High Throughput Screening
Flow Cytometry
LC Subject(s): Proteolytic enzymes--Analysis
Bacterial toxins--Analysis
Flow cytometry
Microspheres (Pharmacy)
Proteolytic enzymes--Inhibitors
Enzyme kinetics
Degree Level: Doctoral
Abstract: Proteases, proteins which cleave peptide bonds in other proteins, are a large and varied group of proteins which regulate a variety of physiological processes. Methodologies to study proteases are often protease specific and often differ greatly from the roles proteases play in vivo. In vitro protease assays often use peptide based substrates, which do not take into account highly specific interactions distal from the proteolytic site of peptide cleavage on protease substrates. In the work described here we have developed a microsphere based protease assay, capable of using full length protease substrates, and have successfully measured proteolytic activity via loss of fluorescence as measured by flow cytometry. This assay is capable of being used in high throughput screening for small molecule inhibitors for proteases of medical relevance. Screening of chemical libraries against the Bacillus anthracis Lethal factor metalloprotease and the Clostridium botulinum Neurotoxin type A Light Chain metalloprotease has led to the discovery of small molecule inhibitors for both of these pathogenic proteases. The compound ebselen has been shown to inhibit Botulinum Neurotoxin type A Light Chain with an IC50 value in the low µM range. Additional small molecule inhibitors for Botulinum neurotoxin type A Light Chain as well as for anthrax lethal factor have also been discovered by this methodology. This work shows the potential for microsphere based protease assays in discovery of small molecule protease inhibitors and can be adapted to any protease/substrate system of interest in a multiplex setup. Additional work with these proteases has also led to the discovery of novel solution based kinetics models and shows promise to validate microsphere based protease kinetics using the same system.
Graduation Date: December 2009
URI: http://hdl.handle.net/1928/10270

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