Biomedical Sciences ETDs

Publication Date

12-1-2009

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.

Keywords

High Throughput Screening, Flow Cytometry

Sponsors

The National Flow Cytometry Resource at Los Alamos National Laboratory. The University of New Mexico Center for Molecular Discovery The University of New Mexico department of Chemical and Nuclear Engineering Center for Biomedical Engineering.

Document Type

Dissertation

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Doctoral

Department Name

Biomedical Sciences Graduate Program

First Advisor

Edwards, Bruce

Second Advisor

Graves, Steven

First Committee Member (Chair)

Edwards, Bruce

Second Committee Member

Graves, Steven

Third Committee Member

McGuire, Paul

Fourth Committee Member

Sklar, Larry

Fifth Committee Member

Wilson, Michael

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