{"id":{"repo_id":"unm","oai_identifier":"oai:digitalrepository.unm.edu:nsms_etds-1009"},"canonical_url":"https://search.dev.ndltd.org/etd/unm/oai:digitalrepository.unm.edu:nsms_etds-1009","repository":{"repo_id":"unm","name":"University of New Mexico","base_url":"https://digitalrepository.unm.edu/do/oai/"},"display":{"title":"DEVELOPMENT AND KINETIC MODELING OF MULTIPLEX MICROSPHERE ASSAYS FOR HIGH-THROUGHPUT DISCOVERY OF PROTEASE ACTIVE SMALL MOLECULE COMPOUNDS","abstract":"In this dissertation, we have designed a series of recombinant fluorescent full-length protease substrates containing the cleavage sites separated by a biotinylation tag and a GFP domain, which can be associated with streptavidin-coated microspheres/nanospheres. We have developed microsphere-based platforms to detect the cleavage activities of several proteases simultaneously. In addition, we have performed high-throughput screening (HTS) to identify potential inhibitors for the Bacillus anthracis lethal factor protease (LF) and the Botulinum neurotoxin type A & F light chain proteases (BoNT/A & F LC). In order to select the efficient inhibitors, a chemical library of 350,000 compounds was screened with the HyperCyt® high-throughput flow cytometry system, which can screen thousands of compounds per day for drug discovery. In order to understand the kinetic mechanisms of these proteases as well as the inhibitory mechanisms of some inhibitor candidates, we have carried out surface-based analysis to characterize the enzymatic reaction in terms of the key kinetic parameters of BoNT/A LC (Km, kcat etc.) for surface-bound substrates by flow cytometry. In my work, we have also created a FRET full-length substrate solution assay to determine the kinetic constants of the proteases and inhibitory mechanisms of some known inhibitors. However, despite the advantages of the microsphere-based assay, the surface area to volume ratio of microspheres limits us to sub-saturating concentrations of substrate (in the low nM range). Therefore, we developed a nanosphere-based protease assay platform to increase substrate concentrations. To demonstrate this approach, we have implemented our nanosphere-based protease assay using the toxin protease models to prove the concept. The surface-based system is a promising method for the use of multiple protease assays to evaluate the kinetic mechanisms and to select inhibitors for several different proteases simultaneously. This provides the advantages of robustness, sensitivity, low substrate cost and reproducibility.","abstract_html":"In this dissertation, we have designed a series of recombinant fluorescent full-length protease substrates containing the cleavage sites separated by a biotinylation tag and a GFP domain, which can be associated with streptavidin-coated microspheres/nanospheres. We have developed microsphere-based platforms to detect the cleavage activities of several proteases simultaneously. In addition, we have performed high-throughput screening (HTS) to identify potential inhibitors for the Bacillus anthracis lethal factor protease (LF) and the Botulinum neurotoxin type A & F light chain proteases (BoNT/A & F LC). In order to select the efficient inhibitors, a chemical library of 350,000 compounds was screened with the HyperCyt® high-throughput flow cytometry system, which can screen thousands of compounds per day for drug discovery. In order to understand the kinetic mechanisms of these proteases as well as the inhibitory mechanisms of some inhibitor candidates, we have carried out surface-based analysis to characterize the enzymatic reaction in terms of the key kinetic parameters of BoNT/A LC (Km, kcat etc.) for surface-bound substrates by flow cytometry. In my work, we have also created a FRET full-length substrate solution assay to determine the kinetic constants of the proteases and inhibitory mechanisms of some known inhibitors. However, despite the advantages of the microsphere-based assay, the surface area to volume ratio of microspheres limits us to sub-saturating concentrations of substrate (in the low nM range). Therefore, we developed a nanosphere-based protease assay platform to increase substrate concentrations. To demonstrate this approach, we have implemented our nanosphere-based protease assay using the toxin protease models to prove the concept. The surface-based system is a promising method for the use of multiple protease assays to evaluate the kinetic mechanisms and to select inhibitors for several different proteases simultaneously. This provides the advantages of robustness, sensitivity, low substrate cost and reproducibility.","abstract_has_math":false,"creators":["Zhu, Jingsh"],"institution":null,"degree_name":"Nanoscience and Microsystems","degree_level":"Dissertation","degree_discipline":"Nanoscience and Microsystems","degree_department":null,"school":null,"contributors":["Graves, Steven","Edwards, Bruce","Sklar, Larry","Shreve, Andrew","Whitten, David"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2014,"date_issued":"2014-07-12T07:00:00Z","date_published":"2014-07-12T07:00:00Z","updated_at":"2026-07-24T05:26:42Z","subjects":["Protease","Microsphere","Multiplex","High-throughput screening","Drug Discovery","Inhibitors","Kinetic mechanism","Nanosphere"],"languages":["English"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["https://digitalrepository.unm.edu/nsms_etds/10"],"render_values":[{"text":"https://digitalrepository.unm.edu/nsms_etds/10","href":"https://digitalrepository.unm.edu/nsms_etds/10","code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/1928/24363","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Graves, Steven","Edwards, Bruce","Sklar, Larry","Shreve, Andrew","Whitten, David"]},{"key":"dc:creator","label":"Author","values":["Zhu, Jingsh"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2016-05-17T07:00:00Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Nanoscience and Microsystems"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation","Doctoral"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Nanoscience and Microsystems"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Protease","Microsphere","Multiplex","High-throughput screening","Drug Discovery","Inhibitors","Kinetic mechanism","Nanosphere"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["English"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/1928/24363","https://digitalrepository.unm.edu/nsms_etds/10"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["In this dissertation, we have designed a series of recombinant fluorescent full-length protease substrates containing the cleavage sites separated by a biotinylation tag and a GFP domain, which can be associated with streptavidin-coated microspheres/nanospheres. We have developed microsphere-based platforms to detect the cleavage activities of several proteases simultaneously. In addition, we have performed high-throughput screening (HTS) to identify potential inhibitors for the Bacillus anthracis lethal factor protease (LF) and the Botulinum neurotoxin type A & F light chain proteases (BoNT/A & F LC). In order to select the efficient inhibitors, a chemical library of 350,000 compounds was screened with the HyperCyt® high-throughput flow cytometry system, which can screen thousands of compounds per day for drug discovery. In order to understand the kinetic mechanisms of these proteases as well as the inhibitory mechanisms of some inhibitor candidates, we have carried out surface-based analysis to characterize the enzymatic reaction in terms of the key kinetic parameters of BoNT/A LC (Km, kcat etc.) for surface-bound substrates by flow cytometry. In my work, we have also created a FRET full-length substrate solution assay to determine the kinetic constants of the proteases and inhibitory mechanisms of some known inhibitors. However, despite the advantages of the microsphere-based assay, the surface area to volume ratio of microspheres limits us to sub-saturating concentrations of substrate (in the low nM range). Therefore, we developed a nanosphere-based protease assay platform to increase substrate concentrations. To demonstrate this approach, we have implemented our nanosphere-based protease assay using the toxin protease models to prove the concept. The surface-based system is a promising method for the use of multiple protease assays to evaluate the kinetic mechanisms and to select inhibitors for several different proteases simultaneously. This provides the advantages of robustness, sensitivity, low substrate cost and reproducibility."]},{"key":"dc:title","label":"Title","values":["DEVELOPMENT AND KINETIC MODELING OF MULTIPLEX MICROSPHERE ASSAYS FOR HIGH-THROUGHPUT DISCOVERY OF PROTEASE ACTIVE SMALL MOLECULE COMPOUNDS"]}]}],"canonical_facts":{"dc:contributor":["Graves, Steven","Edwards, Bruce","Sklar, Larry","Shreve, Andrew","Whitten, David"],"dc:creator":["Zhu, Jingsh"],"dc:date.available":["2016-05-17T07:00:00Z"],"dc:description.abstract":["In this dissertation, we have designed a series of recombinant fluorescent full-length protease substrates containing the cleavage sites separated by a biotinylation tag and a GFP domain, which can be associated with streptavidin-coated microspheres/nanospheres. We have developed microsphere-based platforms to detect the cleavage activities of several proteases simultaneously. In addition, we have performed high-throughput screening (HTS) to identify potential inhibitors for the Bacillus anthracis lethal factor protease (LF) and the Botulinum neurotoxin type A & F light chain proteases (BoNT/A & F LC). In order to select the efficient inhibitors, a chemical library of 350,000 compounds was screened with the HyperCyt® high-throughput flow cytometry system, which can screen thousands of compounds per day for drug discovery. In order to understand the kinetic mechanisms of these proteases as well as the inhibitory mechanisms of some inhibitor candidates, we have carried out surface-based analysis to characterize the enzymatic reaction in terms of the key kinetic parameters of BoNT/A LC (Km, kcat etc.) for surface-bound substrates by flow cytometry. In my work, we have also created a FRET full-length substrate solution assay to determine the kinetic constants of the proteases and inhibitory mechanisms of some known inhibitors. However, despite the advantages of the microsphere-based assay, the surface area to volume ratio of microspheres limits us to sub-saturating concentrations of substrate (in the low nM range). Therefore, we developed a nanosphere-based protease assay platform to increase substrate concentrations. To demonstrate this approach, we have implemented our nanosphere-based protease assay using the toxin protease models to prove the concept. The surface-based system is a promising method for the use of multiple protease assays to evaluate the kinetic mechanisms and to select inhibitors for several different proteases simultaneously. This provides the advantages of robustness, sensitivity, low substrate cost and reproducibility."],"dc:identifier":["http://hdl.handle.net/1928/24363","https://digitalrepository.unm.edu/nsms_etds/10"],"dc:language":["English"],"dc:subject":["Protease","Microsphere","Multiplex","High-throughput screening","Drug Discovery","Inhibitors","Kinetic mechanism","Nanosphere"],"dc:title":["DEVELOPMENT AND KINETIC MODELING OF MULTIPLEX MICROSPHERE ASSAYS FOR HIGH-THROUGHPUT DISCOVERY OF PROTEASE ACTIVE SMALL MOLECULE COMPOUNDS"],"thesis:degree_discipline":["Nanoscience and Microsystems"],"thesis:degree_level":["Dissertation","Doctoral"],"thesis:degree_name":["Nanoscience and Microsystems"]},"updated_at":"2026-07-24T05:26:42Z"}