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Title: Synthesis of novel activity based probes and combinatorial peptide libraries to profile proteases
Keywords: Proteases, substrate fingerprinting, bioimaging, activity based probes, positional scanning combinatorial libraries hydroxamates,
Issue Date: 14-Aug-2008
Citation: RESMI CHANDRASEKHARA PANICKER (2008-08-14). Synthesis of novel activity based probes and combinatorial peptide libraries to profile proteases. ScholarBank@NUS Repository.
Abstract: Proteases are one of the largest groups of enzymes which play a vital role in major human diseases like AIDS, Cancer, Alzheimerb s disease etc, and have been the focus of new enzyme-assay developments in recent years. High-throughput functional studies of proteases involve both the development of potent and selective probes/inhibitors to proteases as well as the studies of the substrate specificity, or b fingerprintingb of various classes of proteases. In the first part of the thesis work, a fluorescently labeled affinity probe capable of targeting a particular class of proteases, specifically caspases (enzymes which play key mediating role in apoptosis), was synthesized. In vitro proteomic experiments clearly demonstrated the potential of this probe in the selective detection of caspases. The approach was extended to the in vivo labeling of caspases expressed inside apoptotic HeLa cells using modified probes which are cell permeable; preliminary results indicated that these probes can be used to identify novel caspase-associating proteins. The second part of the work mainly focuses on investigating the substrate specificity profiles of different classes of proteases using positional scanning combinatorial libraries (PS-SCLs) of peptide substrates/inhibitors. The screening of PS-SCLs, in most instances, allows for the identification of the most active amino acids at each position of a peptide in a single assay. PS-SCLs of 7-amino-4-methyl coumarin (ACC) conjugated peptides to profile proteases, vinyl sulfone-containing peptides to study cysteine proteases, and a library of peptide hydroxamates for profiling metalloproteases were synthesized and the substrate specificity profiles of these classes of enzymes were successfully obtained.
Appears in Collections:Ph.D Theses (Open)

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