Development of Peptidase-Resistant Peptide Substrates for Measurement of Protein Kinase B and Bcr-Abl Kinase Activity

Proctor, Angela

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March 22, 2019

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Proctor, Angela
- Affiliation: College of Arts and Sciences, Department of Chemistry

Abstract

Synthetic peptides are widely used by the biomedical research community as kinase substrates for purified kinases, in cell lysates, and sometimes in intact single cells. Peptides are relatively straightforward to construct, they have a long shelf-life, and they are easily derivatized with labels to facilitate detection. However, despite the benefits of peptides, they suffer from susceptibility to degradation by intracellular peptidases. Peptidolysis is often extremely rapid, yielding peptide fragmentation within minutes of introduction into a cell lysate or single cell. While protease inhibitors can be used to slow degradation, they do not entirely eliminate protease activity and they are difficult to use with intact cells. In order to render peptides more suitable as substrates in in vivo settings, proteolytic degradation needs to be dramatically slowed or halted. The work described in this dissertation develops an iterative strategy to screen rationally designed peptides for their suitability as kinase substrates in cell lysates and single cells. Peptide bonds susceptible to peptidolysis were identified and stabilized by replacement of native residues with non-native residues, which are poor substrates for peptidases. Modified peptides were screened for substrate suitability as well as for resistance to degradation by peptidases. Substrates were designed for two kinases, protein kinase B and Bcr-Abl kinase, because of their roles in cancer. Protein kinase B is involved in regulation of multiple cellular functions, including stress response and programmed cell death, and is upregulated in many cancers, including pancreatic, breast, and prostate tumors. Bcr-Abl is the primary driver of chronic myelogenous leukemia. This dissertation outlines the development of peptidase-resistant substrate reporters for these two kinases. Initial characterization and design was performed in cytosolic lysates. Once suitably designed peptide substrates were synthesized, they were incubated in single cells to characterize substrate metabolism and phosphorylation in the intracellular environment. Finally, protein kinase B activity was measured in single primary cells from a human pancreatic cancer xenograft. The design strategy presented in this dissertation should be applicable for future design of peptidase-resistant peptide substrates for alternative kinases.

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