Kinases play a crucial role in regulating cellular signaling cascades, making them
therapeutic targets for several human diseases. In human cancers, mis-regulation and
mutations of kinases such as EGFR (epidermal growth factor receptor) have been found
to drive malignant transformation. Due to the conserved structural elements of protein
kinases, the majority of kinase inhibitors available have a tendency to inhibit multiple
targets. The biological impact of this promiscuity is insufficiently defined and the
prevalence of cellular compensatory mechanisms additionally varies the clinical
responses to drug treatment. In order to understand the relationship between selectivity
and efficacy, prior to clinical trials, it is essential to characterize how inhibitors interact
with the kinome within a cellular context.
Monitoring inhibitor-target interactions generally involves in vitro assaying with purified
proteins or protein domains, which compromises the native integrity of the kinases. Cellbased
assays either gain outcomes from bulk populations that average out cell variance or
phenotypic assays that lack molecular resolution. To obtain information on drug
interactions on a single cell level, we have developed a method to measure the direct
binding of kinase inhibitors to their targets in situ and in vivo. Kinase inhibitors are
chemically tagged with fluorophores that serve as acceptors to genetically tagged donor
fluorophores on the enzyme and the interaction is measured using FRET-FLIM. With
epidermal growth factor receptor (EGFR) and irreversible EGFR inhibitors as the model
system, this approach has been applied to image inhibitor-kinase interactions in live and
fixed cells. Using this method, a small panel of tyrosine kinase targets, and labeled
inhibitors, we were able to investigate the cross-specificity within the panel. Additionally
it was found that the specificity of inhibitors for specific kinase conformations enables
the distinction between EGFR in the active and inactive conformation by the inhibitor-probes. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/8575 |
| Date | January 2012 |
| Creators | Chuntharpursat, Eulashini. |
| Contributors | Elliott, Edith., Bastiaens, Philippe I. H. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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