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<p>Protein kinases are a highly targeted class of enzymes for cancer therapeutics. All current FDA approved drugs bind within the ATP site and many demonstrate toxicity due to off-target effects. A new avenue is to design drugs that compete with substrate interactions. This requires the knowledge of how kinases interact with their substrates. Current structure information on kinase-substrate interactions is particularly deficient due to the transient nature of tyrosine kinase- substrate complexes. To overcome this deficiency, we used biophysical approaches in the native solution state to characterize structural patterns for substrate binding of Src tyrosine kinase, a well- known cancer-related drug target. We developed a new peptide substrate of Src kinase with SPOT peptide array screening. Binding kinetics and activity information was obtained with various biophysical and biochemical assays. NMR experiments, such as paramagnetic relaxation enhancement and chemical shift perturbation, were exploited to define the peptide pose for Src- peptide complexes. Our data suggests an alternative binding mode for Src substrate recognition. While peptide substrates inform on interactions near the active site, the native protein substrate involves additional contacts. We obtained high-quality NMR spectra of a Src-Csk protein-protein complex that probe for the interaction of the substrate near the kinase catalytic site. Given the strong correlation between protein tyrosine kinase dysfunction and cancer, substrate recognition patterns become critical for drug discovery. Finding of tight binders and clarifying substrate recognition modes would facilitate development of more specific drugs for future cancer treatments. </p>
Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/21684905 |
Date | 07 December 2022 |
Creators | Dan Xie (14227772) |
Source Sets | Purdue University |
Detected Language | English |
Type | Text, Thesis |
Rights | CC BY 4.0 |
Relation | https://figshare.com/articles/thesis/BIOPHYSICAL_INVESTIGATIONS_OF_SRC_TYROSINE_KINASE_SUBSTRATE_RECOGNITION/21684905 |
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