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| Status |
Public on Dec 17, 2021 |
| Title |
Drug resistance profiling of thousands of Src kinase mutants uncovers a regulatory network that couples autoinhibition to catalytic domain dynamics |
| Organisms |
Escherichia coli; Saccharomyces cerevisiae |
| Experiment type |
Other
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| Summary |
Protein kinase inhibitors are effective cancer therapies, but acquired resistance often limits clinical efficacy. Despite the cataloguing of numerous resistance mutations with model studies and in the clinic, we still lack a comprehensive understanding of kinase inhibitor resistance. Here, we measured the resistance of thousands of Src tyrosine kinase mutants to a panel of ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Src’s catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Src’s phosphotransferase activity were prone to the development of resistance. Unexpectedly, a resistance-prone cluster of residues that are on the top face of the N-terminal lobe of the catalytic domain contributes to Src autoinhibition by reducing the dynamics of the catalytic domain, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how comprehensive profiling of drug resistance can be used to understand potential resistance pathways and uncover new mechanisms of kinase regulation.
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| Overall design |
A barcoded plasmid library of Src variants was expressed in S. cerevisiae, treated with different Src kinase inhibitors, and timepoints were sampled throughout growth. Plasmids were extracted and barcodes amplified and counted with Illumina sequencing. Barcodes were related back to their cognate variant using barcode-variant map.
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| Contributor(s) |
Maly DJ, Fowler DM |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
| |
| Submission date |
Dec 08, 2021 |
| Last update date |
Jul 24, 2023 |
| Contact name |
Douglas Fowler |
| E-mail(s) |
dfowler@uw.edu
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| Organization name |
University of Washington
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| Department |
Genome Sciences
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| Lab |
Fowler
|
| Street address |
3720 15th Ave NE, Foege Building, S041
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
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| Platforms (2) |
| GPL16085 |
Illumina MiSeq (Escherichia coli) |
| GPL19756 |
Illumina NextSeq 500 (Saccharomyces cerevisiae) |
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| Samples (5)
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| Relations |
| BioProject |
PRJNA787284 |
| SRA |
SRP349865 |
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