Biophysical and structural characterization of the impacts of MET phosphorylation on tepotinib binding.
In: The Journal of biological chemistry, Jg. 299 (2023-11-01), Heft 11, S. 105328
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Zugriff:
The receptor tyrosine kinase MET is activated by hepatocyte growth factor binding, followed by phosphorylation of the intracellular kinase domain (KD) mainly within the activation loop (A-loop) on Y1234 and Y1235. Dysregulation of MET can lead to both tumor growth and metastatic progression of cancer cells. Tepotinib is a highly selective, potent type Ib MET inhibitor and approved for treatment of non-small cell lung cancer harboring METex14 skipping alterations. Tepotinib binds to the ATP site of unphosphorylated MET with critical π-stacking contacts to Y1230 of the A-loop, resulting in a high residence time. In our study, we combined protein crystallography, biophysical methods (surface plasmon resonance, differential scanning fluorimetry), and mass spectrometry to clarify the impacts of A-loop conformation on tepotinib binding using different recombinant MET KD protein variants. We solved the first crystal structures of MET mutants Y1235D, Y1234E/1235E, and F1200I in complex with tepotinib. Our biophysical and structural data indicated a linkage between reduced residence times for tepotinib and modulation of A-loop conformation either by mutation (Y1235D), by affecting the overall Y1234/Y1235 phosphorylation status (L1195V and F1200I) or by disturbing critical π-stacking interactions with tepotinib (Y1230C). We corroborated these data with target engagement studies by fluorescence cross-correlation spectroscopy using KD constructs in cell lysates or full-length receptors from solubilized cellular membranes as WT or activated mutants (Y1235D and Y1234E/1235E). Collectively, our results provide further insight into the MET A-loop structural determinants that affect the binding of the selective inhibitor tepotinib.
Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of the article.
(Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
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Biophysical and structural characterization of the impacts of MET phosphorylation on tepotinib binding.
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Autor/in / Beteiligte Person: | Grädler, U ; Schwarz, D ; Wegener, A ; Eichhorn, T ; Bandeiras, TM ; Freitas, MC ; Lammens, A ; Ganichkin, O ; Augustin, M ; Minguzzi, S ; Becker, F ; Bomke, J |
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Zeitschrift: | The Journal of biological chemistry, Jg. 299 (2023-11-01), Heft 11, S. 105328 |
Veröffentlichung: | 2021- : [New York, NY] : Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology ; <i>Original Publication</i>: Baltimore, MD : American Society for Biochemistry and Molecular Biology, 2023 |
Medientyp: | academicJournal |
ISSN: | 1083-351X (electronic) |
DOI: | 10.1016/j.jbc.2023.105328 |
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