8AW1

c-MET Y1235D mutant in complex with Tepotinib

Summary for 8AW1

• Entry DOI: 10.2210/pdb8aw1/pdb
• Descriptor: Hepatocyte growth factor receptor, 3-[1-(3-{5-[(1-methylpiperidin-4-yl)methoxy]pyrimidin-2-yl}benzyl)-6-oxo-1,6-dihydropyridazin-3-yl]benzonitrile (3 entities in total)
• Functional Keywords: protein kinase, transferase
• Biological source: Homo sapiens (human)
• Total number of polymer chains: 2
• Total formula weight: 68777.72

Authors

Graedler, U.,Lammens, A. (deposition date: 2022-08-29, release date: 2023-09-06, Last modification date: 2024-03-20)

Primary citation

Gradler, U.,Schwarz, D.,Wegener, A.,Eichhorn, T.,Bandeiras, T.M.,Freitas, M.C.,Lammens, A.,Ganichkin, O.,Augustin, M.,Minguzzi, S.,Becker, F.,Bomke, J.
Biophysical and structural characterization of the impacts of MET phosphorylation on tepotinib binding.
J.Biol.Chem., 299:105328-105328, 2023

PubMed Abstract: 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.

PubMed: 37806493
DOI: 10.1016/j.jbc.2023.105328

Experimental method

X-RAY DIFFRACTION (2.14 Å)