TY - JOUR
T1 - Fluorescence based live cell imaging identifies exon 14 skipped hepatocyte growth factor receptor (MET) degraders
AU - Mallareddy, Jayapal Reddy
AU - Yang, Lin
AU - Lin, Wan Hsin
AU - Feathers, Ryan
AU - Ayers-Ringler, Jennifer
AU - Tolosa, Ezequiel
AU - Kizhake, Amritha G.
AU - Kizhake, Smitha
AU - Kubica, Sydney P.
AU - Boghean, Lidia
AU - Alvarez, Sophie
AU - Naldrett, Michael J.
AU - Singh, Sarbjit
AU - Rana, Sandeep
AU - Zahid, Muhammad
AU - Schaefer-Klein, Janet
AU - Roden, Anja
AU - Kosari, Farhad
AU - Anastasiadis, Panos Z.
AU - Borad, Mitesh
AU - Natarajan, Amarnath
AU - Mansfield, Aaron S.
N1 - Publisher Copyright:
© 2025 The Royal Society of Chemistry.
PY - 2025/4/3
Y1 - 2025/4/3
N2 - Despite ongoing efforts to employ structure-based methods to discover targeted protein degraders (TPD), the prevailing strategy continues to be the synthesis of a focused set of heterobifunctional compounds and screening them for target protein degradation. Here we used a fluorescence based live cell imaging screen to identify degraders that target exon 14 skipped hepatocyte growth factor receptor (MET). MET is a known oncogenic driver. MET exon 14 skipping mutations (METex14Δ) are found in lung cancers and result in the loss of a degron that is required for E3-ligase recognition and subsequent ubiquitination, prolonging the half-life and oncogenicity of MET. Since proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that promote target degradation by the proteosome, we sought to restore degradation of MET lost with METex14Δ using a MET-targeting PROTAC. We generated a library of sixty PROTACs of which 37 used the MET inhibitor capmatinib as the protein of interest targeting ligand. We screened this PROTAC library for targeted degradation of METex14Δ-GFP using live cell imaging. We benchmarked the MET-targeting PROTACs to that of a previously reported MET-targeting PROTAC, SJF8240. Curve fitting live cell imaging data affords determination of time required to degrade 50% of the target protein (DT50), which was used in determining structure activity relationships. A promising candidate, 48-284, identified from the screen, exhibited classic PROTAC characteristics, was >15-fold more potent than SJF8240, had fewer off targets compared to SJF8240, and degraded MET in multiple cell lines.
AB - Despite ongoing efforts to employ structure-based methods to discover targeted protein degraders (TPD), the prevailing strategy continues to be the synthesis of a focused set of heterobifunctional compounds and screening them for target protein degradation. Here we used a fluorescence based live cell imaging screen to identify degraders that target exon 14 skipped hepatocyte growth factor receptor (MET). MET is a known oncogenic driver. MET exon 14 skipping mutations (METex14Δ) are found in lung cancers and result in the loss of a degron that is required for E3-ligase recognition and subsequent ubiquitination, prolonging the half-life and oncogenicity of MET. Since proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that promote target degradation by the proteosome, we sought to restore degradation of MET lost with METex14Δ using a MET-targeting PROTAC. We generated a library of sixty PROTACs of which 37 used the MET inhibitor capmatinib as the protein of interest targeting ligand. We screened this PROTAC library for targeted degradation of METex14Δ-GFP using live cell imaging. We benchmarked the MET-targeting PROTACs to that of a previously reported MET-targeting PROTAC, SJF8240. Curve fitting live cell imaging data affords determination of time required to degrade 50% of the target protein (DT50), which was used in determining structure activity relationships. A promising candidate, 48-284, identified from the screen, exhibited classic PROTAC characteristics, was >15-fold more potent than SJF8240, had fewer off targets compared to SJF8240, and degraded MET in multiple cell lines.
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U2 - 10.1039/d5ra01441g
DO - 10.1039/d5ra01441g
M3 - Article
AN - SCOPUS:105002234366
SN - 2046-2069
VL - 15
SP - 10419
EP - 10425
JO - RSC Advances
JF - RSC Advances
IS - 13
ER -