title: Cell lines expressing recombinant transmembrane domain-activated receptor kinases as tools for drug discovery.
abstract: Many receptor tyrosine kinases (RTKs) represent bona fide drug targets in oncology. Effective compounds are available, but treatment invariably leads to resistance, often due to RTK mutations. The discovery of second-generation inhibitors requires cellular models of resistant RTKs. An approach using artificial transmembrane domains (TMDs) to activate RTKs was explored for the rapid generation of simple, ligand-independent cellular RTK assays, including resistance mutants. The RTKs epidermal growth factor receptor (EGFR), MET, and KIT were chosen in a proof-of-concept study. Their intracellular domains were inserted into a series of expression vectors encoding artificial TMDs, and they were tested for autophosphorylation activity in transient transfection assays. Active constructs could be identified for MET and EGFR, but not for KIT. Rat1 cell pools were generated expressing the MET or EGFR constructs, and their sensitivity to reference tool compounds was compared to that of MKN-45 or A431 cells. A good correlation between natural and recombinant cells led us to build a panel of clinically relevant MET mutant cell pools, based on the wild-type construct, which were then profiled via MET autophosphorylation and soft agar assays. In summary, a platform was established that allows for the rapid generation of cellular models for RTKs and their resistance mutants.
authors.affiliation: ProQinase GmbH, Tumor Biology Center, Freiburg, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Weber
authors.affiliation: ProQinase GmbH, Tumor Biology Center, Freiburg, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Müller
authors.affiliation: ProQinase GmbH, Tumor Biology Center, Freiburg, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Müller
authors.affiliation: ProQinase GmbH, Tumor Biology Center, Freiburg, Germany Department of Cardiology, University of Freiburg, Freiburg, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Ortiz
authors.affiliation: ProQinase GmbH, Tumor Biology Center, Freiburg, Germany.
authors.forename: Marianne
authors.initials: M
authors.lastname: Birkle
authors.affiliation: ProQinase GmbH, Tumor Biology Center, Freiburg, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Umber
authors.affiliation: ProQinase GmbH, Tumor Biology Center, Freiburg, Germany.
authors.forename: Constance
authors.initials: C
authors.lastname: Ketterer
authors.affiliation: ProQinase GmbH, Tumor Biology Center, Freiburg, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Siedentopf
authors.affiliation: ProQinase GmbH, Tumor Biology Center, Freiburg, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Feger
authors.affiliation: ProQinase GmbH, Tumor Biology Center, Freiburg, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Totzke
authors.affiliation: ProQinase GmbH, Tumor Biology Center, Freiburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Kubbutat
authors.affiliation: ProQinase GmbH, Tumor Biology Center, Freiburg, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Schaechtele
authors.affiliation: Paul Scherrer Institute, Biomolecular Research, Villigen, Switzerland.
authors.forename: Kurt
authors.initials: K
authors.lastname: Ballmer-Hofer
authors.affiliation: ProQinase GmbH, Tumor Biology Center, Freiburg, Germany.
authors.forename: Jan Erik
authors.initials: JE
authors.lastname: Ehlert
authors.affiliation: ProQinase GmbH, Tumor Biology Center, Freiburg, Germany (currently) Janssen Pharmaceutical, Beerse, Belgium r.graeser17@gmail.com.
authors.forename: Ralph
authors.initials: R
authors.lastname: Graeser
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D055808:Drug Discovery / Q000379:methods*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D058990:Molecular Targeted Therapy; D009154:Mutation; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics / Q000378:metabolism; D019859:Proto-Oncogene Proteins c-met / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D066246:ErbB Receptors; D019009:Proto-Oncogene Proteins c-kit; D019859:Proto-Oncogene Proteins c-met; D020794:Receptor Protein-Tyrosine Kinases
keywords: cell-based assays; enzyme assays; enzyme kinetics; oncology; receptor tyrosine kinase; transmembrane domain