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.
Keywords: cell-based assays; enzyme assays; enzyme kinetics; oncology; receptor tyrosine kinase; transmembrane domain.
© 2014 Society for Laboratory Automation and Screening.