Vivazine substrate

For the KRAS(G12D):CRAF Ras binding domain (RBD) NanoBiT interaction assay, CMV-based expression constructs were made encoding fusions of LgBiT to KRAS 4B (UNIPROT P01116–2) with the G12D mutation and SmBiT to residues 51–133 of CRAF [UNIPROT P04049–1, CRAF(RBD)). HEK293 cells (~4E6) were transiently transfected in T75 flasks with plasmids encoding a LgBiT-KRAS(G12D) and SmBiT-CRAF(RBD). Plasmids were transfected at 500 ng/construct/flask together with 9 μg of Transfection Carrier DNA (Promega) at a 3:1 lipid:DNA ratio using FuGENE HD (10 ml total volume). Following expression for 24 hours, cells were plated at 20,000 cells/well in Opti-MEM I (Thermo) containing 4% FBS and allowed to attach overnight. Serial dilutions of MRTX-EX185 were made in Opti-MEM I containing 4% FBS and 1X Vivazine substrate (Promega N2581) to generate 1X solutions containing varying concentrations of MRTX-EX185. Existing medium was removed by plate inversion and blotting, and 1X solutions were added to respective wells. Luminescence was measured every 5 minutes in a GloMax Discover luminometer at 37°C for 16 h using a 1 s integration time.