| Assay Method Information | |
| | Inhibition of KRASG12C and PI3Ka Binding |
| Description: | The AlphaScreen technology was used to determine IC50s for compound inhibition of KRAS G1 2C (present as the Cys-light (C51S, C80L and C118S), truncated version comprising amino acids 1-169) and P13Ka interaction. Compounds were diluted in 100% DMSO and each compound concentration was spotted at 200 nl/well onto low volume, white 384 well plates. The KRAS G12C contained a biotin-AviTag and the P13Ka, as Ras-binding domain (amino acids 157-300, RBD), was His-tagged. KRAS G12C was preloaded with the GTP analogue Guanosine 5′-[β, γ-imido]triphosphate (GMPPNP). The KRAS GT2C was diluted in 25 mM Hepes, pH 7.4, 150 mM NaCl, 5 mMv MgCl2, 0.0100 TritonX-100 and 10 μM GMPPNP and added at 10 ul/well to compound-spotted plates resulting in a DMR concentration of 2%. Plates were incubated for 2 hours. A mixture of RBD and the AlphaScreen streptavidin donor and nickel chelate acceptor beads diluted in 25 mMv Hepes, pH 7.4, 150 mMv NaCl, 5 mM MgCl2, 0.0100 TritonX-100 and 2% DMSO was then added at 10 ul/well and incubated for 60-90 minutes before the samples were read for emission at 570 nm after excitation of the donor beads at 680 nm. All incubations were performed at room temperature. The final top compound concentration was 50 μM with 1:3 titrations for 10-point dose response curves. Final assay conditions were 1.5 nM KRAS GT2C, 100 nM RBD, 1.25 ug/ml of AlphaScreen donor beads and 10 μg/ml AlphaLISA acceptor beads. IC50S were determined using nonlinear regression fit of [inhibitor]vs. response (4 parameters). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |