| Assay Method Information | |
| | AlphaScreen Binding Assay |
| Description: | Table 29: The assay measures the inhibitory effect of compounds on KRAS mutant protein-protein interactions using the Alpha Screen technology by Perkin Elmer. This assay can be used to examine the potency with which compounds according to the invention binding to a mutated KRAS inhibit the protein-protein interaction between SOS1 and the mutated KRAS e.g., KRAS G12C, KRAS G12D, or KRAS G12V. This inhibits the GEF functionality of SOS1 and locks the corresponding mutated KRAS protein in its inactive, GDP-bound state. Low IC50 vlues in this assay setting are indicative of strong inhibition of protein-protein interaction between SOS1 and KRAS. The following mutant enzyme forms of KRAS and interacting proteins have been used in these assays at the given concentrations: KRAAS (G12D) 1-169, N-terminal 6His-tag, C-terminal avi-tag (Xtal BioStructures, Inc); final Streptavidin Donor Beads (PerkinElmer Cat No 6760002) in assay buffer at a final assay concentration of 10 ug/ml each. Plates are kept at room temperature in a darkened incubator. After an additional 60 minutes incubation time the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen specs from PerkinElmer. |
| Affinity data for this assay | |
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