| Assay Method Information | |
| | SOS1-KRas(G12C) FRET Assay |
| Description: | Inhibition of the SOS1:KRAS interaction was measured using purified GST-tagged KRAS (res. 1-169, G12C, purified based on Hillig, et al., Proc Natl Acad Sci USA (2019); 116(7):2551-2560) and recombinant His10-SOS1 (res. 564-1049; purified based on Hillig, et al.). The final assay was performed at 20 uL with 0.5 nM SOS1 protein and 2.5 nM KRAS protein in a buffer of PBS, 0.1% BSA, 5 mM MgCl2, 0.0025% Igepal, 100 mM KF, 5 mM DTT in a white 384 square well OptiPlate (PerkinElmer, Cat. 6007290). A 2× KRAS working solution was prepared in an assay buffer containing 5 nM GST-KRAS G12C and 2 nM anti-GST-Eu(K) (Cisbio, Cat. 61GSTKLA) and pre-incubated for 15 minutes at 25° C. Compounds were serially diluted in 100% DMSO from 2 mM (positive control, compound I-13, PCT Publ. No. WO2018/115380) or 20 mM and then diluted 1:20 in assay buffer before incubation with a solution of SOS1 protein mixed 1:5 with anti-6His-XL665 FRET donor (Cisbio, Cat. 61HISXL) for 15 minutes at 25° C. before addition of 2× KRAS working solution. The final DMSO concentration is 0.5%. Plates were incubated at RT for 2 hrs before the FRET signal was measured using Envision at emission 665 nm and 615 nm. |
| Affinity data for this assay | |
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