| Assay Method Information | |
| | SOS1 Binding and Inhibition Assay |
| Description: | Table 23: This Example illustrates that exemplary compounds of the present invention bind to SOS1 and inhibit the SOS1-mediated nucleotide exchange of mantGDP (preloaded into human KRAS) with GTP within a recombinant human KRAS.The ability of an exemplary compound of Formula (I) to bind to SOS1 and inhibit the nucleotide exchange of mantGDP with GTP within recombinant human KRAS was measured using a fluorescence assay. Recombinant human SOS1 polypeptide (corresponding to amino acids 564-1049, expressed in E. coli with a C-terminal StrepII tag. MW=60.59 kDa) in buffer (40 mM HEPES 7.4, 10 mM MgCl2, 1 mM DTT, 0.002% Triton X100, 0.1% DMSO) was incubated with an exemplary compound of Formula (I) (in a DMSO stock solution) at room temperature for 15 minutes. A mixture of preloaded mantGDP recombinant human KRAS polypeptide (corresponding to amino acids 2-169, expressed in E. coli with an N-terminal TEV cleavable his-tag. MW 21.4 kDa) and GTP was incubated for 5 minutes in buffer (40 mM HEPES 7.4, 10 mM MgCl2, 1 mM DTT 0.002%, Triton X100, 0.1% DMSO) at room temperature, then added to the SOS1/compound mixture. Reaction progress was monitored at room temperature for 60 minutes using a Clariostar plate reader (excitation 370±15 nm, emission 450±20 nm) according to the manufacturer's instructions. The slope of the linear portion of the progress curve was calculated using a Clariostar software. Typical analysis interval was 8-30 minutes. Background signals were calculated from well without protein added. The background subtracted signals were converted to % activity relative to DMSO controls. Data were analyzed using GraphPad Prism 4 software with the settings: sigmoidal dose-response (variable slope) ; 4 parameters with Hill Slope (Constraints: Bottom=Constant equal to 0; Top=Must be less than 120). |
| Affinity data for this assay | |
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