| Assay Method Information | |
| | Biological Data of Disclosed Compounds |
| Description: | The capacity of compounds to inhibit SOS1 binding to KRAS-WT (wild-type) was quantified using a FRET-based protein-protein interaction assay. The assay is based on the transfer of energy between two fluorophores, a donor and an acceptor, when in close proximity. In this instance, the donor is a Europium-conjugated α-GST antibody that binds to GST-tagged KRAS-WT, and the acceptor is an XL665-conJugated α-His6 antibody that binds to His6-tagged SOS1. Binding of SOS1 to KRAS-WT results in an increased fluorescent signal at emission wavelength of 665 nm which can be detected on the EnVision plate reader. Compounds that inhibit binding will reduce the 665 nm signal emitted. Recombinant KRAS-WT protein (40 nM; Human KRAS, aal-188 recombinant protein with N-terminal GST-tag) and SOS1 protein (40 nM; Human SOS1 exchange domain, aa564-1049 with N-terminal 6His-tag) were mixed together in assay buffer (5 mM HEPES pH7.3, 150 mM NaCl, 10 mM EDTA, 5 mM MgCl2, 0.05% BSA, 0.0025% NP-40, 1 mM DTT and 100 mM KF) and incubated at room temperature with a dose response of compound in a 384-well low volume white plate and a final volume of 5 ul. After a 60 minute incubation, 5 ul of 4 nM anti-GST-Eu(K) (Cisbio, France) combined with 20 nM anti-6His-XL665 (Cisbio, France), diluted in assay buffer, was added to the plate. Following a further 4 h incubation at room temperature, time-resolved fluorescence was measured on the EnVision plate reader. DMSO (0.05%) and 10 μM reference compound were used to generate the Max and Min assay signals, respectively. Data was analyzed using a four-parameter logistic model to calculate IC50 values, with at least two independent replicates performed for each compound. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |