| Assay Method Information | |
| | Evaluation of Inhibitory Activity of Compounds on KRAS G12C Nucleotide (GDP-GTP) Exchange Reaction |
| Description: | The inhibitory activity of compounds on exchange reaction of GDP into GppNHp in Bodipy (trademark) FL-bound KRAS G12C was examined by fluorescence measurement using human recombinant KRAS G12C and SOS1 proteins.For the preparation of KRAS G12C to which Bodipy FL GDP was bound, first, 50 μMy KRAS G12C (amino acid region: 1-169) and 1 mM Bodipy FL GDP (Invitrogen, G22360) were incubated for 1 hour in a buffer (20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM DTT) in ice in the presence of 2.5 mM EDTA. Thereafter, MgCl2 with a final concentration of 10 mM was added, and the mixture was incubated at room temperature for 30 minutes. The protein was allowed to pass through a NAP-5 column to remove free nucleotides and was used for compound evaluation.For the measurement of the inhibitory activity of compounds on nucleotide exchange reaction, first, the compound of the present invention was diluted stepwise with dimethyl sulfoxide (DMSO). Subsequently, a solution of the gradually diluted compound of the present invention in DMSO (the final concentration of DMSO: 5%) and KRAS G12C (25 nM) bound to Bodipy FL GDP were added to a reaction buffer (20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM MgCl2, 2 mM DTT, 0.1% Tween20), followed by preincubation at 25° C. for 4 hours. Thereafter, Son of Sevenless Homolog 1 (SOS1, amino acid region: 564-1049) and GppNHp (GMPPNP, Jena Bioscience GmbH, NU-401-50) were added such that their final concentration was 100 nM and 1 μM respectively, followed by reaction for 30 minutes. The change in fluorescence intensity of Bodipy FL (excitation wavelength: 485 nm, fluorescence wavelength: 520 nm) immediately after the start of reaction and after 30 minutes from the start of reaction was standardized. The signal value for only DMSO was determined to be 0% inhibition, and the signal value for no addition of GppNHp was determined to be 100% inhibition. The compound concentration at which 50% inhibition is achieved was determined to be IC50 (nM), and was calculated. The following table shows the inhibitory activity IC50 (nM) of the test compounds. |
| Affinity data for this assay | |
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