Fig. 5: Nb22 increases the affinity of the aminopiperidine indole ligand to SOS1•RAS as shown by NMR spectroscopy, ITC and nucleotide exchange assays.

a NMR spectra measuring the binding of the aminopiperidine indole, an allosteric SOS1•RAS compound discovered by Burns et al.⁸, to the SOS1•RAS complex in the presence or absence of Nb22. The different plots show several regions of the 1D 1H reference spectrum of aminopiperidine indole (black), water ligand observed via gradient spectroscopy (water-LOGSY) spectra of aminopiperidine indole in the presence of SOS1-RAS alone (blue) and with 4 molar equivalents of Nb22 (red), and saturation transfer difference (STD) spectra of the compound in the presence of SOS1-RAS alone (green) and with 4 molar equivalents of Nb22 (orange). Estimated from the WaterLOGSY spectra, Nb22 enhances the binding signal of aminopiperidine indole to SOS1-RAS by a factor of 2. All experiments were performed in 20 mM potassium phosphate 75 mM NaCl pH 7.0 at 298 K. b ITC experiments to measure the affinity of SOS1 for the aminopiperidine indole in presence or absence of Nb22. In each case, the sample cell was filled with 30 µM of SOS1 in the presence (50 µM) or absence of Nb22 while the syringe was loaded with 300 µM of ligand. c SOS1•RAS nucleotide exchange assay performed with increasing concentrations of aminopiperidine indole in the presence (red) or absence (black) of an excess of Nb22. Data are presented as mean values +/− SEM. In the presence of Nb22, the maximum activation of SOS1 by aminopiperidine is unchanged (3.5 to 3.7-fold) but the apparent EC50 is lowered approximately two fold. The best fit value of the EC50 is 24.2 μM with the 95% confidence interval ranging from 18.5 μM to 31.6 μM in presence of aminopiperidine and 14.2 µM with the 95% confidence interval ranging from 11.7 μM to 17.2 μM in presence of aminopiperidine and Nb22. The experiments were repeated independently three times. Source data are provided as a Source Data file.