Fig. 2

Cell viability assay and STD NMR experiments. (A) Anti-proliferative activity of the indicated compounds against the RUNX1/ETO-dependent leukemia cell line SKNO-1. Depicted are the mean ± STD IC₅₀ values in µM calculated by using a sigmoid dose-response curve and nonlinear regression of the raw data normalized to the corresponding DMSO controls. Compounds with IC₅₀ values over 1000 µM were classified as inactive (grey color). Data was collected from three independent experiments (n = 3). (B) Compounds from the cell-based assay were counter-screened by STD NMR experiments. For STD NMR experiments, 20–30 µM of ¹³C, ¹⁵N-labelled NHR2 protein were prepared in buffer containing 20 mM sodium phosphate, 50 mM sodium chloride, 0.5 mM tris(2-carboxyethyl)phosphine pH 6.5 (80% (v/v) H₂O, 10% (v/v) DMSO-d₆, 10% (v/v) D₂O). The compound concentration was 1–3 mM in the complex. All compounds with IC₅₀ values ~ 50–650 µM (denoted on the right side of the spectrum) showed STD signals in the NMR spectra, indicating their binding to NHR2. M3 and M6 showed no STD signals. DSS: Sodium 2,2-dimethyl-2-silapentane-5-sulfonate, used for chemical shift referencing (calibrated to zero ppm).