Fig. 6: Design and synthesis of de novo mTOR/MEK1 inhibitors.

a Growth of A549 cells (y-axis, relative to DMSO) treated with increasing concentrations of each of four compounds generated by POLYGON (IDK compounds) versus two negative controls (ChemBridge compounds 32574271 and 91530087, respectively). Error bars are the standard error of the mean cell growth across three independent replicates. b Capillary western blot of P70 S6K (top band), phospho-P70 S6K (middle band), and GAPDH (bottom band, loading control). Four synthesized IDK compounds are shown after treatment at 10 μM (vertical lanes) with MK-8669 treatment at 100 nM as the positive control and DMSO and trametinib treatment at 100 nM as the negative controls. Experiments were repeated three times with similar results. c Capillary western blot of ERK (top band), phospho-ERK (middle band), and GAPDH (bottom band, loading control). As for b, with trametinib as positive control and DMSO and MK-8669 as negative controls. Experiments were repeated three times with similar results. d IC50 from dose-response assays of IDK compounds in A549 cells. Error bars show the standard errors of the mean across at least three independent replicates. The first two rows (trametinib, MK-8669) are positive controls for MEK or mTOR inhibition, respectively. e, f mTOR (e) and MEK1 (f) activity measured by percent reduction of phospho-P70 S6K and phospho-ERK, respectively, upon three-hour treatment of IDK compounds at 1 µM or 10 µM. Controls were treated with MK8669 or trametinib for three hours at 100 nM (top dark gray bars). g Validation of MEK1 and mTOR activity upon three-hour treatment of IDK compounds at 1 µM, as measured by percent reduction of phospho-ERK and phospho-P70, respectively. Bars represent the mean across three independent treatments; error bars show the standard error of the mean; *p < 0.05 by one sample t test. h, i Cell-free in-vitro mTOR/FRBP12 (h) and MEK1 (i) activity quantified with ³³P-labeled phosphate transfer upon three independent treatments of IDK compounds, select Chembridge compounds, trametinib, or MK8669. Error bars are the standard error of the mean cell growth at a given dose across three independent replicates. Source data are provided as a Source Data file.