Figure 2. AC3573 compound inhibits heregulin-induced signalling.

(A,B) Effects of the selected 94 representative compounds of each cluster on heregulin-induced signalling in a cell-based screen. SK-BR-3 cells were serum-starved for 16 h and treated for 1 h with either DMSO or 30 μM of each compound and subjected to 15 min NRG stimulation (10 nM). After lysis, whole-cell extracts were immunoblotted with phospho-HER3 (Tyr1289), Total HER3, phospho-Akt (Ser473) or total Akt primary antibodies. pHER3/Total HER3 and pAkt/Total Akt ratios were quantified relative to the DMSO NRG-stimulated condition using ImageStudioLite software and normalised to α-tubulin. Heat maps illustrate and summarise quantitative Western Blot results as a percentage of inhibition of HER3 (A) and Akt (B) phosphorylation for each compound. Data are means from N = 2 independent experiments. The proof-of-concept compound AC3573 corresponds to well C1 (full line green box, 83.3% inhibition of pHER3 and 96% inhibition of pAKT). The five other hit compounds are indicated by dashed line green boxes (compounds A3, A6, B1, C5 and G3). (C) SK-BR-3 cells were serum-starved for 16 h and treated for 1 h with either DMSO, the indicated concentrations of AC3573 or 1 μM lapatinib and subjected or not to 15 min NRG stimulation (10 nM). After lysis, whole-cell extracts were immunoblotted with the indicated primary antibodies for assessment of effects of AC3573 on NRG-induced signalling. Data shown are representative of three independent experiments. (D) pHER3Tyr1289/Total HER3 ratio was quantified relative to NRG-stimulated using ImageStudioLite software. Blots were normalised to α-tubulin. Data are means ± SD from N = 3 independent experiments. (E) Quantification of pAktSer473/Total Akt ratio as percentage of NRG-stimulated. Data are means ± SD from N = 3 independent experiments. (F) Quantification of pERK1/2/Total ERK ratio as percentage of NRG-stimulated. Data are means ± SD from N = 3 independent experiments.