(A) Overview of the screening strategy and hit rate for each step of the screening cascade. (B) Scatter graph of DSF primary screening shows pools of compounds that either stabilise or destabilise HER3 kinase domain in vitro. ApoHER3 (HER3 kinase domain with no ligand) was used as a control for all DSF analyses (mean of ApoHER3 T_m across the plates set at ΔT_m = 0°C is indicated by the dash green line) and ΔT_m values were calculated for each pool of compounds tested at 30 μM each (N = 1). Cut-off values of ΔT_m>3°C and ΔT_m < −5°C were used to designate hits (shown as dashed pink lines). The pool of compounds containing proof-of-concept compound AC3573 is circled. Gaps indicate the rare plate failures (three plates). Compounds from these plates were retested during the deconvolution screen. (C) Correlation of shift in HER3 T_m value induced by deconvoluted pool compounds tested at 30 μM between the two repeats of deconvolution screening. AC3573 and its three pool compounds are highlighted. (D) Dose-dependent analysis of thermal shifts induced by AC3573 compound tested at 1, 3, 10 and 30 μM in the HER3 potency screen. The change in HER3 T_m value (ΔT_m), compared with ApoHER3 control, is reported as mean ± SD from N = 4 independent experiments. Representative thermal denaturation profiles of recombinant HER3 kinase domain, in the presence of 0.3% DMSO (Apo control), ATP/MgCl2 (ATP control) or AC3573 tested at 1, 3, 10 and 30 μM. (E) Dose-dependent analysis of thermal shifts induced by AC3573 compound tested at 1, 3, 10 and 30 μM in the HER2 counter screen. The change in HER2 T_m value (ΔT_m), compared with ApoHER2 control (HER2 recombinant protein without ligand), is reported as mean ± SD from N = 3 independent experiments. A representative plot of the counter screening thermal denaturation profiles of recombinant HER2 kinase domain, in the presence of 0.3% DMSO (Apo control), 1 μM lapatinib (Lapatinib control) or AC3573 compound tested at 1, 3, 10 and 30 μM is shown. (F) Chemical structure of the proof-of-concept compound AC3573 identified in the DSF screen.