Fig.Figure 3Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(A) Spironolactone, molecular structure shown left, inhibits ERBB4/PIK3R1 split TEV assay activity (black line), with an IC 50 value of 2.76 µM. Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(B-D) The spironolactone metabolites (B) canrenone. Note that only spironolactone bears a thio-ketone group attached to the sterol core structure. All ERBB4/PIK3R1 assays were run in a single-culture assay mode using 10ng/ml EGFld as functional Nrg1 stimulus, and ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids were transfected into PC12 cells (indicated by icon). Fluc, firefly luciferase activity reporting ERBB4-PIK3R1 assay activity (black lines); Rluc, Renilla luciferase activity (grey lines) assessing viability; data are shown as mean, error bars represent SEM; n=6. Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment. (B-D) The spironolactone metabolites(C) 7α-thiomethyl-spironolactone. Note that only spironolactone bears a thio-ketone group attached to the sterol core structure. All ERBB4/PIK3R1 assays were run in a single-culture assay mode using 10ng/ml EGFld as functional Nrg1 stimulus, and ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids were transfected into PC12 cells (indicated by icon). Fluc, firefly luciferase activity reporting ERBB4-PIK3R1 assay activity (black lines); Rluc, Renilla luciferase activity (grey lines) assessing viability; data are shown as mean, error bars represent SEM; n=6. Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(B-D) The spironolactone metabolites (D) eplerenone do not attenuate the ERBB4/PIK3R1 assay activity (black lines). Note that only spironolactone bears a thio-ketone group attached to the sterol core structure. All ERBB4/PIK3R1 assays were run in a single-culture assay mode using 10ng/ml EGFld as functional Nrg1 stimulus, and ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids were transfected into PC12 cells (indicated by icon). Fluc, firefly luciferase activity reporting ERBB4-PIK3R1 assay activity (black lines); Rluc, Renilla luciferase activity (grey lines) assessing viability; data are shown as mean, error bars represent SEM; n=6. Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(E) Schematic representation of the most critical ERBB dimers tested in spironolactone dose-response assays using the split TEV protein-protein interaction detection technique. Note that not all possible combinations are depicted. Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(F) Heat map showing the IC 50 values obtained from individual ERBB dimerization split TEV assays. All single dose-response assays can be found in Fig EV4; the combination ERBB4-ERBB4 is shown in Fig 2E. Fluc, firefly luciferase activity (black lines); Rluc, Renilla luciferase activity (grey lines, indicating toxicity levels); n=6; data are shown as mean, error bars represent SEM. The insets depict IC 50 values in µM.