Fig. 2. Lead optimization. (A and B) Biochemical studies with compound 35 derivatives in MG87 RET/GFRa1 cells assayed 20 μM concentrations of compounds 7, 8, and 9 with a 20-minute exposure time. Representative Western blot shown for RET-pY1062 and its effectors pAkt and pErk. GDNF (G) is GDNF positive control, and vehicle (V/Veh.) is the DMSO negative control. Compound 8 displayed a signaling profile that mirrored that of the parent compounds, whereas compound 7 generated a pErk-biased signaling trend. Compound 9 was completely inactive. (C and D) Biochemical studies with compound 35 derivatives tested in MG87 RET cells. Representative Western blot shown for RET-pY1062 and its effectors pAkt and pErk. GDNF (G) is GDNF positive control, and vehicle (V/Veh.) is the DMSO negative control. Signaling profiles of compounds 7, 8, and 9 were similar to those observed in MG87 GFRa1/RET cells, indicating that these derivatives maintained GFRa1 independence. (E and F) Selectivity screens in MG87 TrkA cells, using 20-μM concentrations of compound 35 derivatives stimulated for 20 minutes. Representative blot for pTrkA and its effectors pAkt and pErk. NGF (N) is positive control, and vehicle (V/Veh.) is the negative control. Compounds 7, 8, and 9 were all inactive in these assays. (G) Compound 8 signaling in E18 mouse cortical neurons. Neurons were treated with compound 9 at 20 μM, GDNF at 100 and 200 ng/ml, and compound 8 at 10 and 20 μM for 20 minutes. Representative blot showing pAkt increases observed with both treatments of compound 8. Actin was used as loading control. (H) RET inhibition blocks compound 8 signaling. MG87 RET/GFRa1 cells were first treated with the RET antagonist SU5416 at 10 μM for 20 minutes and then an additional for 20 minutes with GDNF (G) or compound 8. Vehicle (V) and SU5416 (S) alone are controls. SU5416 pretreatment resulted in clear reductions in the signaling capacity of both GDNF and compound 8. Representative blot showing pAkt and pErk, with Actin as loading control. Vehicle (V) is the negative control. In (B, D, and F), quantification of Western blot data were standardized to total Erk or total Akt and expressed as mean ± S.D. from three independent experiments. For pAkt, *P < 0.05; **P < 0.005; ***P < 0.0005. For pErk, #P < 0.05; ##P < 0.005; ###P < 0.0005 vs. vehicle, Dunnett’s test.