Fig 1.
Structures of compounds 1 (1,2-bis(4-chlorophenyl)disulfide), 2 (4-chloro-N-(4-chlorophenyl)benzamide), 3 ((E)-1,2-bis(4-chlorophenyl)diazene), 4 (1,2-bis(4-chlorophenyl)hydrazine), 5 (1,2-bis(4-methoxyphenyl)disulfide), and 6 (1,2-bis(4-nitrophenyl)disulfide).
Fig 2.
HEK293 cells stably expressing the Pdcd4 stability reporter Pdcd4(39–91)luc were incubated for 8 h with TPA [10 nM] in combination with increasing concentrations (0.1–30 μM) of compounds 1, 2, 3, 4, 5, and 6. Pdcd4 stabilizing activity was determined relative to Δ(RLUDMSO–RLUTPA). Data are presented as means ± SEM of at least 3 independent experiments (* p<0.05, ** p<0.01, *** p<0.01).
Fig 3.
HEK293 cells stably expressing Pdcd4(mut39–91)luc were incubated for 8 h with TPA [10 nM] in combination with increasing concentrations (0.1–30 μM) of compounds 1, 2, 3, 4, 5, and 6. Luciferase activity is given relative to TPA-treated controls. Data are presented as means ± SEM of at least 3 independent experiments (* p<0.05, ** p<0.01, *** p<0.01).
Fig 4.
HEK293 cells stably expressing Pdcd4(39–91)luc were incubated for 5 days with DMSO (blue) or increasing concentrations of compounds 1, 2, 3, 4, 5, and 6 (1 μM (green), 3 μM (violet), 10 μM (yellow), and 30 μM (red)). Confluency was assessed every 4 h in an IncuCyte instrument. Proliferation kinetics shown are representative for at least 3 independent experiments and represent means of 3 wells treated in parallel (* p<0.05, ** p<0.01, *** p<0.01).
Fig 5.
HEK293 cells stably expressing Pdcd4(39–91)luc were incubated for 24 h with DMSO (blue) or increasing concentrations of compounds 1, 2, 3, 4, 5, and 6 (3 μM (violet), 10 μM (yellow), and 30 μM (red)). Cell cycle analysis was performed after permeabilization using 7-AAD. Data are presented as means ± SEM of at least 3 independent experiments (* p<0.05, ** p<0.01, *** p<0.01).
Fig 6.
HEK293 cells stably expressing Pdcd4(39–91)luc were incubated for 24 h with DMSO (blue) or increasing concentrations of compounds 1, 2, 3, 4, 5, and 6 (3 μM (violet), 10 μM (yellow), and 30 μM (red)). Cell death analysis was performed using Annexin V and 7-AAD co-staining. Data are presented as means ± SEM of at least 3 independent experiments (* p<0.05, ** p<0.01, *** p<0.01).
Fig 7.
Conformational analysis of compounds 1, 2 and 4.
(A) A systematic approach was chosen to explore the conformational space around the three rotable bonds. (B-D) Global minimum energy conformations (left panel) and energy plots (right panel) of compounds 1 (B), 2 (C), and 4 (D).