Figure 1.
The ligands used in this study are shown: prodigiosin, obatoclax, ligand number 6 (a rhodanine derivative) and ABT-737.
Figure 2.
(A and B) PG disrupts constitutive MCL-1/BAK interaction. Cell lysates were subjected to immunoprecipitation with anti-MCL-1 or anti-BAK antibody after PG treatment at 1 µM (8 h) and 100 nM (24 h), respectively. (C) PG permeabilizes the outer mitochondrial membrane. BAX was immunoprecipitated from PG (50, 100 and 500 nM), OBX (10 µM) or DMSO- treated cells for 24 h and then BAK/BAX complex formation was analyzed by immunoblot with anti-BAK and anti-BAX antibodies.
Figure 3.
PG induces activation of the intrinsic apoptotic pathway.
(A) PG induces cytochrome c release to the cytosol. Cytochrome c release from SK-MEL-5 isolated mitochondria after PG and OBX treatment. Mitochondria were isolated from PG (100 nM), OBX (10 µM) or DMSO- treated cells for 24 h. Cytochrome c release from mitochondria to cytosol was analyzed by Immunoblot using the mitochondrial marker porin as a quality control of the isolation process. (B) Activation of caspases. Cells were treated with PG (100 nM) or OBX (10 µM) for 24 h and total cell lysates were analyzed by immunoblot. Actin was used as loading control.
Figure 4.
Effect of MCL-1 overexpression on cell viability.
(A) MCL-1 overexpression partially blocks PGs cytotoxicity. SK-MEL-5 cells were transfected with 1 µg of pTOPOMCL1 plasmid and, after 20 h, cells were treated with PGs (2 and 20 µM, respectively) for an addittional 24 h. Then cell viability was assessed by the MTT assay. (B) Analysis of MCL-1 protein levels. After MCL1 overexpression and PGs treatment, MCL-1 protein levels were analyzed by Immunoblot. Vinculin was used as a loading control.
Table 1.
Before and after induced fit docking scores.
Figure 5.
Induced fit docked structures.
Top panels (A): final induce fit structures in MCL-1 (left) BCL-xl (center) and BCL-2 (right). Bottom panles (B): detailed view of the molecular interactions of PG, OBX and 6 with MCL-1.
Figure 6.
(A) Crystal structure of the mNoxaB BH3 peptide bound to MCL-1. In cyan we show Gly82, the 11 helix residue located in the h3 position. Leu267 in blue, Thr266 in red and His224 in green. from MCL-1 are also shown. (B) Pharmacophore analysis of the binding interactions. Residues within 3 Å from the ligand have been included in the anlysis.