Figure 1.
Alignment of the bHLHZip domains of c-MYC and MYCN.
The amino acids in the bHLHZip domains of human c-MYC and human MYCN were aligned, while the secondary structure is indicated above. Identical amino acid are denoted (*), conserved substitution (:) and semi-conserved substitution (.). The two binding sites for the specified compounds as reported by Hammoudeh et al is indicated by a colored square [38]. Each small molecule is positioned under their reported or assumed binding site [38], [39]. For the 10058-F4 analogs #474 and #764 as well as its potential metabolite C-m/z 232 the binding sites have not been determined experimentally [39]. Through the similarity of their chemical structure to 10058-F4, it has been assumed that these compounds bind to the same site as indicated.
Figure 2.
Equilibrium binding response for compounds binding to the bHLHZip of c-MYC and MYCN.
Equilibrium binding response determined by Surface Plasmon Resonance of small molecules to c-MYC (A) or MYCN (B) as a function of the concentration as indicated. The concentrations were plotted versus the response units after solvent correction and subtraction from the reference surface. The plots are from one representative experiment. For each molecule the experiments were performed at least with two different immobilizations followed by at least three independent experiments.
Table 1.
KD values of small molecules binding to the bHLHZip of c-MYC and MYCN.
Figure 3.
Reduction of MYCN/MAX interaction in NB cells after treatment with small molecules.
A) Proximity ligation assay (PLA) for MYCN/MAX interaction after treatment of SMS-KCN69n cells with the different small molecules at the indicated concentrations for 6 hours. Green signals represent the proximity of the MYCN and MAX proteins, while the red signal shows the total MYCN signal in the cell. DNA is stained with DAPI. Scale bar: 10 µM. The pictures are representative from three independent experiments. B) Quantification of the PLA signals per cell after treatment with the different compounds. Error bars indicate standard error of the mean.
Figure 4.
Reduction in MYCN protein levels in NB cells after treatment with small molecules.
Western blot analysis of MYCN expression in SK-N-BE(2) cells treated for 48 hours with the indicated concentrations of 10058-F4, 10058-F4 metabolite C-m/z 232, 10058-F4 analogs #474 and #764 and 10074-G5. Membranes were probed with antibodies recognizing MYCN and GAPDH. The blots are representative from three independent experiments.
Figure 5.
Growth inhibition and apoptosis induction in MYCN amplified cells.
(A) The amount of viable of cells was determined using crystal violet assay following 48 hours treatment of Kelly cells with increasing concentration of the indicated compound. Data are shown as percent of control (DMSO) treated cells and represent the mean of three independent experiments. Error bars indicate standard deviation. (B) Pearson's correlation between the IC50 values in the growth inhibition assay (A, Table 2) and the KD values for binding to MYCN (Table 1). (C–D) Quantification of apoptosis by propidium iodide staining for sub G1 DNA content of SK-N-BE(2) (C) and Kelly (D) cells. Data represent the means of at least three independent experiments. Error bars indicate standard deviation.
Table 2.
IC50 values from the crystal violet assay in Kelly cells.
Figure 6.
10074-G5 induces neuronal differentiation in NB cells.
Morphological differentiation of SK-N-BE(2) cells in response to 15 days culture with 10058-F4 (60 µM) 10074-G5 (30 µM) or DMSO in the presence or absence of NGF (50 ng/ml). Phase contrast micrographs show representative pictures from one out of three independent experiments.
Figure 7.
Lipid accumulation in MYCN-amplified NB cells.
Oil red O staining of SK-N-BE(2) cells treated with 10058-F4 (60 µM), C-m/z 232 (90 µM), 10074-G5 (20 µM), #474 (10 µM), #764 (10 µM) or DMSO for 3 days. The scale bar corresponds to 20 µm. Pictures are representative from four independent experiments.