As targeting its direct or indirect downstream targets. A number of small molecular compounds inhibiting c-MYC-MAX dimerization have been identified and among them 10058-F4 is by far the most studied. Biophysical experiments have shown that it interacts with the C-terminal bHLHZip region of c-MYC. A fluorescence polarization assay was used to determine the affinity as well as to identify the binding site of 10058-F4 on cMYC using different deletions, point mutations and synthetic peptides. NMR measurements confirmed that 10058-F4 binds to amino acid residues 402�C412 in the bHLHZip domain of c-MYC. Furthermore, metadynamic molecular simulations and an ion mobility mass spectrometry using a peptide corresponding to the identified binding site, indicated that the compound binds to an inactive, disordered conformation of cMYC. Together these studies suggest that 10058-F4 inhibits the function of c-MYC in a direct manner by preventing cMYC/MAX hetero-dimerization. Importantly, several reports have shown that 10058-F4 affects c-MYC expression and induces cell cycle arrest, inhibits cell growth, promotes apoptosis and confers chemo-sensitivity in a c-MYC specific manner in various cancer cell types. In addition, GSK2118436 Raf inhibitor treatment of acute myeloid leukemia cells with 10058-F4 leads to myeloid differentiation. The effect of 10058-F4 treatment in vivo has been investigated in xenograft models of prostate cancer but no significant antitumor activity could be observed, probably due to its rapid clearance and low potency. In contrast, we have recently demonstrated anti-tumorigenic effects of 10058-F4 in two tumor models of MYCN-amplified neuroblastoma, suggesting that direct MYC inhibition using a small molecule is achievable in vivo. The structurally unrelated small molecule 10074-G5 was identified simultaneously as 10058-F4 as another substance that inhibits the c-MYC/MAX interaction. This molecule also decreased c-MYC protein levels and inhibited cell growth, but failed to show any antitumor activity in a xenograft model using a Burkitt’s lymphoma cell line. The cognate binding site for 10074-G5 on c-MYC was found to be distinct from that of 10058-F4, spanning amino acid residues 363�C381. Both molecules were found to bind independently of each other, and probably induce only local conformation changes in the bHLHZip domain of c-MYC preventing its interaction with MAX. In order to identify more potent compounds, several analogs of 10058-F4 have been synthesized, some of which, including exhibited improved growth inhibition of c-MYC expressing cells. Since c-MYC and MYCN share structural similarity in the bHLHZip domain we hypothesized that 10058-F4 also targets MYCN. We have previously shown that this compound interferes with the MYCN/MAX interaction leading to cell cycle arrest, apoptosis, and neuronal differentiation in MYCN-overexpressing NB cell lines. In addition, using 10058-F4 as a tool, we found that inhibition of MYCN results in mitochondrial dysfunction leading to lipid accumulation. Importantly, 10058-F4 treatment furthermore increased the survival of TH-MYCN transgenic mice and showed anti-tumor effects in established aggressive NB xenografts. Here, we determined the direct binding of 10058-F4 and additional selected c-MYC-targeting compounds to MYCN by surface plasmon resonance. We found that all molecules previously reported to bind to c-MYC also bound to MYCN. Treatment with the small molecules furthermore interfered with the MYCN/ MAX interaction and caused protein degradation, apoptosis, differentiation and lipid formation to different extents in MYCNamplified NB cells. To date several research PLX4032 Raf inhibitor groups have focused on developing compounds that target c-MYC for cancer therapy, whilst there have been only a few publications that have explored the possibility of targeting MYCN.