Members of the MYC family are the most frequently deregulated oncogenes in human 
cancer and are often correlated with aggressive disease and/or poorly 
differentiated tumors. Since patients with MYCN-amplified neuroblastoma have a 
poor prognosis, targeting MYCN using small molecule inhibitors could represent a 
promising therapeutic approach. We have previously demonstrated that the small 
molecule 10058-F4, known to bind to the c-MYC bHLHZip dimerization domain and 
inhibiting the c-MYC/MAX interaction, also interferes with the MYCN/MAX 
dimerization in vitro and imparts anti-tumorigenic effects in neuroblastoma 
tumor models with MYCN overexpression. Our previous work also revealed that 
MYCN-inhibition leads to mitochondrial dysfunction resulting in accumulation of 
lipid droplets in neuroblastoma cells. To expand our understanding of how small 
molecules interfere with MYCN, we have now analyzed the direct binding of 
10058-F4, as well as three of its analogs; #474, #764 and 10058-F4(7RH), one 
metabolite C-m/z 232, and a structurally unrelated c-MYC inhibitor 10074-G5, to 
the bHLHZip domain of MYCN. We also assessed their ability to induce apoptosis, 
neurite outgrowth and lipid accumulation in neuroblastoma cells. Interestingly, 
all c-MYC binding molecules tested also bind MYCN as assayed by surface plasmon 
resonance. Using a proximity ligation assay, we found reduced interaction 
between MYCN and MAX after treatment with all molecules except for the 10058-F4 
metabolite C-m/z 232 and the non-binder 10058-F4(7RH). Importantly, 10074-G5 and 
10058-F4 were the most efficient in inducing neuronal differentiation and lipid 
accumulation in MYCN-amplified neuroblastoma cells. Together our data 
demonstrate MYCN-binding properties for a selection of small molecules, and 
provide functional information that could be of importance for future 
development of targeted therapies against MYCN-amplified neuroblastoma.