Max has the additional capacity to form homodimers that can bind to the same DNA sequences but generally repress gene expression . We therefore performed an electrophoresis gel mobility shift assay to determine if our inhibitors had selectivity for inhibiting the binding of Myc:Max heterodimers to DNA over Max:Max homodimers. Incubation of Max protein with the E-box oligonucleotide resulted in a DNA band shift indicative of Max:Max homodimers . Addition of Myc resulted in a decrease in the amount of Max:Max homodimers and the appearance of Myc:Max heterodimers in complex with DNA. Neither of the two monomers, E07 or N12, had any noticeable effect on the Myc: Max or Max:Max complexes, however E07+N12 caused a dose-dependent decrease in the levels of the Myc:Max complex. Notably the decrease in Myc:Max complex is inversely correlated with an increase in the levels of the Max:Max complex, suggesting the dimer is specifically blocking the Myc:Max interaction, freeing Max to homodimerize and bind to the DNA. As further evidence that the formation of the dimeric inhibitor was critical for the 1252003-15-8 supplier inhibitory activity, we performed similar ZM241385 experiments with the non-dimerizable control monomer C12 . In contrast to E07+N12, the combination of E07+C12 had no effect on the binding of the Myc:Max or Max:Max complexes to DNA. These data are consistent with the effects of these compounds in the SPR assay and ELISA. Collectively these cell-free assay data demonstrate that the self-assembling dimeric inhibitors identified in the cell assays can directly bind to Myc and inhibit its interaction with Max, supporting an on-target mechanism of action for these inhibitors. Further, the use of nondimerizable control monomers confirms that the ability to form a large molecular weight dimeric inhibitor is key to the ability to target the Myc protein. Our cell-free experiments had clearly shown that the ability for self-assembly of the dimer was important in driving the improved inhibitory effect versus the Myc protein. To test this in a cellular context we compared the effect on cell viability of the E08+N11 dimer versus its nondimerizable control combinatio
