Compounds that selectively prevent or disrupt the association between the c-Myc oncoprotein and its obligate heterodimeric partner Max (Myc-Max compounds) have been identified previously by high-throughput screening of chemical libraries. Although these agents specifically inhibit the growth of c-Myc -expressing cells, their clinical applicability is limited by their low potency. We describe here several chemical modifications of one of these original compounds, 10058-F4, which result in significant improvements in efficacy. Compared with the parent structure, these analogues show enhanced growth inhibition of c-Myc -expressing cells in a manner that generally correlates with their ability to disrupt c-Myc-Max association and DNA binding. Furthermore, we show by use of a sensitive fluorescence polarization assay that both 10058-F4 and its active analogues bind specifically to monomeric cMyc. These studies show that improved Myc-Max compounds can be generated by a directed approach involving deliberate modification of an index compound. They further show that the compounds specifically target c-Myc, which exists in a dynamic and relatively unstructured state with only partial and transient A-helical content. [Mol Cancer Ther 2007;6(9):2399 -408]

Deregulation of the c-Myc transcription factor is involved in many types of cancer, making this oncoprotein an attractive target for drug discovery. One approach to its inhibition has been to disrupt the dimeric complex formed between its basic helix-loop-helix leucine zipper (bHLHZip) domain and a similar domain on its dimerization partner, Max. As monomers, bHLHZip proteins are intrinsically disordered (ID). Previously we showed that two c-Myc-Max inhibitors, 10058-F4 and 10074-G5, bound to distinct ID regions of the monomeric c-Myc bHLHZip domain. Here, we use circular dichroism, fluorescence polarization, and NMR to demonstrate the presence of an additional binding site located between those for 10058-F4 and 10074-G5. All seven of the originally identified Myc inhibitors are shown to bind to one of these three discrete sites within the 85-residue bHLHZip domain of c-Myc. These binding sites are composed of short contiguous stretches of amino acids that can selectively and independently bind small molecules. Inhibitor binding induces only local conformational changes, preserves the overall disorder of c-Myc, and inhibits dimerization with Max. NMR experiments further show that binding at one site on c-Myc affects neither the affinity nor the structural changes taking place upon binding to the other sites. Rather, binding can occur simultaneously and independently on the three identified sites. Our results suggest the widespread existence of peptide regions prone to small-molecule binding within ID domains. A rational and generic approach to the inhibition of protein-protein interactions involving ID proteins may therefore be possible through the targeting of ID sequence.

The basic-helix-loop-helix-leucine-zipper domains of the c-Myc oncoprotein and its obligate partner Max are intrinsically disordered (ID) monomers that undergo coupled folding and binding upon heterodimerization. We have identified the binding sites and determined the structural means by which two unrelated small molecules, 10058-F4 and 10074-G5, bind c-Myc and stabilize the ID monomer over the highly ordered c-Myc-Max heterodimer. In solution, the molecules bind to distinct regions of c-Myc and thus limit its ability to interact with Max and assume a more rigid and defined conformation. The identification of multiple, specific binding sites on an ID domain suggests that small molecules may provide a general means for manipulating the structure and function of ID proteins, such as c-Myc.