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.