Multiple Independent Binding Sites for Small-Molecule Inhibitors on the Oncoprotein c-Myc

Abstract

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.

In Vitro Cytotoxicity and In Vivo Efficacy, Pharmacokinetics, and Metabolism of 10074-G5, a Novel Small-Molecule Inhibitor of c-Myc/Max Dimerization

“…It is also possible that much of the decline in overall c-Myc levels simply reflects a secondary reduction in its synthesis as a result of cell cycle arrest in G 0 /G 1 (Yin et al, 2003). On the other hand, the invariant levels of Max probably reflect its much greater stability (t 1/2 ; Ͼ24 h), its lack of interaction with small molecules such as 10074-G5 or 10058-F4 (Wang et al, 2007;Follis et al, 2008;Hammoudeh et al, 2009), and its ability to interact with numerous other bHLH-ZIP factors (Baudino and Cleveland, 2001). 10074-G5 was nontoxic to Daudi tumor-bearing mice at 20 mg/kg i.v., its maximum soluble dose.…”

“…Because c-Myc must heterodimerize with its obligatory bHLH-ZIP partner Max to exhibit transcriptional activity (Walhout et al, 1997), several groups have designed smallmolecule inhibitors to interfere with this protein-protein dimerization and thereby inactivate c-Myc function (Berg et al, 2002;Yin et al, 2003;Kiessling et al, 2006;Xu et al, 2006;Wang et al, 2007;Hammoudeh et al, 2009;Shi et al, 2009). Berg et al (2002) showed that small molecules can interfere with c-Myc/Max dimerization and inhibit c-Mycdependent transformation of chicken embryo fibroblasts.…”

“…In vitro, 10058-F4 exhibited high binding affinity to the bHLH-ZIP domain of c-Myc, promoted dissociation of the c-Myc/Max dimer, prevented binding of the heterodimer to DNA targets, induced cell cycle arrest, and caused apoptosis (Yin et al, 2003;Gomez-Curet et al, 2006;Huang et al, 2006;Lin et al, 2007;Sampson et al, 2007;Wang et al, 2007;Follis et al, 2008). Hammoudeh et al (2009) examined in detail the interactions of seven small-molecule inhibitors identified by Yin et al (2003) and found that 10058-F4 and 10074-G5 inhibited c-Myc/Max heterodimer formation similarly, but bound to different residues in the c-Myc bHLH-ZIP domain. 10058-F4 bound to residues 402 to 409, and 10074-G5 bound to residues 366 to 375.…”

“…Moreover, 10058-F4 and 10074-G5 could bind simultaneously and independently . Based on NMR structures of these small molecules in association with synthetic peptides, it has been postulated that they distort the bHLH-ZIP domain of the unstructured c-Myc monomer, thereby preventing or reversing its heterodimeric association with Max Hammoudeh et al, 2009). Based on the activity of 10058-F4 in vitro, Guo et al, (2009) evaluated 10058-F4 in C.B-17 SCID mice bearing PC3 and DU145 human androgen-independent prostate xenografts.…”

Hey Factors at the Crossroad of Tumorigenesis and Clinical Therapeutic Modulation of Hey for Anticancer Treatment

“…For example, some small-molecule inhibitors were isolated to specifically inhibit Myc-Max dimerization and block the binding of Myc-Max to DNA without affecting other structure-like bHLH factor dimerization (165,166). By using Myc bHLH-Zip domain fragments, researchers also discovered local conformational changes and formation of hydrophobic cavities at the specific peptide sequences of the fragments upon binding with these small-molecule inhibitors (167). This makes it possible to design specific inhibitors simply through protein sequence analysis because the small-molecule binding sites have certain peptide sequence criteria.…”