Exploiting Structural Disorder to Enhance Small Molecule Inhibition of the Oncoprotein c-Myc DImerization with its Partner Max
Viacava Follis, Ariele Giorgio G.
Metallo, Steven J.
Yang, David C.
The transcription factor c-Myc, in its normal function, is involved in cell cycle regulation. The uncontrolled cell proliferation consequent to c-Myc deregulation is typical of cancer and neoplastic diseases. Most known functions of c-Myc depend upon the dimerization between its basic-helix-loop-helix-leucine zipper (bHLHZip) domain and a similar domain within the partner protein Max. Small molecules capable of specifically and selectively disrupting the c-Myc-Max interaction have been reported. Because of c Myc's involvement in cancer, the inhibition of its dimer formation with Max is a promising way to down-regulate its activity for therapeutic purposes. Small molecules interfering with c-Myc-Max dimer formation must directly interact with one or both protein monomers. These monomers are intrinsically disordered and lack a stable structure, as they undergo coupled folding and binding and they assume a defined structure only upon dimerization. The possibility of directly targeting disordered proteins with small molecules has not yet been broadly considered. In the studies described here, recombinant c-Myc, Max and derived peptides were employed in purified component assays based on several biophysical techniques, including fluorescence polarization, circular dichroism, and gel electrhophoresis, to elucidate the mechanism of action of small molecule inhibitors of c-Myc-Max dimer formation originally described by the Prochownik lab at the Children's Hospital, Pittsburgh. The synthesis of several modified small molecules further allowed for structure-activity studies. It was found that these compounds bind in a plastic mode to short segments of the c-Myc bHLHZip domain. NMR spectroscopy was employed to collect structural information about the studied complexes, confirming their dynamic nature. It is hypothesized that the combined presence of hydrophobic residues and low sequence conservation make such sites prone to specific small molecule binding. The different location of binding sites on c-Myc was found to result in different mechanisms of disruption of c-Myc-Max dimers. The presence of HLZip dimerization equilibria competing with c-Myc-Max dimerization was found to facilitate the inhibition of the latter. The multiple binding sites on c-Myc were further exploited in the design of bivalent inhibitors which interact with two such sites and display enhanced affinity for c-Myc.
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