INVESTIGATION OF PROTEIN AND SMALL-MOLECULE SELF-ASSOCIATION IN THE CONTEXT OF C-MYC/MAX INHIBITION

Abstract

The intrinsically disordered c-Myc oncoprotein is deregulated in ~70% of human cancers and is considered a promising but challenging drug target. To regulate transcription, c-Myc undergoes coupled folding and binding with another intrinsically disordered protein, Max, forming a basic-helix-loop-helix leucine zipper (bHLHZip) motif that interacts with the E-box DNA consensus sequence. In the early c-Myc literature, homo-oligomerization of c-Myc was observed but dismissed as only occurring at high (>20 µM) concentrations. Although it has been demonstrated in a mouse model that interfering with the c-Myc/Max interaction can lead to complete tumor regression, a clinically viable small-molecule inhibitor of the c-Myc/Max interaction remains elusive.

In this work, it is found that the c-Myc bHLHZip domain dramatically quenches the fluorescent probe Alexa488 upon conjugation. Quenching occurs irrespective of c-Myc labeling position and it is determined through specific cleavage of the bHLHZip that the residues responsible for quenching are located at the N-terminus. It is shown that the quenching occurs intramolecularly and the c-Myc bHLHZip forms a disordered, compact state in solution. Unexpectedly, quenching by the bHLHZip was concentration dependent, with relief occurring at higher c-Myc concentrations, providing evidence for an intermolecular interaction between c-Myc molecules. This association begins at ~100 nM, which is much lower than the previously identified concentration for c-Myc homo-oligomerization. The formation of higher order c-Myc bHLHZip oligomers displays a pH and salt dependence.

In addition to describing c-Myc self-association, the c-Myc-Max inhibitor 10074-A4 is investigated. This small-molecule was previously shown to interact enantiospecifically with the bHLHZip of c-Myc. By synthesizing the individual enantiomers of 10074-A4, the enantiospecific interaction with c-Myc is further characterized. The small-molecule undergoes formation of a chiral self-assembly which may have interfered with previous affinity measurements. The affinities of R-10074-A4 and S-10074-A4 for Myc353-437 are tenfold stronger than previously reported (Kd=1.9±0.2 µM and 2.6±0.5 µM respectively). Synthesis of 10074 A4 derivatives an initial structure activity relationship for 10074-A4/c-Myc binding is reported. Although this system is prone to challenges stemming from the self-association of both small-molecule inhibitors and the c-Myc protein, these studies elaborate upon an example of an IDP/small-molecule interaction with single-digit micromolar affinity.