Discovery of Methyl 4′‐Methyl‐5‐(7‐nitrobenzo[c][1,2,5]oxadiazol‐4‐yl)‐[1,1′‐biphenyl]‐3‐carboxylate, an Improved Small‐Molecule Inhibitor of c‐Myc–Max Dimerization

Abstract

c-Myc is a bHLH-ZIP transcription factor that is responsible for the transcription of a wide range of target genes involved in many cancer-related cellular processes, such as proliferation, differentiation, apoptosis and metabolism. Over-expression of c-Myc has been observed in, and directly contributes to, a variety of human cancers including those of the hematopoietic system, lung, prostate and colon. To become transcriptionally active, c-Myc must first dimerize with Max via its own bHLH-ZIP domain. A proven strategy towards the inhibition of c-Myc oncogenic activity is to interfere with the structural integrity of the c-Myc–Max heterodimer. The small-molecule 10074-G5 is an inhibitor of c-Myc–Max dimerization (IC50 = 146 μM) that operates by binding and stabilizing c-Myc in its monomeric form. Herein, we report on our on-going efforts to optimize the c-Myc–Max inhibitory activity of 10074-G5-related molecules in vitro and in cancer cells that over-express c-Myc. Specifically, we have identified a congener of 10074-G5, termed 3jc48-3, that is about five times as potent (IC50 = 34 μM) at inhibiting c-Myc–Max dimerization as the parent compound. In addition, 3jc48-3 exhibited an approximate two-fold selectivity for c-Myc–Max heterodimers over Max–Max dimers, suggesting that, like its predecessor, its mode of action is through binding c-Myc. 3jc48-3 inhibited the proliferation of c-Myc-over-expressing HL60 and Daudi cells with single-digit micromolar IC50 values by causing growth arrest at the G0/G1 phase. Furthermore, co-immunoprecipitation studies indicated that 3jc48-3 inhibits c-Myc–Max dimerization in cells, which was further substantiated by the specific silencing of a c-Myc-driven luciferase reporter gene. Finally, unlike previously described 10074-G5 analogues, which are rapidly released and/or metabolized by cells following their uptake, 3jc48-3’s intracellular half-life was >17 h. Collectively, these data demonstrate 3jc48-3 to be one of the most potent cellularly active c-Myc inhibitors reported to date.

Perturbation of the c-Myc–Max Protein–Protein Interaction via Synthetic α-Helix Mimetics

“…45,46 We have also recently completed a structure–activity relationship (SAR) analysis of the direct c-Myc inhibitor 2 , 47 which binds one of these sites centered around residues 363–381 corresponding to the junction between the basic domain and helix 1. NMR analysis of the interaction of 2 with a c-Myc 363–381 synthetic peptide allowed the delineation of a more refined binding site, and our SAR work was largely in agreement with this model.…”

“…Since this hydrophobic domain is flanked by arginines Arg 366 /Arg 367 /Arg 372 and Arg 378 at the N- and C-terminal ends, respectively, we incorporated groups into the periphery of 4 that would complement these basic side chains. For inspiration, we looked to previous small-molecules that also bind this region: the electron rich nitro group of 2 is believed to interact with Arg 366 , Arg 367 and Arg 372 , 45,46 whereas the carboxylic acid in the second-generation 2 congener JY-3-094 47 possibly interacts with Arg 378 . Since the nitro group of 2 and that of JY-3-094 along with its carboxylic acid were instrumental in furnishing the small-molecules with c-Myc inhibitory activities, we, therefore, selected a nitro group to recognize Arg 366 , Arg 367 and/or Arg 372 and a carboxylic acid to recognize Arg 378 .…”

“…37,38,47,48,62 Briefly, cells at >90% viability were seeded into 96 well plates (2×10 3 cells/well) in fresh growth medium containing 10-point serial dilutions of the compounds of interest. Incubations were allowed to proceed for three days at which time cell viability was assed using the MTT assay as previously described.…”

A quantitative, surface plasmon resonance-based approach to evaluating DNA binding by the c-Myc oncoprotein and its disruption by small molecule inhibitors

“…Two full-length isoforms of Max, termed Max(L) (160 residues) and Max(S) (151 residues), varying only by an N-terminal insertion of 9 residues in the former protein, were used for different purposes [7]. In the absence of Myc, Max(L) forms a DNA-binding homodimer that serves as an excellent control to test the specificity of small molecule Myc inhibitors [14,15,19]. Like Max(L), Max(S) also forms low-affinity homodimers [29] but binds DNA poorly, if at all.…”

“…The first, represented by the compounds 10058-F4 and 10074-G5 and their analogs [14–19] bind to distinct regions of monomeric Myc’s intrinsically disordered bHLH-ZIP domain and create a localized distortion that prevents heterodimerization with Max [20,21]. Due to the high free energy of Myc-Max association, these compounds are much less able to promote the dissociation of pre-formed heterodimers [8,9].…”