Disruption of Myc-Max Heterodimerization with Improved Cell-Penetrating Analogs of the Small Molecule 10074-G5

Abstract

The c-Myc (Myc) oncoprotein is a high-value therapeutic target given that it is deregulated in multiple types of cancer. However, potent small molecule inhibitors of Myc have been difficult to identify, particularly those whose mechanism relies on blocking the association between Myc and its obligate heterodimerization partner, Max. We have recently reported a structure-activity relationship study of one such small molecule, 10074-G5, and generated an analog, JY-3-094, with significantly improved ability to prevent or disrupt the association between recombinant Myc and Max proteins. However, JY-3094 penetrates cells poorly. Here, we show that esterification of a critical para-carboxylic acid function of JY-3-094 by various blocking groups significantly improves cellular uptake although it impairs the ability to disrupt Myc-Max association in vitro. These pro-drugs are highly concentrated within cells where JY-3-094 is then generated by the action of esterases. However, the pro-drugs are also variably susceptible to extracellular esterases, which can deplete extracellular reservoirs. Furthermore, while JY-3-094 is retained by cells for long periods of time, much of it is compartmentalized within the cytoplasm in a form that appears to be less available to interact with Myc. Our results suggest that persistently high extracellular levels of pro-drug, without excessive susceptibility to extracellular esterases, are critical to establishing and maintaining intracellular levels of JY-3-094 that are sufficient to provide for long-term inhibition of Myc-Max association. Analogs of JY-3-094 appear to represent promising small molecule Myc inhibitors that warrant further optimization.

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

“…2F), thus attesting to the specificity of the compound as previously noted and documented by both NMR spectroscopy and other methods [22]. Finally, although numerous factors can influence the cellular effects of Myc inhibitors [15,18], we would note that the results obtained with SPR were in good agreement with JKY-2-169 being a more potent inhibitor of Myc-over-expressing cancer cells than 10074-G5 [18,22]. …”

“…They include fluorescence polarization, electrophoretic mobility shift assays (EMSA), circular dichroism, and NMR spectroscopy [15,18,20,21]. While these methods are specific and variably quantitative, each is subject to factors that can compromise or restrict its use.…”

Small-molecule inhibitors of the Myc oncoprotein

“…Although JY-3-094 exhibited no cytotoxicity to HL60 and Daudi cells, this was presumably due to its ionizable carboxylic acid. The observed lack of activity of JY-3-094 was remedied by esterification of its carboxylic acid to afford a panel of ester prodrugs that exhibited IC 50 s in the low micromolar range in both HL60 and Daudi cells (145). LC/MS quantification revealed that the ester pro-drugs were metabolized to the Myc inhibitor JY-3-094 in cells thus confirming that the cytotoxicities, at least in part, stem from JY-3-094.…”

The progress of angiogenic factors in the development of leukemias

“…It is an interesting target for novel drug therapies because of its oncogenic activities and overexpression in a majority of human cancers (47). Binding inhibition of c-myc on gene promoters by some small molecule inhibitors has been tried (48,49), and found that some of them were effective in disrupting essential protein-DNA interactions, for example, Pyrroleimidazole (PI) polyamides, specific sequence DNAbinding small molecules (50)(51)(52), and can inhibit a part of E-box-mediated c-myc downstream gene expression (47). EGFR variant III (EGFRvIII), which strongly induces neovascularization in tumors, could induce Angptl4 expression through the ERK/c-myc pathway.…”