Abstract
MYC is a key player in tumor development, but unfortunately no specific MYC-targeting drugs are clinically available. MYC is strictly dependent on heterodimerization with MAX for transcription activation. Aiming at targeting this interaction, we identified MYCMI-6 in a cell-based protein interaction screen for small inhibitory molecules. MYCMI-6 exhibits strong selective inhibition of MYC:MAX interaction in cells and in vitro at single-digit micromolar concentrations, as validated by split Gaussia luciferase, in situ proximity ligation, microscale thermophoresis and surface plasmon resonance (SPR) assays. Further, MYCMI-6 blocks MYC-driven transcription and binds selectively to the MYC bHLHZip domain with a KD of 1.6 ± 0.5 μM as demonstrated by SPR. MYCMI-6 inhibits tumor cell growth in a MYC-dependent manner with IC50 concentrations as low as 0.5 μM, while sparing normal cells. The response to MYCMI-6 correlates with MYC expression based on data from 60 human tumor cell lines and is abrogated by MYC depletion. Further, it inhibits MYC:MAX interaction, reduces proliferation and induces massive apoptosis in tumor tissue from a MYC-driven xenograft tumor model without severe side effects. Since MYCMI-6 does not affect MYC expression, it is a unique molecular tool to specifically target MYC:MAX pharmacologically and it has good potential for drug development.
Highlights
Were transfected into the cells together with the CMV-Luc plasmid and treated with the indicated compounds for 17 hours and analyzed in a dual luciferase assay
Establishment of a cell-based Bimolecular Fluorescence Complementation (BiFC) assay for screening for small molecules interfering with MYC:MAX protein interactions
To optimize the system for screening purposes, MYC-yellow fluorescent protein (YFP)-C and MAX-YFP-N were transiently coexpressed in HEK293 cells together with a full-length cyan fluorescent protein (CFP) vector as internal control (Fig. 1C, upper panels), after which the ratio of BiFC/CFP fluorescence intensities was calculated
Summary
Were transfected into the cells together with the CMV-Luc plasmid and treated with the indicated compounds for 17 hours and analyzed in a dual luciferase assay. A number of small molecules have been reported to target the MYC:MAX or MYC:MAX:DNA interaction[15,16,22,24,25,26,27,28,29,30,31,32,33] None of these compounds have made their way for clinical studies due to a number of limitations including low potency in vitro or in cells, poor specificity or inadequate bioavailability in vivo[15,16,26,34]. In a cellular MYC:MAX PPI inhibitor screen, we identified the compound MYCMI-6 that fulfills all these criteria
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