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.
Deregulated expression of MYC family oncogenes occurs frequently in human cancer and is often associated with aggressive disease and poor prognosis. While MYC is a highly warranted target, it has been considered "undruggable", and no specific anti-MYC drugs are available in the clinic. We recently identified molecules named MYCMIs that inhibit the interaction between MYC and its essential partner MAX. Here we show that one of these, MYCMI-7, efficiently and selectively inhibits MYC:MAX and MYCN:MAX interactions in cells, binds directly to recombinant MYC and reduces MYC-driven transcription. In addition, MYCMI-7 induces degradation of MYC and MYCN proteins. MYCMI-7 potently induces growth arrest/apoptosis in tumor cells in a MYC/MYCN-dependent manner and downregulates the MYC pathway on a global level as determined by RNA-seq. Sensitivity to MYCMI-7 correlates with MYC expression in a panel of 60 tumor cell lines and MYCMI-7 shows high efficacy towards a collection of patient-derived primary glioblastoma and acute myeloid leukemia (AML) ex vivo cultures. Importantly, a variety of normal cells become G1 arrested without signs of apoptosis upon MYCMI-7 treatment. Finally, in mouse tumor models of MYC-driven AML, breast cancer and MYCN-amplified neuroblastoma, treatment with MYCMI-7 downregulates MYC/MYCN, inhibits tumor growth and prolongs survival through apoptosis with little side effects. In conclusion, MYCMI-7 is a potent and selective MYC inhibitor that is highly relevant for the development into clinically useful drugs for treatment of MYC-driven cancer.
Restoration of the p53 tumor suppressor for personalised cancer therapy is a promising treatment strategy. However, several high-affinity MDM2 inhibitors have shown substantial side effects in clinical trials. Thus, elucidation of the molecular mechanisms of action of p53 reactivating molecules with alternative functional principle is of the utmost importance. Here, we report a discovery of a novel allosteric mechanism of p53 reactivation through targeting the p53 N-terminus which promotes inhibition of both p53/MDM2 (murine double minute 2) and p53/MDM4 interactions. Using biochemical assays and molecular docking, we identified the binding site of two p53 reactivating molecules, RITA (reactivation of p53 and induction of tumor cell apoptosis) and protoporphyrin IX (PpIX). Ion mobility-mass spectrometry revealed that the binding of RITA to serine 33 and serine 37 is responsible for inducing the allosteric shift in p53, which shields the MDM2 binding residues of p53 and prevents its interactions with MDM2 and MDM4. Our results point to an alternative mechanism of blocking p53 interaction with MDM2 and MDM4 and may pave the way for the development of novel allosteric inhibitors of p53/MDM2 and p53/MDM4 interactions.
The MYC family oncoproteins/transcription factors MYC, MYCN and MYCL (here referred to as MYC) are key players in tumor development and are particularly associated with aggressive disease and poor prognosis. Efficient and specific MYC-targeting drugs are therefore highly warranted, but no such drugs are available in the clinic at present. MYC is strictly dependent on heterodimerization with MAX for activation of transcription. In a cell-based Bimolecular Fluorescence Complementation protein-protein interaction screen for small molecule inhibitors we identified a molecule that exhibits strong selective inhibition of MYC-MAX interaction in cells as validated by Gaussia luciferase protein complementation assay, coimmunoprecipitation and in situ proximity ligation (isPLA) assay, reaching an IC50 at single-digit micromolar concentrations. The molecule was shown to inhibit MYC-MAX interactions in a biochemical FRET assay and binds selectively to the MYC bHLHZip domain with affinity in the single digit micromolar range as demonstrated by Microscale Thermophoresis and Surface Plasmon Resonance. Further, within the same concentration range, this molecule blocks MYC-driven transcription and efficiently inhibits tumor cell growth in a MYC-dependent manner, but spares normal cells. Moreover, the growth inhibitory responses to the molecule correlated significantly with MYC expression levels in a cohort of 60 human tumor cell lines. Importantly, utilizing a mouse tumor model of MYCN-amplified neuroblastoma, treatment with the molecule resulted in significant inhibition of the MYC-MAX interaction in tumor tissue, as shown by isPLA, and massive induction of apoptosis in the tumors. Since this molecule, unlike many experimental MYC inhibitors, is selective, has high affinity for MYC, has high efficacy in cells, reaches its target in vivo and does not affect MYC expression levels, it can be used as a chemical tool to specifically study the role of the MYC-MAX complex in MYC biology in normal and cancerous cells, and it has potential for drug development. Citation Format: Alina Castell, Qinzi Yan, Karin Fawkner, Per Hydbring, Fan Zhang, Vasiliki Verschut, Marcela Franco, Giovanna Zinzalla, Lars-Gunnar Larsson. Selective high affinity MYC-binding compound inhibits MYC-MAX interaction and MYC-dependent tumor cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3952.
Given the immense significance of p53 restoration for anti-cancer therapy, elucidation of the mechanisms of action of p53-activating molecules is of the utmost importance. Here we report a discovery of novel allosteric modulation of p53 by small molecules, which is an unexpected turn in the p53 story. We identified a structural element involved in p53 regulation, whose targeting by RITA, PpIX and licofelone block the binding of p53 inhibitors, MDM2 and MDMX. Deletion and mutation analysis followed by molecular modeling, identified the key p53 residues S33 and S37 targeted by RITA and PpIX. We propose that the binding of small molecules to the identified site induces a conformational trap preventing p53 from the interaction with MDM2 and MDMX. These results point to a high potential of allosteric activators. Our study provides the basis for the development of therapeutics with a novel mechanism of action, thus extending the p53 pharmacological potential.
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