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Stabilizers of the Max homodimer identified in virtual ligand screening inhibit Myc function.

ABSTRACT: Many human cancers show constitutive or amplified expression of the transcriptional regulator and oncoprotein Myc, making Myc a potential target for therapeutic intervention. Here we report the down-regulation of Myc activity by reducing the availability of Max, the essential dimerization partner of Myc. Max is expressed constitutively and can form unstable homodimers. We have isolated stabilizers of the Max homodimer by applying virtual ligand screening (VLS) to identify specific binding pockets for small molecule interactors. Candidate compounds found by VLS were screened by fluorescence resonance energy transfer, and from these screens emerged a potent, specific stabilizer of the Max homodimer. In vitro binding assays demonstrated that the stabilizer enhances the formation of the Max-Max homodimer and interferes with the heterodimerization of Myc and Max in a dose-dependent manner. Furthermore, this compound interferes with Myc-induced oncogenic transformation, Myc-dependent cell growth, and Myc-mediated transcriptional activation. The Max-Max stabilizer can be considered a lead compound for the development of inhibitors of the Myc network.

SUBMITTER: Jiang H

PROVIDER: S-EPMC2730382 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Stabilizers of the Max homodimer identified in virtual ligand screening inhibit Myc function.

Jiang Hao H Bower Kristen E KE Beuscher Albert E AE Zhou Bin B Bobkov Andrey A AA Olson Arthur J AJ Vogt Peter K PK

Molecular pharmacology 20090604 3

Many human cancers show constitutive or amplified expression of the transcriptional regulator and oncoprotein Myc, making Myc a potential target for therapeutic intervention. Here we report the down-regulation of Myc activity by reducing the availability of Max, the essential dimerization partner of Myc. Max is expressed constitutively and can form unstable homodimers. We have isolated stabilizers of the Max homodimer by applying virtual ligand screening (VLS) to identify specific binding pocket ...[more]

PMID: 19498040

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Project description:Introduction Differentiation therapy is a promising strategy for cancer treatment. The translationally controlled tumor protein (TCTP) is an encouraging target in this context. By now, this field of research is still at its infancy, which motivated us to perform a large-scale screening for the identification of novel ligands of TCTP. We studied the binding mode and the effect of TCTP blockade on the cell cycle in different cancer cell lines. Methods Based on the ZINC-database, we performed virtual screening of 2,556,750 compounds to analyze the binding of small molecules to TCTP. The in silico results were confirmed by microscale thermophoresis. The effect of the new ligand molecules was investigated on cancer cell survival, flow cytometric cell cycle analysis and protein expression by Western blotting and co-immunoprecipitation in MOLT-4, MDA-MB-231, SK-OV-3 and MCF-7 cells. Results Large-scale virtual screening by PyRx combined with molecular docking by AutoDock4 revealed five candidate compounds. By microscale thermophoresis, ZINC10157406 (6-(4-fluorophenyl)-2-[(8-methoxy-4-methyl-2-quinazolinyl)amino]-4(3H)-pyrimidinone) was identified as TCTP ligand with a KD of 0.87 ± 0.38. ZINC10157406 revealed growth inhibitory effects and caused G0/G1 cell cycle arrest in MOLT-4, SK-OV-3 and MCF-7 cells. ZINC10157406 (2 × IC50) downregulated TCTP expression by 86.70 ± 0.44% and upregulated p53 expression by 177.60 ± 12.46%. We validated ZINC10157406 binding to the p53 interaction site of TCTP and replacing p53 by co-immunoprecipitation. Discussion ZINC10157406 was identified as potent ligand of TCTP by in silico and in vitro methods. The compound bound to TCTP with a considerably higher affinity compared to artesunate as known TCTP inhibitor. We were able to demonstrate the effect of TCTP blockade at the p53 binding site, i.e. expression of TCTP decreased, whereas p53 expression increased. This effect was accompanied by a dose-dependent decrease of CDK2, CDK4, CDK, cyclin D1 and cyclin D3 causing a G0/G1 cell cycle arrest in MOLT-4, SK-OV-3 and MCF-7 cells. Our findings are supposed to stimulate further research on TCTP-specific small molecules for differentiation therapy in oncology.

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Stabilizers of the Max homodimer identified in virtual ligand screening inhibit Myc function.

Publications

Stabilizers of the Max homodimer identified in virtual ligand screening inhibit Myc function.

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