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Pharmacophore-guided discovery of CDC25 inhibitors causing cell cycle arrest and tumor regression.

ABSTRACT: CDC25 phosphatases play a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational methods, we defined a minimal common pharmacophore in established CDC25 inhibitors and performed virtual screening of a proprietary library. Based on the availability of crystal structures for CDC25A and CDC25B, we implemented a molecular docking strategy and carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, the compounds induced differentiation, accompanied by decreased stemness properties, in intestinal crypt stem cell-derived Apc/K-Ras-mutant mouse organoids, and led to tumor regression and reduction of metastatic potential in zebrafish embryo xenografts used as in vivo model.

SUBMITTER: Kabakci Z

PROVIDER: S-EPMC6362118 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Pharmacophore-guided discovery of CDC25 inhibitors causing cell cycle arrest and tumor regression.

Kabakci Zeynep Z Käppeli Simon S Cantù Claudio C Jensen Lasse D LD König Christiane C Toggweiler Janine J Gentili Christian C Ribaudo Giovanni G Zagotto Giuseppe G Basler Konrad K Pinna Lorenzo A LA Cozza Giorgio G Ferrari Stefano S

Scientific reports 20190204 1

CDC25 phosphatases play a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational methods, we defined a minimal common pharmacophore in established CDC25 inhibitors and performed virtual screening of a proprietary library. Based on the availability of crystal structures for CDC25A and CDC25B, we implemented a molecular docking strategy and carried out hit expansion/optimization. Enz ...[more]

PMID: 30718768

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Project description:Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell cycle G(2) arrest in fission yeast (Schizosaccharomyces pombe) and mammalian cells, suggesting the cellular pathway(s) targeted by Vpr is conserved among eukaryotes. Our previous studies in fission yeast demonstrated that Vpr induces G(2) arrest in part through inhibition of Cdc25, a Cdc2-specific phosphatase that promotes G(2)/M transition. The goal of this study was to further elucidate molecular mechanism underlying the inhibitory effect of Vpr on Cdc25. We show here that, similar to the DNA checkpoint controls, expression of vpr promotes subcellular relocalization of Cdc25 from nuclear to cytoplasm and thereby prevents activation of Cdc2 by Cdc25. Vpr-induced nuclear exclusion of Cdc25 appears to depend on the serine/threonine phosphorylation of Cdc25 and the presence of Rad24/14-3-3 protein, since amino acid substitutions of the nine possible phosphorylation sites of Cdc25 with Ala (9A) or deletion of the rad24 gene abolished nuclear exclusion induced by Vpr. Interestingly, Vpr is still able to promote Cdc25 nuclear export in mutants defective in the checkpoints (rad3 and chk1/cds1), the kinases that are normally required for Cdc25 phosphorylation and nuclear exclusion of Cdc25, suggesting that others kinase(s) might modulate phosphorylation of Cdc25 for the Vpr-induced G(2) arrest. We report here that this kinase is Srk1. Deletion of the srk1 gene blocks the nuclear exclusion of Cdc25 caused by Vpr. Overexpression of srk1 induces cell elongation, an indication of cell cycle G(2) delay, in a similar fashion to Vpr; however, no additive effect of cell elongation was observed when srk1 and vpr were coexpressed, indicating Srk1 and Vpr are likely affecting the cell cycle G(2)/M transition through the same cellular pathway. Immunoprecipitation further shows that Vpr and Srk1 are part of the same protein complex. Consistent with our findings in fission yeast, depletion of the MK2 gene, a human homologue of Srk1, either by small interfering RNA or an MK2 inhibitor suppresses Vpr-induced cell cycle G(2) arrest in mammalian cells. Collectively, our data suggest that Vpr induces cell cycle G(2) arrest at least in part through a Srk1/MK2-mediated mechanism.

Project description:BackgroundNew approaches for the prevention of colon cancer perseveres an essential necessity. Though, resistance to existing chemo-preventive drugs is moderately predominant in colon carcinogenesis. Taxifolin (dihydroquercetin) is a flavononol, have shown virile biological activities against few cancers. The current study was designed to investigate and equate antitumor activity of Taxifolin (TAX) in colorectal cancer cell lines and in HCT116 xenograft model in a comprehensive approach.MethodsTwo human colorectal cancer cell lines HCT116 and HT29, were used. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide (MMT) protocol was performed to elucidate the impact of TAX and β- catenin inhibitor (FH535) on the viability of HCT116 and HT29 cell lines. Apoptosis /cell cycle assay was performed. Data interpretation was done with a FACScan (Becton Dickinson, NJ). About 1 × 104 cells per sample were harvested. Histograms of DNA were analyzed with ModiFitLT software (verity Software House, ME, USA). Western blotting and RT-PCR were performed for protein and gene expression respectively in in vitro and in vivo.ResultsWe found that TAX induced cytotoxicity in colorectal cells in a dose-dependent manner and time dependent approach. Further, our data validated that administration of TAX to human colorectal cancer HCT116 and HT29 cells resulted in cell growth arrest, variation in molecules controlling cell cycle operative in the G2 phase of the cell cycle and apoptosis in a concentration dependent approach. Further our results concluded that TAX administration decreases expression of β-catenin gene, AKT gene and Survivin gene and protein expression in in vitro and in vivo.ConclusionOur findings proposed that targeting β-catenin gene may encourage the alterations of cell cycle and cell cycle regulators. Wnt/β-catenin signaling pathway possibly takes part in the genesis and progression of colorectal cancer cells through regulating cell cycle and the expression of cell cycle regulators.

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Pharmacophore-guided discovery of CDC25 inhibitors causing cell cycle arrest and tumor regression.

Publications

Pharmacophore-guided discovery of CDC25 inhibitors causing cell cycle arrest and tumor regression.

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