-
Journal of Natural Products Aug 2019Aphidicolin, a potent DNA polymerase α inhibitor, has been explored in clinical trials for the treatment of cancer. So far, about 300 modified aphidicolins have been...
Aphidicolin, a potent DNA polymerase α inhibitor, has been explored in clinical trials for the treatment of cancer. So far, about 300 modified aphidicolins have been discovered. However, none have shown a stronger effect. Herein, we report 71 new (aphidicolins A1-A71, -) and eight known (-) aphidicolin congeners from MCCC 3A00494, a fungus isolated from the western Pacific Ocean (-5572 m). The structures of - were determined through extensive spectroscopic analysis, X-ray crystallography, chemical derivatization, modified Mosher's method, and the ECD exciton chirality method. Compounds - and - are novel 6/6/5/6/5 pentacyclic aphidicolins featuring tetrahydrofuran and dihydrofuran rings, respectively, while compounds - are rare noraphidicolins. Aphidicolin A8 () significantly induced apoptosis in T24 (IC = 2.5 μM) and HL-60 (IC = 6.1 μM) cancer cells by causing DNA damage. By docking its structure to the human DNA polymerase α binding pocket, was found to form tight intermolecular contacts, elaborating aphidicolin A8 as a potently cytotoxic lead compound.
Topics: Aphidicolin; Botrytis; Carbon-13 Magnetic Resonance Spectroscopy; Crystallography, X-Ray; Marine Biology; Molecular Structure; Proton Magnetic Resonance Spectroscopy; Spectrometry, Mass, Electrospray Ionization
PubMed: 31403790
DOI: 10.1021/acs.jnatprod.9b00705 -
Phytochemistry Dec 2023Natural products that inhibit cell cycle progression may have potential as anticancer agents. In this study, cell cycle inhibition of microbial culture extracts was...
Natural products that inhibit cell cycle progression may have potential as anticancer agents. In this study, cell cycle inhibition of microbial culture extracts was screened by fluorescent images using HeLa/Fucci2 cells. The culture extract of a fungus, Akanthomyces sp., inhibited the cell cycle progression at the S/G2/M phases, and bioassay-guided fractionation of the extract afforded three previously undescribed aphidicolin derivatives, namely akanthomins A-C, and an undescribed chromone glycoside, specifically 9-hydroxyeugenetin 9-O-β-d-(4-O-methyl)glucopyranoside, in addition to aphidicolin. The chemical structures of these compounds were elucidated by spectroscopic analysis and chemical derivatization. Using a flow cytometer, akanthomin A and aphidicolin were found to inhibit cell cycle progression at the S phase.
Topics: Aphidicolin; Cell Cycle; Cell Division; Hypocreales; Plant Extracts
PubMed: 37806468
DOI: 10.1016/j.phytochem.2023.113885 -
Cancer Letters Dec 1992Aphidicolin is a tetracyclic diterpene antibiotic which is known to inhibit the growth of eucaryotic cells by reversible binding to DNA polymerase alpha without...
Aphidicolin is a tetracyclic diterpene antibiotic which is known to inhibit the growth of eucaryotic cells by reversible binding to DNA polymerase alpha without significant effect on cell viability in most common human cell lines. We observed that aphidicolin at a concentration of 5 x 10(-7) M kills all cells of four human neuroblastoma cell lines. In contrast, viability of normal human embryonal cells and of human continuous cell lines including HeLa, H9, A549 and Caco-2 was influenced only moderately by aphidicolin. In addition, neuroblastoma cells were killed after treatment with 5 x 10(-7) M aphidicolin in cocultures with normal embryonal cells which continued to proliferate after removal of aphidicolin. These results show that aphidicolin provides an agent which selectively kills neuroblastoma cells in vitro.
Topics: Antineoplastic Agents; Aphidicolin; Cell Division; Cell Line; Cell Survival; DNA; Drug Screening Assays, Antitumor; Embryo, Mammalian; Humans; Neuroblastoma; Tumor Cells, Cultured
PubMed: 1483268
DOI: 10.1016/0304-3835(92)90144-k -
International Journal of Molecular... Feb 2022Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA...
Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated. The effect of incubation with aphidicolin was tested with CRISPR/Cas9 at different concentrations and different delivery methodologies. As a result, the reversible inhibition of DNA replication was observed, and it was concentration dependent. An optimal concentration of 0.5 μM was established and used for subsequent experiments. Following the use of this drug with CRISPR/Cas9, a halving of mosaicism was observed together with a detrimental effect on embryo development. In conclusion, the use of reversible inhibition of DNA replication offers a way to reduce mosaicism. Nevertheless, due to the reduction in embryo development, it would be necessary to reach a balance for its use to be feasible.
Topics: Animals; Animals, Genetically Modified; Aphidicolin; CRISPR-Cas Systems; Cell Nucleus; DNA Replication; Embryo, Mammalian; Embryonic Development; Eukaryota; Gene Editing; Mosaicism; Swine; Zygote
PubMed: 35216252
DOI: 10.3390/ijms23042135 -
The Journal of Biological Chemistry May 2001DNA replication origins are located at random with respect to DNA sequence in Xenopus early embryos and on DNA replicated in Xenopus egg extracts. We have recently shown...
DNA replication origins are located at random with respect to DNA sequence in Xenopus early embryos and on DNA replicated in Xenopus egg extracts. We have recently shown that origins fire throughout the S phase in Xenopus egg extracts. To study the temporal regulation of origin firing, we have analyzed origin activation in sperm nuclei treated with the DNA polymerase inhibitor aphidicolin. Sperm chromatin was incubated in Xenopus egg extracts in the presence of aphidicolin and transferred to a fresh extract, and digoxigenin-dUTP and biotin-dUTP were added at various times after aphidicolin release to selectively label early and late replicating DNA. Molecular combing analysis of single DNA fibers showed that only a fraction of potential origins were able to initiate in the presence of aphidicolin. After release from aphidicolin, the remaining origins fired asynchronously throughout the S phase. Therefore, initiation during the S phase depends on the normal progression of replication forks assembled at earlier activated origins. Caffeine, an inhibitor of the checkpoint kinases ATR and ATM, did not relieve the aphidicolin-induced block to origin firing. We conclude that a caffeine-insensitive intra-S phase checkpoint regulates origin activation when DNA synthesis is inhibited in Xenopus egg extracts.
Topics: Animals; Aphidicolin; Caffeine; Cell Nucleus; Chromatin; DNA Replication; Female; Kinetics; Male; Nucleic Acid Synthesis Inhibitors; Oocytes; Replication Origin; S Phase; Spermatozoa; Tissue Extracts; Xenopus laevis
PubMed: 11279043
DOI: 10.1074/jbc.M100271200 -
Nitric Oxide : Biology and Chemistry May 2021Aphidicolin represses DNA replication by inhibiting DNA polymerase α and δ, which leads to cell cycle arrest and cell damage. Nitric oxide (NO) generated by...
Aphidicolin represses DNA replication by inhibiting DNA polymerase α and δ, which leads to cell cycle arrest and cell damage. Nitric oxide (NO) generated by endothelial NO synthase (eNOS) plays an essential role in maintenance of endothelial integrity including endothelial cell (EC) survival. Previously, we reported that aphidicolin increases NO production in bovine aortic ECs (BAECs). However, the role of aphidicolin-induced NO on EC viability and its molecular mechanism remain to be elucidated. Treatment with 20 μM aphidicolin for 24 h reduced BAEC viability by ~40%, which was accompanied by increased NO production, phosphorylation of eNOS at Ser1179 (p-eNOS-Ser), and eNOS protein expression. The aphidicolin-increased eNOS expression and p-eNOS-Ser were not altered by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), a cell permeable and specific intracellular Ca chelator. Co-treatment with 2-phenyl-4, 4, 5, 5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), an NO scavenger, or Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME), a NOS inhibitor, exacerbated aphidicolin-stimulated BAEC death. Knockdown of eNOS gene expression using siRNA aggravated aphidicolin-induced BAEC death. However, exogenous NO donors including S-nitroso-l-glutathione (GSNO) or diethylenetriamine NONOate (DETA NO) had no effect on aphidicolin-decreased BAEC viability and aggravated BAEC viability at higher doses. Interestingly, aphidicolin accumulated eNOS protein in the active form, p-eNOS-Ser, in the nucleus. When cells were ectopically transfected with a wild-type (WT)-eNOS gene, aphidicolin induced significant localization of the protein product in the nucleus. Additionally, aphidicolin-elicited cell death was significantly reversed in WT-eNOS gene-transfected BAECs. Furthermore, overexpression of the eNOS gene containing nuclear localization signal (NLS) but not nuclear export signal (NES) significantly attenuated aphidicolin-induced BAEC death. When G2A-eNOS mutant lacking myristoylation at Gly2 was transfected, its intracellular distribution became diffuse and included the nucleus. Finally, expression of N-myristoyltransferase 2 (NMT2) but not NMT1 significantly decreased in aphidicolin-treated BAECs. Taken together, our results suggest that aphidicolin attenuates BAEC death in part by increasing nuclear eNOS localization and NO production.
Topics: Acyltransferases; Animals; Aorta; Aphidicolin; Cattle; Cell Death; Cell Nucleus; Cell Survival; Endothelial Cells; Nitric Oxide; Nitric Oxide Synthase Type III
PubMed: 33592314
DOI: 10.1016/j.niox.2021.02.001 -
Bioscience, Biotechnology, and... Jan 2018The secondary metabolite aphidicolin has previously been produced by Aspergillus oryzae after the heterologous expression of four biosynthetic enzymes isolated from...
The secondary metabolite aphidicolin has previously been produced by Aspergillus oryzae after the heterologous expression of four biosynthetic enzymes isolated from Phoma betae. In this study, we examined the subcellular localization of aphidicolin biosynthetic enzymes in A. oryzae. Fusion of green fluorescent protein to each enzyme showed that geranylgeranyl diphosphate synthase and terpene cyclase are localized to the cytoplasm and the two monooxygenases (PbP450-1 and PbP450-2) are localized to the endoplasmic reticulum (ER). Protease protection assays revealed that the catalytic domain of both PbP450s was cytoplasmic. Deletion of transmembrane domains from both PbP450s resulted in the loss of ER localization. Particularly, a PbP450-1 mutant lacking the transmembrane domain was localized to dot-like structures, but did not colocalize with any known organelle markers. Aphidicolin biosynthesis was nearly abrogated by deletion of the transmembrane domain from PbP450-1. These results suggest that ER localization of PbP450-1 is important for aphidicolin biosynthesis.
Topics: Aphidicolin; Aspergillus oryzae; Cytoplasm; Endoplasmic Reticulum; Farnesyltranstransferase; Polyisoprenyl Phosphates
PubMed: 29191129
DOI: 10.1080/09168451.2017.1399789 -
Nucleic Acids Research Sep 1991Highly compacted (40S) SV40 DNA replication intermediates formed in vivo during aphidicolin exposure and immediately broke down in two stages. In the rapid initial...
Highly compacted (40S) SV40 DNA replication intermediates formed in vivo during aphidicolin exposure and immediately broke down in two stages. In the rapid initial stage, single strand DNA breaks caused loss of superhelicity in the 40S replication intermediates. This DNA breakage was accompanied by the formation of strong, permanent protein-DNA crosslinks which reached a maximum as nicking of the aberrant DNA replication intermediates was completed. These protein-associated DNA strand breaks were not repaired. In the slower second stage of breakdown, the aberrant DNA replication intermediates remained nicked and strongly associated with protein as they underwent DNA replication fork breakage and recombinational changes to produce high molecular weight forms.
Topics: Aphidicolin; Chloroquine; DNA Replication; DNA, Viral; Electrophoresis, Gel, Two-Dimensional; Kinetics; Molecular Weight; Proteins; Simian virus 40; Virus Replication
PubMed: 1656388
DOI: 10.1093/nar/19.18.5065 -
European Journal of Cell Biology Nov 1996The unicellular red alga Cyanidioschyzon merolae possesses one chloroplast, one mitochondrion, and one cell nucleus. Since the division of these organelles and...
The unicellular red alga Cyanidioschyzon merolae possesses one chloroplast, one mitochondrion, and one cell nucleus. Since the division of these organelles and cytokinesis occur in a coordinated manner, mitosis and the organelle division cycles must be tightly coupled. We report here that aphidicolin, a specific inhibitor of DNA polymerase alpha, uncouples the chloroplast division cycle from the mitotic cycle. The effects of aphidicolin on C. merolae cells were examined by both epifluorescence and electron microscopy. When cells at the S phase in synchronous culture were treated with aphidicolin, mitosis and cytokinesis did not occur, while chloroplast division did. Moreover, both of the chloroplasts in these cells continued to divide and then generated four or more chloroplasts per cell. The inhibition of cell-nuclear DNA synthesis by aphidicolin was confirmed using microfluorometry. In addition, microfluorometry revealed that the total size and the amount of DNA in chloroplasts in aphidicolin-treated cells remained constant during uncoupled chloroplast division. As a result, the size and amount of DNA per chloroplast decreased stepwise during chloroplast division. Electron microscopic examination of aphidicolin-treated cells showed that the second division of chloroplasts uses a chloroplast-dividing ring similar to that in cells undergoing normal chloroplast division. These results suggest that chloroplast division by the dividing ring is free from a checkpoint control that inhibits the progression of mitosis and cytokinesis in the absence of the completion of cell-nuclear DNA synthesis, and also that chloroplasts lack a checkpoint control mechanism that inhibits its division without growth or DNA synthesis of itself.
Topics: Aphidicolin; Cell Division; Chloroplasts; DNA Replication; Microscopy, Electron; Mitosis; Rhodophyta
PubMed: 8929569
DOI: No ID Found -
Oncology Reports 1999Aphidicolin is a fungal derived tetracyclic diterpene antibiotic. It is selectively toxic for neuroblastoma (NB) cells in vitro but has no significant effects on the...
Aphidicolin is a fungal derived tetracyclic diterpene antibiotic. It is selectively toxic for neuroblastoma (NB) cells in vitro but has no significant effects on the viability of normal human cells and a variety of other tumor entities. We evaluated the antitumoral effects of the water soluble ester aphidicolin glycinate (AphiG) on established human NB xenografts from UKF-NB-3 cells in athymic (nude) mice. Furthermore, we explored the efficacy of direct intraneoplastic and systemic delivery of AphiG. Systemic administration of AphiG (60 mg/kg intraperitoneally, twice per day on 10 consecutive days) significantly suppressed tumor growth but was not able to induce any cures. In contrast, intratumoral AphiG injections (60 or 40 mg/kg/twice a day for 4 days) induced complete tumor regression. Two weeks after the end of treatment no tumor cells were microscopically detectable. Animals were free of tumor for more than 90 days. Histologic examination of inner organs and bone marrow did not reveal any apparent toxic effects of AphiG. These data strongly indicate that AphiG deserves further evaluation as a specific treatment for neuroblastoma.
Topics: Animals; Antineoplastic Agents; Aphidicolin; Cell Division; Female; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Neuroblastoma; Transplantation, Heterologous; Tumor Cells, Cultured
PubMed: 10203592
DOI: 10.3892/or.6.3.563