-
Biochimica Et Biophysica Acta Dec 1987The binding of rhizoxin, a potent inhibitor of mitosis and in vitro microtubule assembly, to porcine brain tubulin was studied. Tubulin possesses one binding site for...
The binding of rhizoxin, a potent inhibitor of mitosis and in vitro microtubule assembly, to porcine brain tubulin was studied. Tubulin possesses one binding site for rhizoxin per molecule with a dissociation constant (Kd) of 1.7.10(-7) M. Ansamitocin P-3, a homologue of maytansine, was a competitive inhibitor of rhizoxin binding, with an inhibition constant of 1.3.10(-7) M. Vinblastine also inhibited rhizoxin binding, but was not fully competitive, and the inhibition constant was 2.9.10(-6) M. In contrast, both rhizoxin and ansamitocin P-3 were potent inhibitors of vinblastine binding. Rhizoxin inhibited tau-promoted tubulin assembly, but it, differing from vinblastine, did not induce tubulin aggregation into spirals, even at a concentration as high as 2.10(-5) M. In addition, rhizoxin strongly inhibited vinblastine-induced tau-dependent tubulin aggregation. Rhizoxin binding to tubulin was completely independent from colchicine binding. These effects resemble those of maytansine. The results suggested that rhizoxin binds to the maytansine-binding site and that the binding sites of rhizoxin and vinblastine are not the same.
Topics: Animals; Binding Sites; Binding, Competitive; Colchicine; Kinetics; Lactones; Macrolides; Maytansine; Microscopy, Electron; Microtubule-Associated Proteins; Microtubules; Oxazines; Swine; Tubulin; Vinblastine; tau Proteins
PubMed: 3120782
DOI: 10.1016/0304-4165(87)90206-6 -
Molecular & General Genetics : MGG Dec 1989Rhizoxin and ansamitocin P-3 (a maytansinoid compound), potent inhibitors of mammalian brain tubulin assembly, inhibit growth of a variety of fungi including Aspergillus...
Rhizoxin and ansamitocin P-3 (a maytansinoid compound), potent inhibitors of mammalian brain tubulin assembly, inhibit growth of a variety of fungi including Aspergillus nidulans. Mutants of A. nidulans, benA10 which is a benomyl resistant beta-tubulin gene mutant and tubA1 which is a benomyl supersensitive alpha-tubulin gene mutant, were both sensitive to rhizoxin and ansamitocin P-3 to the same extent as wild-type strains. We isolated 18 rhizoxin resistant mutants of A. nidulans. All of these mutants were cross-resistant to ansamitocin P-3, but not to benzimidazole antimitotic drugs. These mutants mapped to two loci, rhiA and rhiB, and all of those with high resistance mapped to rhiA. The fact that the protein extracts of rhiA mutants lost rhizoxin binding affinity and that rhiA was closely linked to benA, the major beta-tubulin gene in A. nidulans, indicated that rhiA must be a structural gene for beta-tubulin and that rhiA mutants are a new class of beta-tubulin gene mutants. All of this suggested that, in A. nidulans, these antimitotic drugs bind to beta-tubulin, and that rhizoxin and ansamitocin P-3 share the same binding site but the site does not overlap with the benzimidazole binding site. Protein extracts from a rhiB mutant retained rhizoxin binding affinity, therefore this rhizoxin resistance mechanism should not be a tubulin mediated process.
Topics: Antifungal Agents; Aspergillus nidulans; Benomyl; Benzimidazoles; Cell Division; Dose-Response Relationship, Drug; Drug Resistance, Microbial; Genes, Fungal; Lactones; Macrolides; Mutation; Tubulin
PubMed: 2691873
DOI: 10.1007/BF00260855 -
Yakugaku Zasshi : Journal of the... Aug 1997The first total synthesis of the antitumor macrolide rhizoxin in a highly stereo-controlled manner was described. The construction of the key building fragments required... (Review)
Review
The first total synthesis of the antitumor macrolide rhizoxin in a highly stereo-controlled manner was described. The construction of the key building fragments required for the total synthesis of the antitumor macrolide rhizoxin, that arose from our retrosynthetic analysis of rhizoxin in an optically pure form by concise and efficient sequence analyses was described. Synthesis of the right-wing was started from the chiral half-ester generated by asymmetric hydrolysis of the corresponding meso-diester using pig liver esterase. The remaining chiral centers of the fragment was constracted by cyclic hydroboration. Synthesis of the left-wing was also accomplished starting from (S)-methyl 3-hydroxy-2-methylpropionate which had been prepared by enzyme mediated transformation. Coupling of the right-wing and the left-wing was accomplished by Julia coupling, and the macrocyclic lactone was constructed by the intramolecular Horner-Emmons reaction. The control of the stereoselective epoxidations was well achieved after the formation of an unsaturated 16-membered macrocyclic lactone. Chromophore-side-chain moiety was constructed at the final stage by the reaction of the phosphineoxide in 80% yield with high selectivity (E/Z = > 20/1). The present methodology will be useful for the synthesis of the homologues and man-made rhizoxin.
Topics: Anti-Bacterial Agents; Antibiotics, Antineoplastic; Epoxy Compounds; Esterases; Hydrolysis; Lactones; Macrolides; Molecular Conformation; Stereoisomerism
PubMed: 9306725
DOI: 10.1248/yakushi1947.117.8_486 -
Pharmacology & Therapeutics 1992This paper summarizes published data on the interactions of tubulin with antimitotic compounds that inhibit the binding of vinca alkaloids to the protein. These are all... (Review)
Review
This paper summarizes published data on the interactions of tubulin with antimitotic compounds that inhibit the binding of vinca alkaloids to the protein. These are all relatively complex natural products isolated from higher plants, fungi and marine invertebrate animals. These agents are maytansine, rhizoxin, phomopsin A, dolastatins 10 and 15 and halichondrin B and their congeners. Effects on tubulin polymerization, ligand binding interactions and structure-activity relationships are emphasized.
Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Depsipeptides; Ethers, Cyclic; Lactones; Macrolides; Maytansine; Mycotoxins; Oligopeptides; Tubulin; Vinca Alkaloids
PubMed: 1287674
DOI: 10.1016/0163-7258(92)90028-x -
Molecular Plant-microbe Interactions :... Mar 2015Two strains of Pseudomonas sp., Os17 and St29, were newly isolated from the rhizosphere of rice and potato, respectively, by screening for 2,4-diacetylphloroglucinol...
Two strains of Pseudomonas sp., Os17 and St29, were newly isolated from the rhizosphere of rice and potato, respectively, by screening for 2,4-diacetylphloroglucinol producers. These strains were found to be the same species and were the closest to but different from Pseudomonas protegens among the sequenced pseudomonads, based on 16S ribosomal RNA gene and whole-genome analyses. Strain Os17 was as effective a biocontrol agent as reported for P. protegens Cab57, whereas strain St29 was less effective. The whole-genome sequences of these strains were obtained: the genomes are organized into a single circular chromosome with 6,885,464 bp, 63.5% G+C content, and 6,195 coding sequences for strain Os17; and with 6,833,117 bp, 63.3% G+C content, and 6,217 coding sequences for strain St29. Comparative genome analysis of these strains revealed that the complete rhizoxin analog biosynthesis gene cluster (approximately 79 kb) found in the Os17 genome was absent from the St29 genome. In an rzxB mutant, which lacks the polyketide synthase essential for the production of rhizoxin analogs, the growth inhibition activity against fungal and oomycete pathogens and the plant protection efficacy were attenuated compared with those of wild-type Os17. These findings suggest that rhizoxin analogs are important biocontrol factors of this strain.
Topics: Anti-Bacterial Agents; Antibiosis; Bacillus; Bacterial Proteins; Base Sequence; Biological Control Agents; Cucumis sativus; Fusarium; Genes, Reporter; Genome, Bacterial; Macrolides; Molecular Sequence Data; Multigene Family; Phloroglucinol; Plant Diseases; Plant Roots; Polyketide Synthases; Pseudomonas; Pythium; Recombinant Fusion Proteins; Rhizosphere; Sequence Analysis, DNA; Species Specificity
PubMed: 25496595
DOI: 10.1094/MPMI-09-14-0294-FI -
MBio Oct 2022The fungus Rhizopus microsporus harbors a bacterial endosymbiont () for the production of the antimitotic toxin rhizoxin. Although rhizoxin is the causative agent of...
The fungus Rhizopus microsporus harbors a bacterial endosymbiont () for the production of the antimitotic toxin rhizoxin. Although rhizoxin is the causative agent of rice seedling blight, the toxinogenic bacterial-fungal alliance is, not restricted to the plant disease. It has been detected in numerous environmental isolates from geographically distinct sites covering all five continents, thus raising questions regarding the ecological role of rhizoxin beyond rice seedling blight. Here, we show that rhizoxin serves the fungal host in fending off protozoan and metazoan predators. Fluorescence microscopy and coculture experiments with the fungivorous amoeba revealed that ingestion of R. microsporus spores is toxic to . This amoebicidal effect is caused by the dominant bacterial rhizoxin congener rhizoxin S2, which is also lethal toward the model nematode Caenorhabditis elegans. By combining stereomicroscopy, automated image analysis, and quantification of nematode movement, we show that the fungivorous nematode Aphelenchus avenae actively feeds on R. microsporus that is lacking endosymbionts, whereas worms coincubated with symbiotic R. microsporus are significantly less lively. This study uncovers an unexpected ecological role of rhizoxin as shield against micropredators. This finding suggests that predators may function as an evolutionary driving force to maintain toxin-producing endosymbionts in nonpathogenic fungi. The soil community is a complex system characterized by predator-prey interactions. Fungi have developed effective strategies to defend themselves against predators. Understanding these strategies is of critical importance for ecology, medicine, and biotechnology. In this study, we shed light on the defense mechanisms of the phytopathogenic - symbiosis that has spread worldwide. We report an unexpected role of rhizoxin, a secondary metabolite produced by the bacterium residing within the hyphae of R. microsporus. We show that this bacterial secondary metabolite is utilized by the fungal host to successfully fend off fungivorous protozoan and metazoan predators and thus identified a fundamentally new function of this infamous cytotoxic compound. This endosymbiont-dependent predator defense illustrates an unusual strategy employed by fungi that has broader implications, since it may serve as a model for understanding how animal predation acts as an evolutionary driving force to maintain endosymbionts in nonpathogenic fungi.
Topics: Animals; Burkholderia; Antimitotic Agents; Macrolides; Symbiosis; Oryza; Toxins, Biological; Seedlings; Soil
PubMed: 36005392
DOI: 10.1128/mbio.01440-22 -
British Journal of Cancer Dec 1996Rhizoxin is a macrocyclic lactone compound that binds to tubulin and inhibits microtubule assembly. Rhizoxin demonstrated preclinical anti-tumour activity against a... (Clinical Trial)
Clinical Trial
Rhizoxin is a macrocyclic lactone compound that binds to tubulin and inhibits microtubule assembly. Rhizoxin demonstrated preclinical anti-tumour activity against a variety of human tumour cell lines and xenograft models. Phase I evaluation found a maximum tolerated rhizoxin dose of 2.6 mg m-2, with reversible, but dose-limiting, mucositis, leucopenia and diarrhoea. Clinical trials were then initiated by the EORTC ECSG in melanoma, breast, head and neck, and non-small-cell lung cancers with the recommended phase II rhizoxin dose of 2 mg m-2. Pharmacological studies were instituted with the phase II trials to complement the limited pharmacokinetic data available from the phase I trial. Blood samples were obtained from 69 of 103 eligible patients enrolled in phase II rhizoxin studies, and these were evaluable for pharmacokinetic analysis in 36 patients. Plasma rhizoxin concentrations were determined by high-performance liquid chromatography (HPLC), and post-distribution pharmacokinetic parameters were estimated by a one-compartment model. Rhizoxin was rapidly eliminated from plasma, with a median systemic clearance of 8.41 min-1 m-2 and an elimination half-life of 10.4 min. Rhizoxin area under the concentration-time curve (AUC) was higher in patients obtaining a partial response or stable disease than in those with progressive disease (median 314 vs 222 ng ml-1 min; P = 0.03). As predicted from previous studies, haematological and gastrointestinal toxicity was observed, but could not be shown to be related to rhizoxin AUC. This study demonstrated the rapid and variable elimination of rhizoxin from the systemic circulation. The presence of pharmacodynamic relationships and the low level of systemic toxicity suggest that future trials of rhizoxin with alternative dosage or treatment schedules are warranted.
Topics: Antibiotics, Antineoplastic; Area Under Curve; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Female; Head and Neck Neoplasms; Humans; Injections, Intravenous; Lactones; Lung Neoplasms; Macrolides; Male; Melanoma; Treatment Outcome
PubMed: 8980394
DOI: 10.1038/bjc.1996.657 -
Environmental Microbiology Oct 2016Pseudomonas protegens strain Pf-5 is a soil bacterium that was first described for its capacity to suppress plant diseases and has since been shown to be lethal to...
Pseudomonas protegens strain Pf-5 is a soil bacterium that was first described for its capacity to suppress plant diseases and has since been shown to be lethal to certain insects. Among these is the common fruit fly Drosophila melanogaster, a well-established model organism for studies evaluating the molecular and cellular basis of the immune response to bacterial challenge. Pf-5 produces the insect toxin FitD, but a ΔfitD mutant of Pf-5 retained full toxicity against D. melanogaster in a noninvasive feeding assay, indicating that FitD is not a major determinant of Pf-5's oral toxicity against this insect. Pf-5 also produces a broad spectrum of exoenzymes and natural products with antibiotic activity, whereas a mutant with a deletion in the global regulatory gene gacA produces none of these exoproducts and also lacks toxicity to D. melanogaster. In this study, we made use of a panel of Pf-5 mutants having single or multiple mutations in the biosynthetic gene clusters for seven natural products and two exoenzymes that are produced by the bacterium under the control of gacA. Our results demonstrate that the production of rhizoxin analogs, orfamide A, and chitinase are required for full oral toxicity of Pf-5 against D. melanogaster, with rhizoxins being the primary determinant.
Topics: Animals; Bacterial Proteins; Chitinases; Drosophila melanogaster; Genes, Regulator; Lipopeptides; Mutation; Peptides, Cyclic; Pseudomonas; Virulence
PubMed: 27130686
DOI: 10.1111/1462-2920.13369 -
Journal of Cancer Research and Clinical... 1997Rhizoxin is an antineoplastic drug that inhibits tubulin polymerization. In this study, we demonstrated that rhizoxin was approximately twice as active in vitro against...
Rhizoxin is an antineoplastic drug that inhibits tubulin polymerization. In this study, we demonstrated that rhizoxin was approximately twice as active in vitro against a human small-cell lung cancer cell line with non-P-glycoprotein-mediated resistance to vindesine, H69/VDS, as against its parental line, H69. Tubulin polymerization in H69/VDS, demonstrated by Western blot analysis, was inhibited markedly by rhizoxin compared with that in H69, in a concentration-dependent manner. A drug-accumulation study showed that the intracellular rhizoxin level in H69/VDS was 15% lower than that in H69, whereas efflux from H69/VDS was enhanced slightly. These results indicate that enhanced inhibition of tubulin polymerization rather than increased intracellular drug concentration accounted for the higher sensitivity of H69/VDS to rhizoxin. In an experiment using mice with severe combined immunodeficiency and inoculated subcutaneously with H69/VDS, in vivo tumor growth was reduced markedly by three intermittent intraperitoneal doses of rhizoxin compared with that in mice inoculated with H69. Three weeks after the last rhizoxin dose, the relative treated/untreated tumor volumes were 0.29 for H69, but only 0.06 for H69/VDS, indicating that H69/VDS regrowth was minimal even after a 3-week treatment-free period. In conclusion, rhizoxin conquers vindesine resistance of a human small-cell lung cancer cell line in vitro and in vivo.
Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Agents, Phytogenic; Drug Resistance; Female; Humans; Lactones; Macrolides; Mice; Mice, SCID; Tubulin; Tumor Cells, Cultured; Vindesine
PubMed: 9177491
DOI: 10.1007/BF01240315 -
Journal of the American Chemical Society Sep 2006The potent antimitotic polyketide macrolide rhizoxin, the causal agent of rice seedling blight, is not produced by the fungus Rhizopus microsporus, as has been believed...
The potent antimitotic polyketide macrolide rhizoxin, the causal agent of rice seedling blight, is not produced by the fungus Rhizopus microsporus, as has been believed for over two decades, but by endosymbiotic bacteria that reside within the fungal mycelium. Here we report the successful isolation and large-scale fermentation of the bacterial endosymbiont ("Burkholderia rhizoxina") in pure culture, which resulted in a significantly elevated (10x higher) production of antimitotics. In addition to several known rhizoxin derivatives, numerous novel natural and semisynthetic variants were isolated, and their structures were fully elucidated. Cell-based assays as well as tubulin binding experiments revealed that methylated seco-rhizoxin derivatives are 1000-10000 times more active than rhizoxin and thus rank among the most potent antiproliferative agents known to date. Furthermore, more stable didesepoxy rhizoxin analogues were obtained by efficiently inhibiting a putative P-450 monooxygenase involved in macrolide tailoring.
Topics: Animals; Antimitotic Agents; Burkholderia; Cell Proliferation; Cell Survival; Chromatography, High Pressure Liquid; Fibroblasts; HeLa Cells; Humans; K562 Cells; Macrolides; Mice; Microtubule Proteins; Molecular Structure; Oryza; Rhizopus; Structure-Activity Relationship; Symbiosis
PubMed: 16939276
DOI: 10.1021/ja062953o