WF-1360F

A tritiated derivative of the sponge-derived natural product spongistatin 1 was prepared, and its interactions with tubulin were examined. [3H]Spongistatin 1 was found to bind rapidly to tubulin at a single site (the low specific activity of the [3H]spongistatin 1, 0.75 Ci/mmol, prevented our defining an association rate), and the inability of spongistatin 1 to cause an aberrant assembly reaction was confirmed. Spongistatin 1 bound to tubulin very tightly, and we could detect no significant dissociation reaction from tubulin. The tubulin-[3H]spongistatin 1 complex did dissociate in 8 M urea, so there was no evidence for covalent bond formation. Apparent KD values were obtained by Scatchard analysis of binding data and by Hummel-Dreyer chromatography (3.5 and 1.1 μM, respectively). The effects of a large cohort of vinca domain drugs on the binding of [3H]spongistatin 1 to tubulin were evaluated. Compounds that did not cause aberrant assembly reactions (halichondrin B, eribulin, maytansine, and rhizoxin) caused little inhibition of [3H]spongistatin 1 binding. Little inhibition also occurred with the peptides dolastatin 15, its active pentapeptide derivative, vitilevuamide, or diazonamide A, nor with the vinca alkaloid vinblastine. Strong inhibition was observed with dolastatin 10, hemiasterlin, and cryptophycin 1, all of which cause aberrant assembly reactions that might actually mask the spongistatin 1 binding site. Spongistatin 5 was found to be a competitive inhibitor of [3H]spongistatin 1 binding, with an apparent Ki of 2.2 μM. We propose that the strong picomolar cytotoxicity of spongistatin 1 probably derives from its extremely tight binding to tubulin.

The fungus Rhizopus microsporus harbors a bacterial endosymbiont (Mycetohabitans rhizoxinica) 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 Protostelium aurantium revealed that ingestion of R. microsporus spores is toxic to P. aurantium. 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. IMPORTANCE 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 Rhizopus-Mycetohabitans symbiosis that has spread worldwide. We report an unexpected role of rhizoxin, a secondary metabolite produced by the bacterium M. rhizoxinica 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.

The role of viral infection in developing cancer was determined in the start of 20th century. Until now, 8 different virus-associated cancers have been discovered and most of them progressed in immunosuppressed individuals. The aim of the present study is to look into the benefits of natural products in treating virally infected cancers. The study focuses on bioactive compounds derived from natural sources. Numerous pharmaceutical agents have been identified from plants (vincristine, vinblastine, stilbenes, combretastatin, and silymarin), marine organisms (bryostatins, cephalostatin, ecteinascidins, didemnin, and dolastatin), insects (cantharidin, mastoparan, parectadial, and cecropins), and microorganisms (vancomycin, rhizoxin, ansamitocins, mitomycin, and rapamycin). Beside these, various compounds have been observed from fruits and vegetables which can be utilized in anticancer therapy. These include curcumin in turmeric, resveratrol in red grapes, S-allyl cysteine in allium, allicin in garlic, catechins in green tea, and β-carotene in carrots. The present study addresses various types of virally infected cancers, their mechanism of action, and the role of different cell surface molecules elicited during viral binding and entry into the target cell along with the anticancer drugs derived from natural products by targeting screening of bioactive compounds from natural sources.