Role of calmodulin inhibition in the mode of action of ophiobolin a

Calmodulin has been isolated from the root of Zea mays. It activates the bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase and has electrophoretic mobility very similar to that of bovine brain calmodulin. Ophiobolin A, a fungal toxin, interacts with the maize calmodulin. The interaction is not reversed by dilution or denaturation in SDS and results in the loss of ability of the calmodulin to activate the phosphodiesterase. The inhibition is much faster in the presence than in the absence of Ca²⁺. The electrophoretic mobility of ophiobolin A-treated calmodulin is less than that of untreated calmodulin. Several similarities are found between the inhibition of maize calmodulin by ophiobolin A in vitro and the effects of ophiobolin A on excised roots. Both are irreversible and time-dependent. The concentration of ophiobolin A for half-maximal inhibition of calmodulin in the phosphodiesterase assay is similar to that for phytotoxicity. In both cases ophiobolin A derivatives behave similarly, i.e. 18-bromo-19-methoxyophiobolin A is as potent as ophiobolin A, while 3-anhydro-ophiobolin A and 6-epi-ophiobolin A are less potent. A smaller amount of active calmodulin was measured in the extract from ophiobolin A-treated roots than in those from untreated roots. The present study suggests that calmodulin is a target molecule in the root for the toxicity of ophiobolin A.     

Identification of the Binding and Inhibition Sites in the Calmodulin Molecule for Ophiobolin A by Site-Directed Mutagenesis

Ophiobolin A, a fungal toxin that affects maize and rice, has previously been shown to inhibit calmodulin by reacting with the lysine (Lys) residues in the calmodulin. In the present study we mutated Lys-75, Lys-77, and Lys-148 in the calmodulin molecule by site-directed mutagenesis, either by deleting them or by changing them to glutamine or arginine. We found that each of these three Lys residues could bind one molecule of ophiobolin A. Normally, only Lys-75 and Lys-148 bind ophiobolin A. Lys-77 seemed to be blocked by the binding of ophiobolin A to Lys-75. Lys-75 is the primary binding site and is responsible for all of the inhibition of ophiobolin A. When Lys-75 was removed, Lys-77 could then react with ophiobolin A to produce inhibition. Lys-148 was shown to be a binding site but not an inhibition site. The Lys-75 mutants were partially resistant to ophiobolin A. When both Lys 75 and Lys-77 or all three Lys residues were mutated, the resulting calmodulins were very resistant to ophiobolin A. Furthermore, Lys residues added in positions 86 and/or 143 (which are highly conserved in plant calmodulins) did not react with ophiobolin A. None of the mutations seemed to affect the properties of calmodulin. These results show that ophiobolin A reacts quite specifically with calmodulin.     

Initial kinetics of the inactivation of calmodulin by the fungal toxin ophiobolin A

Ophiobolin A, a fungal toxin that affects rice and maize, inhibits calmodulin by reacting with the lysine residues in calmodulin. Previous studies have shown that lysines 75, 77 and 148 in the calmodulin molecule were the binding sites for ophiobolin A, and that lysine 75 was the primary inhibitory site. In this study, we used kinetic analysis and mutated calmodulins to further characterize the inhibition process. The inhibition of bovine-brain calmodulin by ophiobolin A in the presence of excess ophiobolin A occurred rapidly and followed pseudo-first-order kinetics with a second-order rate constant of 3470 M(-1) min(-1). The kinetics data indicated that the binding of a single ophiobolin A molecule was enough to inactivate a calmodulin molecule. Mutant calmodulins in which two of the three aforementioned binding sites for ophiobolin A had been removed by site-directed mutagenesis were examined for the role of each of the three lysines in the inhibition. It was found that when lysine 75 or 77 in the mutant calmodulin was reacted with ophiobolin A, the resulting calmodulin became a poor activator of phosphodiestease. These results provide further evidence that lysine 75 in calmodulin is the primary inhibitory site for ophiobolin A.     

Characterization of the interaction of ophiobolin A and calmodulin

1. The fungal toxin ophiobolin A reacts with the epsilon-amino group of lysine to give a conjugated enamine produce with lambda max at 272 nm and a molar extinction of 19,200 per M/cm. 2. Bovine brain calmodulin reacts with ophiobolin A to give a lambda max at 272 nm. 3. One mol of calmodulin reacts with two moles of ophiobolin A. Reaction of 1 mol of ophiobolin A inactivates 1 mol of calmodulin. 4. Ophiobolin A-treated calmodulin is resistant to tryptic cleavage at lysine 77. 5. Ophiobolin A also inhibits Dictyostelium calmodulin which has glutamine instead of lysine at residue 77.     

Effects of ophiobolin a on ion leakage and hexose uptake by maize roots

Ophiobolin A, a sesterterpene metabolite of Helminthosporium maydis, Nisikado and Miyake, stimulates net leakage of electrolytes and glucose from maize (Zea mays L.) seedling roots. Treatment of the roots with ophiobolin A at a concentration of 10 mug/ml (25 mum) inhibits uptake of 10 mm 2-deoxyglucose by 50% and of 0.5 mm 2-deoxyglucose by 85%. Compartmental analysis of the efflux of 3-O-methylglucose failed to show a similar effect of ophiobolin A on the rate of efflux of hexose. The inhibition of uptake is not reversible by washing. There is no difference in the effects on roots from cytoplasmic male sterile or normal cytoplasm plants, and exposure of carrot (Daucus carota L.) root discs to ophiobolin A also causes inhibition of 2-deoxyglucose uptake by this tissue.     

The novel agent ophiobolin O induces apoptosis and cell cycle arrest of MCF-7 cells through activation of MAPK signaling pathways

Ophiobolin O is a natural compound that has been isolated from Aspergillus ustus 094102. This is the first study to demonstrate the anti-proliferative effect of ophiobolin O in human breast cancer MCF-7 cells. The results of present study show that ophiobolin O induced cycle G(0)/G(1) phase arrest in MCF-7 cells using a cell cycle analysis. In addition, we demonstrated that ophiobolin O reduced the viability of human breast cancer MCF-7 cells in a time- and dose-dependent manner and efficiently induced apoptosis in MCF-7 cells using the Annexin V/PI binding assay. Ophiobolin O also caused the activation of JNK (c-Jun NH(2)-terminal kinase), p38 MAPK (mitogen activated protein kinase) and ERK (extracellular signal-regulated kinase) as well as the degradation of Bcl-2 phosphorylation (Ser70). Bax protein expression was not changed in ophiobolin O-treated cells. Taken together, ophiobolin O may be considered as a novel therapeutic agent in breast cancer.     

Screening and identification of insertion mutants from Bipolaris eleusines by mutagenesis based on restriction enzyme-mediated integration

Ophiobolin A is sesterterpenoid-type phytotoxin and may be an important candidate for development of new crop protection and pharmaceutical products. The restriction enzyme-mediated integration (REMI) method was used to introduce the plasmid pSH75 into the ophiobolin A-producing filamentous fungus Bipolaris eleusines. A total of 323 stable transformants were obtained, all of which were capable of growing on potato-dextrose agar medium containing 200?μg?mL(-1) hygromycin B. The transformation frequency was about 4-5 transformants?μg(-1) plasmid DNA. An ophibolin A-deficient transformant (B014) was assessed and the presence of the hph gene in this transformant was confirmed by PCR. The cell-free cultural filtrates of this transformant showed significantly less inhibition on mycelial growth of the fungal pathogen Rhizoctoni solani but little effect on barnyard grass as opposed to that of the wild-type B.?eleusines. There was no detectable amount of ophiobolin A in B014 samples measured with HPLC. This research suggests REMI as a potential approach for improving the production of ophiobolin A by B.?eleusines via genetic engineering to upregulate certain genes responsible for desired biosynthetic pathways.     

The biology of ophiobolins

This review article aims at summarizing the research findings on the biological aspects of ophiobolins, phytotoxins produced by the pathogenic fungi Bipolaris species, which usually infect rice, maize and sorghum. The topics covered include the organisms that produce the various ophiobolins, the structural variations of ophiobolins, the biological actions of ophiobolins in plants, animals and microorganisms, and the mode of action and the possible use of ophiobolin A as a calmodulin antagonist.     

Ophiobolin E and 8-epi-ophiobolin J produced by Drechslera gigantea, a potential mycoherbicide of weedy grasses

Drechslera gigantea, a fungal pathogen isolated from large crabgrass (Digitaria sanguinalis) and proposed as a potential mycoherbicide of grass weeds, produces phytotoxic metabolites in liquid and solid cultures. Ophiobolin A and three minor ophiobolins i.e., 6-epi-ophiobolin A, 3-anhydro-6-epi-ophiobolin A and ophiobolin I were obtained from the liquid culture broths. Interestingly and unexpectedly, ophiobolins also appeared in cultures of this fungus and they were isolated together with the known ophiobolins B and J, and designed as ophiobolin E and 8-epi-ophiobolin J. They were characterized using essentially spectroscopic methods. It is noteworthy that D. gigantea produces such a plethora of bioactive organic substances. Some structure-activity relationship results are also discussed in this report.     

A Survey of Phytotoxic Microbial and Plant Metabolites as Potential Natural Products for Pest Management

Phytotoxic microbial metabolites produced by certain phytopathogenic fungi and bacteria, and a group of phytotoxic plant metabolites including Amaryllidacea alkaloids and some derivatives of these compounds were evaluated for algicide, bactericide, insecticide, fungicide, and herbicide activities in order to discover natural compounds for potential use in the management and control of several important agricultural and household structural pests. Among the various compounds evaluated: i) ophiobolin A was found to be the most promising for potential use as a selective algicide; ii) ungeremine was discovered to be bactericidal against certain species of fish pathogenic bacteria; iii) cycasin caused significant mortality in termites; iv) cavoxin, ophiobolin A, and sphaeropsidin A were most active towards species of plant pathogenic fungi; and v) lycorine and some of its analogues (1‐O‐acetyllycorine and lycorine chlorohydrate) were highly phytotoxic in the herbicide bioassay. Our results further demonstrated that plants and microbes can provide a diverse and natural source of compounds with potential use as pesticides.     

Effect of the sesterterpene-type metabolites, ophiobolins A and B, on zygomycetes fungi

Ophiobolins are sesterterpene-type phytotoxins produced by fungi belonging mainly to the genus Bipolaris. In this study, the antifungal effect of ophiobolins A and B on different zygomycetes has been examined. Depending on the zygomycete tested, MIC values of 3.175-50 μg mL(-1) were found for ophiobolin A and 25-50 μg mL(-1) for ophiobolin B. Ophiobolin A inhibited sporangiospore germination of Mucor circinelloides and caused morphological changes; the fungus formed degenerated, thick or swollen cells with septa. Cytoplasm effusions from the damaged cells were also observed. Fluorescence microscopy after annexin and propidium iodide staining of the treated cells suggested that the drug induced an apoptosis-like cell death process in the fungus.     

Isolation from sugarcane cultures of variants resistant to antimetabolites

Variants resistant to antimetabolites are useful for investigating metabolic regulation and biochemical genetics in organisms. In this study, suspensions of mutagenized sugarcane ( Saccharum sp.) cells, originating from a stalk parenchyma explain of the Hawaiian variety 50–7209, were used to investigate the feasibility of isolating variants resistant to l -canavanine, glyphosate [N-(phosphonomethyl)glycine], ophiobolin A and orthovanadate. Rigorous retesting of clones which grew on selection media led to the identification of three cell lines, two of which were resistant to glyphosate and one to orthovanadate. No variants were isolated which showed a persistent resistance either to l -canavanine or ophiobolin A.
The results demonstrate that resistant variants do occur, or can be induced, in sugarcane cell suspensions and that they can be rescued and cultivated.     

Unique antimicrobial spectrum of ophiobolin K produced by Aspergillus ustus

A co-cultivation study of two fungal strains showed that Aspergillus ustus could inhibit Aspergillus repens growth. The bioactive compound responsible for the observed activity was purified and identified as a sesterterpene, ophiobolin K. Ophiobolin K exhibited marked inhibition against both fungi and bacteria, especially A. repens, A. glaucus and gram-positive bacteria including Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus.     

3-Anhydro-6-hydroxy-ophiobolin A,a new sesterterpene inhibiting the growth of methicillin-resistant Staphylococcus aureus and inducing the cell death by apoptosis on K562, from the phytopathogenic fungus Bipolaris oryzae

A new ophiobolin derivative, 3-anhydro-6-hydroxy-ophiobolin A (1), as well as two known ophiobolin derivatives 3-anhydro-ophiobolin A (2) and 3-anhydro-6-epi-ophiobolin A (3) were isolated from the PDB culture of a phytopathogenic fungus Bipolaris oryzae. The structure of 1 was elucidated through 2D NMR and other spectroscopic techniques. Compound 1 exhibited strong antimicrobial activity against Bacille Calmette–Guerin, Bacillus subtilis, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus with MIC value of 12.5 μg/mL, and potent antiproliferative activity against cell lines HepG2 and K562 with IC₅₀ of 6.49 μM and 4.06 μM, respectively. Further studies on the cytotoxicity of compound 1 against K562 cells demonstrated that it induced apoptosis, observed by flow cytometric method. Preliminary structure–activity relationships of these ophiobolins and the mechanism of apoptosis induced by 1 were analyzed.     

Calmodulin signals capacitation and triggers the agonist-induced acrosome reaction in mouse spermatozoa

Capacitated acrosome-intact spermatozoa interact with specific sugar residues on neoglycoproteins (ngps) or solubilized zona pellucida (ZP), the egg's extracellular glycocalyx, prior to the initiation of a signal transduction cascade that results in the fenestration and fusion of the sperm plasma membrane and the outer acrosomal membrane at multiple sites and exocytosis of acrosomal contents (i.e., induction of the acrosome reaction (AR)). The AR releases acrosomal contents at the site of sperm-zona binding and is thought to be a prerequisite event that allows spermatozoa to penetrate the ZP and fertilize the egg. Since Ca(2+)/calmodulin (CaM) plays a significant role in several cell signaling pathways and membrane fusion events, we have used a pharmacological approach to examine the role of CaM, a calcium-binding protein, in sperm capacitation and agonist-induced AR. Inclusion of CaM antagonists (calmodulin binding domain, calmidazolium, compound 48/80, ophiobolin A, W5, W7, and W13), either in in vitro capacitation medium or after sperm capacitation blocked the npg-/ZP-induced AR. Purified CaM largely reversed the AR blocking effects of antagonists during capacitation. Our results demonstrate that CaM plays an important role in priming (i.e., capacitation) of mouse spermatozoa as well as in the agonist-induced AR. These data allow us to propose that CaM regulates these events by modulating sperm membrane component(s).     

Calmodulin and lipid binding to synaptobrevin regulates calcium-dependent exocytosis

Neurotransmitter release involves the assembly of a heterotrimeric SNARE complex composed of the vesicle protein synaptobrevin (VAMP 2) and two plasma membrane partners, syntaxin 1 and SNAP-25. Calcium influx is thought to control this process via Ca(2+)-binding proteins that associate with components of the SNARE complex. Ca(2+)/calmodulin or phospholipids bind in a mutually exclusive fashion to a C-terminal domain of VAMP (VAMP(77-90)), and residues involved were identified by plasmon resonance spectroscopy. Microinjection of wild-type VAMP(77-90), but not mutant peptides, inhibited catecholamine release from chromaffin cells monitored by carbon fibre amperometry. Pre-incubation of PC12 pheochromocytoma cells with the irreversible calmodulin antagonist ophiobolin A inhibited Ca(2+)-dependent human growth hormone release in a permeabilized cell assay. Treatment of permeabilized cells with tetanus toxin light chain (TeNT) also suppressed secretion. In the presence of TeNT, exocytosis was restored by transfection of TeNT-resistant (Q(76)V, F(77)W) VAMP, but additional targeted mutations in VAMP(77-90) abolished its ability to rescue release. The calmodulin- and phospholipid-binding domain of VAMP 2 is thus required for Ca(2+)-dependent exocytosis, possibly to regulate SNARE complex assembly.     

Ophiobolin A induces paraptosis-like cell death in human glioblastoma cells by decreasing BKCa channel activity

Glioblastoma multiforme (GBM) is the most lethal and common malignant human brain tumor. The intrinsic resistance of highly invasive GBM cells to radiation- and chemotherapy-induced apoptosis accounts for the generally dismal treatment outcomes. This study investigated ophiobolin A (OP-A), a fungal metabolite from Bipolaris species, for its promising anticancer activity against human GBM cells exhibiting varying degrees of resistance to proapoptotic stimuli. We found that OP-A induced marked changes in the dynamic organization of the F-actin cytoskeleton, and inhibited the proliferation and migration of GBM cells, likely by inhibiting big conductance Ca²⁺-activated K⁺ channel (BKCa) channel activity. Moreover, our results indicated that OP-A induced paraptosis-like cell death in GBM cells, which correlated with the vacuolization, possibly brought about by the swelling and fusion of mitochondria and/or the endoplasmic reticulum (ER). In addition, the OP-A-induced cell death did not involve the activation of caspases. We also showed that the expression of BKCa channels colocalized with these two organelles (mitochondria and ER) was affected in this programmed cell death pathway. Thus, this study reveals a novel mechanism of action associated with the anticancer effects of OP-A, which involves the induction of paraptosis through the disruption of internal potassium ion homeostasis. Our findings offer a promising therapeutic strategy to overcome the intrinsic resistance of GBM cells to proapoptotic stimuli.     

Activation of porcine oocytes using cyclopiazonic acid, an inhibitor of calcium-dependent ATPases

Cyclopiazonic acid (CPA), a potent inhibitor of endogenous calcium-dependent ATPases, is able to induce parthenogenetic activation in pig oocytes matured in vitro. Sixty-four percent of matured pig eggs cultured with 100 nM CPA for 4 hr were activated. A similar activation rate was observed in oocytes treated with thapsigargin, another inhibitor of calcium-dependent ATPases. The parthenogenetic development of CPA-activated eggs did not proceed beyond the 8-cell stage. The blockage of calcium channels by verapamil only slightly decreased the proportion of CPA-activated pig oocytes. This indicates that the release of calcium from intracellular stores is sufficient for oocyte activation and calcium influx from extracellular sources has no significant role. The significant decrease in CPA-activated oocytes (100 nM of CPA for 4 hr) after a microinjection of heparin indicated that the mobilization of intracellular calcium stores is mediated through inositol trisphosphate receptors. On the other hand, the only slightly depressed activation rate in oocytes microinjected with ruthenium red and procaine indicates that CPA mobilizes a much smaller amount of calcium through the ryanodine receptors. The marked inhibitory effect of ophiobolin A and W7 on the activation of CPA-treated pig oocytes suggests that the calcium signal, as the second messenger, acts downstream through calmodulin. J. Exp. Zool. 287:304-315, 2000.     

Effects of ophiobolin B on cell enlargement and H+/K+ exchange in maize coleoptile tissues

Ophiobolin B (OPH B), a sesterpene metabolite of Helminthosporium oryzae, inhibits proton extrusion from maize coleoptiles. Moreover OPH B counteracts the biological activity of fusicoccin (FC), another terpenoid toxin produced by Fusicoccum amygdali having a similar basic chemical structure: OPH B inhibits FC-promoted proton extrusion, potassium uptake and cell enlargement.The findings suggest that the effect of OPH B in stimulating electrolites, glucose and aminoacid leakage, reported in a previous paper, can be explained by the capacity of the toxin to inhibit proton extrusion.Abbreviations FC fusicoccin - OPH B ophiobolin B     

The phytotoxic ophiobolins produced by Drechslera oryzae,their structures and biological activity on rice

Drechslera oryzae causes brown leaf spot disease of rice (Oryza sativa L.). This fungal pathogen produces a series of ophiobolins which cause some of the characteristic symptoms of the disease when applied to rice leaves at 10⁻⁴ and 10⁻⁵ M. Although ophiobolin A & B were described from D. oryzae nearly 20 years ago, several novel ophiobolins recently discovered in D. maydis and D. sorghicola, are also produced by D. oryzae. The production of ophiobolins is controlled, in part by the prior conditions of culturing the fungus. Various isolates of D. oryzae differ in their ability to produce ophiobolins. Although unstable to heating, the ophiobolins can be incorporated into an agar-based medium and a potential exists for them to be used in screening plant tissues for toxin insensitivity.