Cyclopiazonic acid

Detection of cyclopiazonic acid (CPA) in maize by immunoassay.[Pubmed:28378232]

Mycotoxin Res. 2017 May;33(2):157-165.

Cyclopiazonic acid (alpha-CPA) is a tremorgenic mycotoxin that is commonly produced by certain species of the aspergilli, in particular Aspergillus flavus, which is more widely known for production of the aflatoxins. Despite the fact that alpha-CPA may co-occur with aflatoxins, immunoassay-based methods for monitoring for CPA have not been widely developed. We report the development and evaluation of several monoclonal antibodies (mAbs) for alpha-CPA. Two mAbs in particular were very sensitive, with IC50s of 1.1 and 1 ng/mL (clones 1418 and 1231, respectively). Tolerances to aqueous methanol or acetonitrile were good, which permitted the development of an antigen-immobilized competitive enzyme-linked immunosorbent assay (CI-ELISA) for detection of CPA in maize. Spiked or naturally contaminated maize, extracted with aqueous methanol, was diluted with buffer for analysis. The working range for the assay (IC20 to IC80) was from 5 to 28 mug/kg. Recoveries from maize spiked over the range from 2 to 50 mug/kg averaged 88.6 +/- 12.6%. Twenty-eight samples of maize were tested by both the CI-ELISA and a liquid chromatography-fluorescence (LC-FLD) method. For the five samples above the limits of quantitation of both methods, CI-ELISA tended to overestimate CPA content, a difference that we speculate may be due to related metabolites or perhaps "masked" derivatives of CPA. The antibodies developed and the resulting CI-ELISA will be useful tools for further evaluation of the prevalence of this mycotoxin in maize.

Determination of cyclopiazonic acid in white mould cheese by liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) using a novel internal standard.[Pubmed:27283720]

Food Chem. 2016 Nov 15;211:978-82.

For the determination of Cyclopiazonic acid (CPA) in food and feed samples a simple and accurate LC-MS/MS method, which does not require extensive sample clean-up steps, was developed and validated. A fully carbon-13-labelled internal standard was used to compensate for matrix effects. Briefly, the samples were extracted with 1% formic acid in acetonitrile and directly analysed with HPLC-MS/MS. The following MS/MS transitions were used: m/z 337/196 and 337/182 for Cyclopiazonic acid; m/z 357/210 and 357/191 for (13)C20-Cyclopiazonic acid. Applying this optimised method, LODs down to 0.2mugkg(-1) and LOQs down to 0.5mugkg(-1) were determined in the validated matrices. The focus of this study was testing different types of white mould cheese but other complex samples could also be analyzed successfully with this method. It was interesting to find out, that in some commercially available white mould cheese, high concentrations of CPA (up to 3700mugkg(-1)) could be found.

Unravelling the Diversity of the Cyclopiazonic Acid Family of Mycotoxins in Aspergillus flavus by UHPLC Triple-TOF HRMS.[Pubmed:28098779]

Toxins (Basel). 2017 Jan 13;9(1). pii: toxins9010035.

Cyclopiazonic acid (alpha-Cyclopiazonic acid, alpha-CPA) is an indole-hydrindane-tetramic acid neurotoxin produced by various fungal species, including the notorious food and feed contaminant Aspergillus flavus. Despite its discovery in A. flavus cultures approximately 40 years ago, its contribution to the A. flavus mycotoxin burden is consistently minimized by our focus on the more potent carcinogenic aflatoxins also produced by this fungus. Here, we report the screening and identification of several CPA-type alkaloids not previously found in A. flavus cultures. Our identifications of these CPA-type alkaloids are based on a dereplication strategy involving accurate mass high resolution mass spectrometry data and a careful study of the alpha-CPA fragmentation pattern. In total, 22 CPA-type alkaloids were identified in extracts from the A. flavus strains examined. Of these metabolites, 13 have been previously reported in other fungi, though this is the first report of their existence in A. flavus. Two of our metabolite discoveries, 11,12-dehydro alpha-CPA and 3-hydroxy-2-oxo CPA, have never been reported for any organism. The conspicuous presence of CPA and its numerous derivatives in A. flavus cultures raises concerns about the long-term and cumulative toxicological effects of these fungal secondary metabolites and their contributions to the entire A. flavus mycotoxin problem.

Cyclopiazonic acid, an inhibitor of calcium-dependent ATPases with antiviral activity against human respiratory syncytial virus.[Pubmed:27210812]

Antiviral Res. 2016 Aug;132:38-45.

Human respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in infants and young children worldwide, yet no vaccine or effective antiviral treatment is available. To search for new anti-RSV agents, we developed a cell-based assay that measures inhibition of RSV-induced cytopathic effect (CPE) and identified Cyclopiazonic acid (CPA), an intracellular calcium ATPase inhibitor as a RSV inhibitor (EC50 values 4.13 muM) by screening of natural product library. CPA inhibited the replication of RSV strains belonging to both A and B subgroups and human parainfluenza virus type 3, but not Enterovirus 71. Mechanism of action study by time-of-addition assay and minigenome assay revealed that CPA acts at the step of virus genome replication and/or transcription. Moreover, two other calcium ATPase inhibitors (Thapsigargin and BHQ) and calcium ionophores (A23187 and ionomycin), but not calcium channel blockers (nifedipine, nimodipine, and tetrandrine), also had similar effect. These results indicate that an increase in intracellular calcium concentration is detrimental to RSV replication. Thus, our findings provide a new strategy for anti-RSV therapy via increasing intracellular calcium concentration.

Cyclopiazonic acid effect on Ca2+-dependent conformational states of the sarcoplasmic reticulum ATPase. Implication for the enzyme turnover.[Pubmed:9521749]

Biochemistry. 1998 Mar 24;37(12):4266-74.

The affinity of sarcoplasmic reticulum Ca2+-ATPase for Cyclopiazonic acid is dependent on the conformational state of the enzyme. It is high in the absence of Ca2+ but low in its presence. When Ca2+ was added to the enzyme in the presence of equimolar toxin, the apparent rate constant for Ca2+ binding was 0.6 min-1 when measured at 37 degrees C. The apparent equilibrium constant for Ca2+ dissociation increased from 0.2 to 0.6 microM at neutral pH, and from 5.9 to 37 microM at pH 6.0. The apparent equilibrium constant for Ca2+ dissociation increased progressively as the amount of toxin increased above an equimolar level. Cyclopiazonic acid decreased phosphorylation by ATP and Ca2+ when the enzyme in the absence of Ca2+ was incubated in the presence of toxin, although no effect was observed after a preliminary incubation with Ca2+ at 37 degrees C. Cyclopiazonic acid incubated with the enzyme in the presence of Ca2+ could be eliminated with a Sephadex column. However, the toxin could not be removed when it was incubated with the enzyme in the absence of Ca2+. In the latter case, Cyclopiazonic acid was eliminated when the enzyme in the presence of toxin was incubated with Ca2+ at 37 degrees C. Under turnover conditions and in the presence of 10 microM ATP, the toxin-enzyme interaction can be characterized by an apparent Kd of 7 nM. With an ATP concentration of 1 mM, the enzyme was inhibited completely at a toxin/enzyme molar ratio of approximately 10. Furthermore, enzyme activity was observed to recover at a toxin/enzyme molar ratio of 1 when the Ca2+ concentration was raised, which is consistent with the competitive character of Cyclopiazonic acid and Ca2+. It is concluded that ATP and Ca2+ can protect against Cyclopiazonic acid inhibition.

Comparison of contractions produced by carbachol, thapsigargin and cyclopiazonic acid in the guinea-pig tracheal muscle.[Pubmed:9723957]

Br J Pharmacol. 1998 Aug;124(7):1449-54.

1. Thapsigargin (TPG, 3 microM) and Cyclopiazonic acid (CPA, 10 microM) slowly increased muscle tone in the guinea-pig isolated tracheal muscle. A large sustained contraction was produced when 2.4 mM Ca2+ was readmitted after 10 min exposure to Ca2+-free solution following 30 min treatment with TPG or CPA. 2. The sustained contraction after Ca2+ readmission was partially inhibited by nifedipine (3 microM) and highly dependent on external Ca2+. The TPG- and CPA-induced sustained contractions were 75% and 67%, respectively, of the sustained contraction produced by carbachol (Cch, 1 microM, EC80) in the presence of nifedipine. 3. The contractions produced by Cch, TPG and CPA were all inhibited by isoprenaline (ISO) and sodium nitroprusside (SNP). In the presence of nifedipine, the IC50 of ISO was 11, 17, and 23 nM and that of SNP was 0.5, 1, 0.8 microM for Cch-, TPG-, and CPA-induced contractions, respectively. The contraction produced by 60 mM K+ was only weakly inhibited by ISO and SNP. As with ISO and SNP, the Cch-, TPG- and CPA-induced contractions were also similarly inhibited by SKF 96365 (100 microM) and cadmium (Cd2+, 100 microM). 4. It was concluded that TPG and CPA increased Ca2+ influx probably via a mechanism activated by Ca2+ depletion of the sarcoplasmic reticulum. The susceptibility of the contraction produced by TPG, CPA and Cch to inhibition by ISO and SNP and also by SKF-96365 and Cd2+ suggests that the contractions use common pathways for increasing intracellular Ca2+, and that the contractions produced by K+ involve a different mechanism.

On the inhibition mechanism of sarcoplasmic or endoplasmic reticulum Ca2+-ATPases by cyclopiazonic acid.[Pubmed:9006919]

J Biol Chem. 1997 Jan 31;272(5):2794-800.

Ca2+-ATPase inhibition by stoichiometric and substoichiometric concentrations of Cyclopiazonic acid was studied in sarcoplasmic reticulum preparations from rabbit fast-twitch muscle. The apparent affinity of the nonphosphorylated enzyme for ATP showed a Kd of approximately 3 microM in the absence of Cyclopiazonic acid and approximately 28 microM in the presence of the drug. Fractional saturation of the enzyme by Cyclopiazonic acid was accompanied by the appearance of two ATP-binding populations (enzyme with and without drug) and a progressive increase in the half-maximal concentration for saturating the ATP-binding sites. Enzyme turnover in the presence of stoichiometric concentrations of Cyclopiazonic acid displayed lower apparent affinity for ATP and lower maximal hydrolytic activity than in the absence of the drug. When Cyclopiazonic acid is in the substoichiometric range, the observed kinetic parameters will correspond to the simultaneous contribution of two different reaction cycles sustained by the enzyme with and without drug. The inhibition could be elicited by adding ATP to allow the enzyme turnover when Cyclopiazonic acid was preincubated with the enzyme in the presence of Ca2+. The onset of inhibition during enzyme cycling was observed over a period of seconds, revealing the existence of a low inhibition rate constant. It is concluded that Cyclopiazonic acid decreases enzyme affinity for ATP in non-turnover conditions by approximately one order of magnitude. This allows enzyme cycling after drug binding, provided that a high ATP concentration is used. Cyclopiazonic acid and ATP do not compete for the same binding site.

Cyclopiazonic acid (CPA), a neurotoxic secondary metabolite (SM) made by A. flavus, is a nanomolar inhibitor of endoplasmic reticulum calcium ATPase (Ca2+ATPase; SERCA) and a potent inducer of cell death in plants.

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