Rómulo Aráoz

Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism

Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Analysis of 13-desmethyl spirolide C and gymnodimine A by binding and voltage-clamp recordings on muscle-type α12βγδ and neuronal α3β2 and α4β2 nicotinic acetylcholine receptors reveals subnanomolar affinities, potent antagonism, and limited subtype selectivity. Their binding to acetylcholine-binding proteins (AChBP), as soluble receptor surrogates, exhibits picomolar affinities governed by diffusion-limited association and slow dissociation, accounting for apparent irreversibility. Crystal structures of the phycotoxins bound to Aplysia-AChBP (≈2.4Å) show toxins neatly imbedded within the nest of ar-omatic side chains contributed by loops C and F on opposing faces of the subunit interface, and which in physiological conditions accommodates acetylcholine. The structures also point to three major features: (i) the sequence-conserved loop C envelops the bound toxins to maximize surface complementarity; (ii) hydrogen bonding of the protonated imine nitrogen in the toxins with the carbonyl oxygen of loop C Trp147 tethers the toxin core centered within the pocket; and (iii) the spirolide bis-spiroacetal or gymnodimine tetrahydrofuran and their common cyclohexene-butyrolactone further anchor the toxins in apical and membrane directions, along the subunit interface. In contrast, the se-quence-variable loop F only sparingly contributes contact points to preserve the broad receptor subtype recognition unique to phycotoxins compared with other nicotinic antagonists. These data offer unique means for detecting spiroimine toxins in shellfish and identify distinctive ligands, functional determinants and binding regions for the design of new drugs able to target several receptor subtypes with high affinity.

Neurotoxic cyanobacterial toxins.

Worldwide development of cyanobacterial blooms has significantly increased in marine and continental waters in the last century due to water eutrophication. This phenomenon is favoured by the ability of planktonic cyanobacteria to synthesize gas vesicles that allow them to float in the water column. Besides, benthic cyanobacteria that proliferate at the bottom of lakes, rivers and costal waters form dense mats near the shore. Cyanobacterial massive proliferation is of public concern regarding the capacity of certain cyanobacterial strains to produce hepatotoxic and neurotoxic compounds that can affect public health, human activities and wild and stock animals. The cholinergic synapses and voltage-gated sodium channels constitute the targets of choice of cyanobacterial neurotoxins. Anatoxin-a and homoanatoxin-a are agonists of nicotinic acetylcholine receptors. Anatoxin-a(s) is an irreversible inhibitor of acetylcholinesterase. Saxitoxin, kalkitoxin and jamaicamide are blockers of voltage-gated sodium channels, whereas antillatoxin is an activator of such channels. Moreover the neurotoxic amino acid l-beta-N-methylamino-l-alanine was shown to be produced by diverse cyanobacterial taxa. Although controversial, increasing in vivo and in vitro evidence suggest a link between the ingestion of l-beta-N-methylamino-l-alanine and the development of amyotrophic lateral sclerosis/Parkinsonism-dementia complex, a neurodegenerative disease. This paper reviews the occurrence of cyanobacterial neurotoxins, their chemical properties, mode of action and biosynthetic pathways.

Total Synthesis of Pinnatoxins A and G and Revision of the Mode of Action of Pinnatoxin A

Pinnatoxins belong to an emerging class of potent marine toxins of the cyclic imine group. Detailed studies of their biological effects have been impeded by unavailability of the complex natural product from natural sources. This work describes the development of a robust, scalable synthetic sequence relying on a convergent strategy that delivered a sufficient amount of the toxin for detailed biological studies and its commercialization for use by other research groups and regulatory agencies. A central transformation in the synthesis is the highly diastereoselective Ireland-Claisen rearrangement of a complex α,α-disubstituted allylic ester based on a unique mode for stereoselective enolization through a chirality match between the substrate and the lithium amide base. With synthetic pinnatoxin A, a detailed study has been performed that provides conclusive evidence for its mode of action as a potent inhibitor of nicotinic acetylcholine receptors selective for the human neuronal α7 subtype. The comprehensive electrophysiological, biochemical, and computational studies support the view that the spiroimine subunit of pinnatoxins is critical for blocking nicotinic acetylcholine receptor subtypes, as evidenced by analyzing the effect of a synthetic analogue of pinnatoxin A containing an open form of the imine ring. Our studies have paved the way for the production of certified standards to be used for mass-spectrometric determination of these toxins in marine matrices and for the development of tests to detect these toxins in contaminated shellfish.

Detection of gymnodimine-A and 13-desmethyl C spirolide phycotoxins by fluorescence polarization.

The gymnodimines and spirolides are phycotoxins classified into a heterogeneous group of marine biocompounds called cyclic imines. Although there is no clear evidence of their toxicity to humans, gymnodimines and spirolides are highly toxic to rodents and constitute a source of false positives in lipophilic toxin detection by the mouse bioassay. Using nicotinic acetylcholine receptor-enriched membranes of Torpedo, and fluorescent alpha-bungarotoxin, we developed a fluorescence polarization assay to detect and quantify gymnodimine-A and 13-desmethyl C spirolide. The presence of these cyclic imines in solution inhibited the interaction of fluorescent-labeled alpha-bungarotoxin with nicotinic acetylcholine receptors in a concentration-dependent manner. The sensitivity of the assay is in the order of nanomolar concentrations of gymnodimine and 13-desmethyl C spirolide. Okadaic acid, yessotoxin, and brevetoxin-2, three lipophilic marine toxins, did not interfere with this assay. A suitable extraction method in shellfish was also developed. The gymnodimine-A and 13-desmethyl C spirolide recovery rates of mussel matrix extraction with acetone/chloroform were 63.6% +/- 3.5% and 87.4% +/- 5.3%, respectively. In summary, this inhibition assay is capable of gymnodimine-A and 13-desmethyl C spirolide detection in mussel extracts with enough sensitivity and specificity to quantify these toxins in the range of 50-2000 microg/kg and 70-700 microg/kg of shellfish meat, respectively.

Pinnatoxin G is responsible for atypical toxicity in mussels (Mytilus galloprovincialis) and clams (Venerupis decussata) from Ingril, a French Mediterranean lagoon

Following a review of official control data on shellfish in France, Ingril Lagoon had been identified as a site where positive mouse bioassays for lipophilic toxins had been repeatedly observed. These unexplained mouse bioassays, also called atypical toxicity, coincided with an absence of regulated toxins and rapid death times in mice observed in the assay. The present study describes pinnatoxin G as the main compound responsible for the toxicity observed using the mouse bioassay for lipophilic toxins. Using a well-characterised standard for pinnatoxin G, LC-MS/MS analysis of mussel samples collected from 2009 to 2012 revealed regular occurrences of pinnatoxin G at levels sufficient to account for the toxicity in the mouse bioassays. Baseline levels of pinnatoxin G from May to October usually exceeded 40 μg kg(-1) in whole flesh, with a maximum in September 2010 of around 1200 μg kg(-1). These concentrations were much greater than those at the other 10 sites selected for vigilance testing, where concentrations did not exceed 10 μg kg(-1) in a 3-month survey from April to July 2010, and where rapid mouse deaths were not typically observed. Mussels were always more contaminated than clams, confirming that mussel is a good sentinel species for pinnatoxins. Profiles in mussels and clams were similar, with the concentration of pinnatoxin A less than 2% that of pinnatoxin G, and pteriatoxins were only present in non-quantifiable traces. Esters of pinnatoxin G could not be detected by analysis of extracts before and after alkaline hydrolysis. Analysis with a receptor-binding assay showed that natural pinnatoxin G was similarly active on the nicotinic acetylcholine receptor as chemically synthesized pinnatoxin G. Culture of Vulcanodinium rugosum, previously isolated from Ingril lagoon, confirmed that this alga is a pinnatoxin G producer (4.7 pg cell(-1)). Absence of this organism from the water column during prolonged periods of shellfish contamination and the dominance of non-motile life stages of V. rugosum both suggest that further studies will be required to fully describe the ecology of this organism and the accumulation of pinnatoxins in shellfish.

Feasibility of gymnodimine and 13-desmethyl C spirolide detection by fluorescence polarization using a receptor-based assay in shellfish matrixes.

The detection of toxins in shellfish through reliable methods is essential for human health preservation and prevention of economic losses in the aquaculture industry. Although no human intoxication has been unequivocally linked to gymnodimines or spirolides, these phycotoxins are highly toxic by intraperitoneal injection causing false positives in lipophilic toxin detection by the mouse bioassay. Based on the detection of molecular interactions by fluorescence polarization an inhibition assay was developed using fluorescent alpha-bungarotoxin and nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata to detect gymnodimine and 13-desmethyl C spirolide. Both toxins, classified into the cyclic imine group, inhibit the interaction of alpha-bungarotoxin with Torpedo nicotinic acetylcholine receptors in the nM range. In this study we analyze the matrix effect of four shellfish species on the fluorescence polarization assay. Mussels, clams, cockles and scallops were extracted with acetone and sequentially partitioned with n-hexane and chloroform. The interference of these shellfish extracts with the alpha-bungarotoxin fluorescence or its binding to the nicotinic acetylcholine receptor was lower than 11%. The average recovery rates of gymnodimine and 13-desmethyl C spirolide using these solvents were 90.6+/-7.8% and 89.6+/-3.2%, respectively with variations among species. The quantification range of this fluorescence polarization assay for gymnodimine and 13-desmethyl C spirolide in all tested species was 80-2000 microg kg(-1) and 85-700 microg kg(-1) of shellfish meat, respectively. This assay format can be used to detect gymnodimine and 13-desmethyl C spirolide in shellfish as a screening assay.

Detection of 13,19-didesmethyl C spirolide by fluorescence polarization using Torpedo electrocyte membranes.

Fluorescence polarization (FP) is a powerful tool for studying molecular interactions by monitoring changes in the apparent size of fluorescent molecules. In this paper, a previously described fluorescence polarization assay was used to detect 13,19-didesmethyl C spirolide. The assay is based on the competition of cyclic imine marine biotoxins with alpha-bungarotoxin for binding to nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata. The 13,19-didesmethyl C spirolide was detected in buffer and mussel matrix. The sensitivity of the assay for the 13,19-didesmethyl C spirolide and the 13-desmethyl C spirolide was similar. After an acetone/chloroform extraction of spiked mussel meat, the average recovery rate of 13,19-didesmethyl C spirolide was 77.7 +/- 1.9%. The quantification range for this toxin in mussel was 40-200 microg/kg of shellfish meat. This assay can be used to detect the spirolides 13,19-didesmethyl C spirolide and 13-desmethyl C spirolide, in shellfish as a screening assay.

First direct fluorescence polarization assay for the detection and quantification of spirolides in mussel samples

In 2009, we achieve the first inhibition FP assay to detect imine cyclic toxins. In the present paper we propose a new FP assay for direct quantify spirolides. This new method has resulted in significant improvement of sensitivity, rapidity and accessibility. In the method design, nicotinic acetylcholine receptor from Torpedo marmorata membranes labelled with a derivative of fluorescein was used. Spirolides, 13-desmethyl spirolide C (13-desMeC) and 13,19-didesmethyl spirolide C (13,19-didesMeC) were extracted and purified from cultures of the Alexandrium ostenfeldii dinoflagellate. Data showed the decrease of FP when toxin concentration was increased. Thus, a relationship between the FP units and the spirolides amount present in a sample was obtained. This direct assay is a reproducible, simple and very sensitive method with a detection limit about 25 nM for 13-desMeC and 150 nM for 13,19-didesMeC. The procedure was used to measure spirolides in mussel samples using an extraction and clean up protocol suitable for the FP assay. Results obtained show that this method is able to quantify 13-desMeC in the range of 50-350 μg kg(-1) meat. Other liposoluble toxins did not interfere with the assay, proving a specific method. Moreover, the matrix do not affect in the range of toxin concentrations that involving risk of spirolides intoxication.

Coupling the Torpedo microplate-receptor binding assay with mass spectrometry to detect cyclic imine neurotoxins.

Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.

Fluorescent Agonists for the Torpedo Nicotinic Acetylcholine Receptor

We have synthesized a series of fluorescent acylcholine derivatives carrying different linkers that vary in length and structure and connect the acylcholine unit to the environment‐sensitive fluorophores 7‐(diethylamino)coumarin‐3‐carbonyl (DEAC) or N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐yl) (NBD). The pharmacological properties of the fluorescent analogues were investigated on heterologously expressed nicotinic acetylcholine receptor (nAChR) from Torpedo californica and on oocytes transplanted with nAChR‐rich Torpedo marmorata membranes. Agonist action strongly depends on the length and the structure of the linker. One particular analogue, DEAC‐Gly‐C6‐choline, showed partial agonist behavior with about half of the maximum response of acetylcholine, which is at least 20 times higher than those observed with previously described fluorescent dansyl‐ and NBD‐acylcholine analogues. Binding of DEAC‐Gly‐C6‐choline to Torpedo nAChR induces a strong enhancement of fluorescence intensity. Association and displacement kinetic experiments revealed dissociation constants of 0.5 nM for the αδ‐binding site and 15.0 nM for the αγ‐binding site. Both the pharmacological and the spectroscopic properties of this agonist show great promise for characterizing the allosteric mechanism behind the function of the Torpedo nAChR, as well as for drug‐screening studies.