Anatoxins

Anatoxin-a is a naturally occurring homotropane alkaloid produced by freshwater cyanobacteria of the genera Anabama (A. flos-ctquae and A. circinalis), Aphanizome-non, Cylindrospermum, Planktothrix, Microcystis aeruginosa [5-7], and Phormidium favosum [8]. Fatal intoxications have typically included cattle and birds [9], and, more recently, dogs [8] and flamingos [10]. 

Anatoxin-a, the first highly potent cyanotoxin to have its structure and absolute stereochemistry elucidated, was originally isolated from a unialgal clone of Anabaena Jlos-aquae (NRC-44h) [5]. The structure was confirmed by X-ray crystallographic data for the N-acetyl derivative [11] and additional studies have since provided further proof for the structure and stereochemistry (for example, [12]). Anatoxin-a is an unsymmetrical bicyclic secondary amine, and was the first naturally occurring alkaloid discovered to contain a 9-azabicyclo[4,2,l]nonane (homotropane) skeleton. Homotropanes are one-carbon analogs of the tropanes and, as such, are structurally closely related to the well-known alkaloid cocaine. 

The unique structure of anatoxin-a, and its potential as a pharmacological tool, has inspired many different chemical syntheses of anatoxin-a and analogs. The earliest synthesis, in 1977, used (—)-cocaine as starting material [23]. Subsequently, there 

The SARs of anatoxin-a and analogs have been extensively studied [33-36], and only a few important findings dealing with chirality, conformation, and other structural features of anatoxin-a and homologs vfill be summarized here. A recent review that addresses the chemistry and pharmacology of anatoxin-a and analogs provides a more detailed treatment of this topic [37]. 

The naturally occurring enantiomer, (+)-anatoxin-a, is much more potent than (—)-anatoxin-a [38]. For example, using conditions that preferentially labeled rat brain a4p2 nACh receptors with [ H]nicotine, the natural enantiomer was found to be 1000-fold more potent than (—[-anatoxin [39]. This enantiospecificity of anatoxin-a has provided an excellent tool for probing the stereospedfidty of the ACh binding site on the nicotinic receptor. 

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The cyanobacterial neiirotoxins, anatoxins and saxitoxins have been responsible... 

Daphnia assay, the brine shrimps are exposed to different concentrations of toxicant, and the toxicity is expressed as the LCjo value. Extracts of cyanobacterial blooms and laboratory cultures, containing microcystins or anatoxin-a, have been found to be toxic towards brine shrimp," and fractionation of such extracts resulted in brine shrimp fatalities only with fractions containing microcystins." " ... 

An enzymatic assay can also be used for detecting anatoxin-a(s). " This toxin inhibits acetylcholinesterase, which can be measured by a colorimetric reaction, i.e. reaction of the acetyl group, liberated enzymatically from acetylcholine, with dithiobisnitrobenzoic acid. The assay is performed in microtitre plates, and the presence of toxin detected by a reduction in absorbance at 410 nm when read in a plate reader in kinetic mode over a 5 minute period. The assay is not specific for anatoxin-a(s) since it responds to other acetylcholinesterase inhibitors, e.g. organophosphoriis pesticides, and would need to be followed by confirmatory tests for the cyanobacterial toxin. 

HPLC analysis of anatoxin-a was first carried out by Astrachan and Archer, " who extracted the toxin from Anabaenaflos-aquae using chloroform followed by hydrochloric acid. The HPLC analysis was carried out on an ODS column using hypochlorate-methanol. Other systems used since include acetic acid extraction and analysis on a reversed-phase C g column using methanol-water mobile phase, and extraction in water after ultrasonication and analysis on reversed-phase... 

Mass speetrometry has been used to eharaeterize mieroeystins using the method of fast-atom bombardment (FAB) ionization and MS/MS. Anatoxin-a has been analysed by MS in eombination with gas ehromatography in bloom and water samples, and in benthie eyanobaeterial material and stomaeh eontents of poisoned animals.Reeently, liquid ehromatography (LC) linked to MS has been employed to analyse mieroeystins, where FAB-MS and atmospherie-pressiire ionization (API-MS) have been used, and anatoxin-a, where thermospray (TSP-MS) was iised. ... 

The ability to identify and quantify cyanobacterial toxins in animal and human clinical material following (suspected) intoxications or illnesses associated with contact with toxic cyanobacteria is an increasing requirement. The recoveries of anatoxin-a from animal stomach material and of microcystins from sheep rumen contents are relatively straightforward. However, the recovery of microcystin from liver and tissue samples cannot be expected to be complete without the application of proteolytic digestion and extraction procedures. This is likely because microcystins bind covalently to a cysteine residue in protein phosphatase. Unless an effective procedure is applied for the extraction of covalently bound microcystins (and nodiilarins), then a negative result in analysis cannot be taken to indicate the absence of toxins in clinical specimens. Furthermore, any positive result may be an underestimate of the true amount of microcystin in the material and would only represent free toxin, not bound to the protein phosphatases. Optimized procedures for the extraction of bound microcystins and nodiilarins from organ and tissue samples are needed. 

Of the known cyanobacterial toxins only anatoxin-fl(s) is detected and is, therefore, able to be screened for by acetylcholinesterase inhibition. 

Anatoxins. Neurotoxins produced by filamentous Anabaena flos-aquae are called anatoxins (ANTXS) 15). Currently two anatoxins, from different strains of A. flos-aquae, have been isolated and at least partially characterized. ANTX-A from strain (single filament isolate) NRC-44-1 is the first toxin from a freshwater... 

A. flos-aquae Strain NRC-44-1 (Canada, Saskatchewan) Anatoxin-A Secondary amine alkaloid, MW 165 200... 

Strain NRC-525-17 (Canada, Saskatchewan) Anatoxin-A(S) N-hydvoxy guanidine methyl-phosphate ester, MW 252 20... 

Figure 1. Left Anatoxin-a (ANTX-A) hydrochloride. Produced by the freshwater filamentous cyanobacterium Anabaena flos-aquae NRC-44-1. Right Anatoxin-a(s). Produced by the freshwater filamentous cyanobacterium Anabaena flos-aquae NRC-525-17. Bottom Aphantoxin-I (neosaxitoxin) and Aphantoxin-II (saxitoxin) produced by certain strains of the filamentous cyanobacterium Aphantomenon flos-aquae.

Nicotinic Acetylcholine Receptor Function Studied with Synthetic (+)-Anatoxin-a and Derivatives... 

Anatoxin-a is the most potent and most stereospecific nicotinic acetylcholine receptor agonist thus far identified. It is also highly selective for nicotinic receptors over muscarinic receptors. The molecular parameters which influence the binding affinity, channel activation, channel blockade, and receptor desensitization are being studied. Modifications of the carbonyl and amine moieties can reduce or nearly eliminate the receptor agonist potency of the compounds and also determine the channel blocking characteristics. 

The debut of the selective AChR agonist (+)-anatoxin-a has provided a new tool for AChR physiology and pharmacology. (+)-Anatoxin not only has high affinity for the nicotinic AChR but it also has high selectivity for nicotinic over muscarinic receptors in the mammalian CNS. Recently, the use of (+)-anatoxin-a was essential to the identification of nicotinic receptors on cultured neurons (4), We are studying the features which allow it to bind with high affinity to the peripheral and central nicotinic receptors and the kinetic effects on receptor conformational... 

Using synthetic enantiomers, we found that anatoxin-a is highly stereospecific with the (+) isomer having 150-fold greater potency than the (-) isomer (Figure 2) 19). The semi-rigid nature of anatoxin-a undoubtedly facilitates its stereospecificity. 

Figure 1. Structure of (+)-anatoxin-a and two analogs with agonist and noncompetitive antagonist activity, respectively.

Figure 2. Potency of anatoxin-a analogs to induce contracture in frog rectus abdominis muscle. The data from two experiments are combined in this figure. In one, anatoxinmethylester was found to be equipo-tent with carbamylcholine. In the other, the anatoxin isomers were assayed against ACh [after cholinesterse inhibition by diisopropylfluoro-phosphate (DFP) followed by washing of the preparation] (19). Maximal contracture was measured by depolarization with KCl at the end of each experiment.

Allosteric Antagonism of AChR Function by (+)-Anatoxin-a Analogs... 

Figure 3. (+)-Anatoxin-a (AnTx) and ACh induced single ion channel currents in isolated frog muscle fibers. Open channels with 32 pS conductance are downward deflections (inward current at hyperpolarized potentials). The currents shown on the left are all at one potential. The duration of channel open events had a similar voltage-dependence for both ACh and (+)-anatoxin-a. With ACh, the events were most often singular, while with (+)-anatoxin-a the events were shorter and were more frequently paired so that the mean duration of the exponentially distributed open times and selected membrane holding potentials was approximately one-half, independent of the concentration of the agonist applied.

The (+)-anatoxin-a analogs (/ )- or (5)-iV-methylanatoxinol, both possess the ability to block the ion channel [the voltage-dependence of their characteristics differ in a way which is the topic of another report (34). As is often the case, the blocked state induced by (/ )-7Y-methylanatoxinol and the closed state differed significantly in duration, such that the blocked state was associated with short closed periods within groups of openings called bursts (Figure 4). The number of short closed periods per burst increased with the concentration of the drug because the likelihood of channel blockade increased relative to the likelihood of channel closure. 

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