Interactions of Nereistoxin and Its Analogs with Vertebrate Nicotinic Acetylcholine Receptors and Molluscan ACh Binding Proteins

Abstract

Nereistoxin (NTX) is a marine toxin isolated from an annelid worm that lives along the coasts of Japan. Its insecticidal properties were discovered decades ago and this stimulated the development of a variety of insecticides such as Cartap that are readily transformed into NTX. One unusual feature of NTX is that it is a small cyclic molecule that contains a disulfide bond. In spite of its size, it acts as an antagonist at insect and mammalian nicotinic acetylcholine receptors (nAChRs). The functional importance of the disulfide bond was assessed by determining the effects of inserting a methylene group between the two sulfur atoms, creating dimethylaminodithiane (DMA-DT). We also assessed the effect of methylating the NTX and DMA-DT dimethylamino groups on binding to three vertebrate nAChRs. Radioligand receptor binding experiments were carried out using washed membranes from rat brain and fish (Torpedo) electric organ; [3H]-cytisine displacement was used to assess binding to the predominantly high affinity alpha4beta2 nAChRs and [125I]-alpha-bungarotoxin displacement was used to measure binding of NTX and analogs to the alpha7 and skeletal muscle type nAChRs. While the two quaternary nitrogen analogs, relative to their respective tertiary amines, displayed lower α4β2 nAChR binding affinities, both displayed much higher affinities for the Torpedo muscle nAChR and rat alpha7 brain receptors than their respective tertiary amine forms. The binding affinities of DMA-DT for the three nAChRs were lower than those of NTX and MeNTX. An AChBP mutant lacking the C loop disulfide bond that would potentially react with the NTX disulfide bond displayed an NTX affinity very similar to the parent AChBP. Inhibition of [3H]-epibatidine binding to the AChBPs was not affected by exposure to NTX or MeNTX for up to 24 hr prior to addition of the radioligand. Thus, the disulfide bond of NTX is not required to react with the vicinal disulfide in the AChBP C loop for inhibition of [3H]-epibatidine binding. However, a reversible disulfide interchange reaction of NTX with nAChRs might still occur, especially under reducing conditions. Labeled MeNTX, because it can be readily prepared with high specific radioactivity and possesses relatively high affinity for the nAChR-rich Torpedo nAChR, would be a useful probe to detect and identify any nereistoxin adducts.

The Cyclic Imine Core Common to the Marine Macrocyclic Toxins Is Sufficient to Dictate Nicotinic Acetylcholine Receptor Antagonism

“…These data also contribute to challenge a conclusion built over the years that small molecules containing a quaternary nitrogen and strictly occupying the agonist binding pocket at the subunit interface, without extending substituents outside of this core, cannot act as nicotinic antagonists. Initial insights against this "dogma" include the small cyclic, disulfide-containing marine molecule, nereistoxin (aka N,N-dimethyl-1,2-dithiolan-4-amine), shown to act as a nicotinic antagonist with sub-millimolar IC50 values [93]. Availability of a crystal structure of a nereistoxin-AChBP complex would be of interest to complement the current study.…”

Marine natural products

“…A set of analogues, comprised of quaternised N -methyl-nereistoxin, 5-dimethylamino-1,3-dithiane and its quaternised N -methyl derivative, were prepared and evaluated against three mammalian nAChR's. 631 The quaternised analogues, while displaying lower affinity for α4β2 nAChRs, bound with higher affinities than their secondary amine variants to Torpedo muscle nAChR and rat α7 brain receptors.…”

Application of Marine Natural Products against Alzheimer’s Disease: Past, Present and Future

“…Marine toxins are naturally toxic chemicals from the ocean environment [ 129 ]. Nereistoxin (NFX) was found to have a strong binding affinity with nicotinic acetylcholine receptor (nAChR) in mouse brain [ 130 ], as was Spirolides. Spirolides (SPX) are cycloimine lipophilic marine toxins, mainly produced by Alexandrium ostenfeldii ( A. ostenfeldii ) [ 71 ].…”