Affinities of bispyridinium non-oxime compounds to [3H]epibatidine binding sites of Torpedo californica nicotinic acetylcholine receptors depend on linker length

• We investigated bispyridinium compounds in binding experiments with nAChRs.
• We compared [3H]epibatidine affinity profiles to the inhibition of human AChE.
• nAChR affinity and AChE inhibition depended on linker length.
• Divalent cations enhanced [3H]epibatidine binding.

The toxicity of organophosphorus nerve agents or pesticides arises from accumulation of acetylcholine and overstimulation of both muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs) due to inhibition of acetylcholinesterase (AChE). Standard treatment by administration of atropine and oximes, e.g., obidoxime or pralidoxime, focuses on antagonism of mAChRs and reactivation of AChE, whereas nicotinic malfunction is not directly treated. An alternative approach would be to use nAChR active substances to counteract the effects of accumulated acetylcholine. Promising in vitro and in vivo results were obtained with the bispyridinium compounds SAD-128 (1,1′-oxydimethylene bis(4-tert-butylpyridinium) dichloride) and MB327 (1,1′-(propane-1,3-diyl)bis(4-tert-butylpyridinium) di(iodide)), which were partly attributed to their interaction with nAChRs. In this study, a homologous series of unsubstituted and 4-tert-butyl-substituted bispyridinium compounds with different alkane linker lengths was investigated in competition binding experiments using [3H]epibatidine as a reporter ligand. Additionally, the effect of the well-characterised MB327 on the [3H]epibatidine equilibrium dissociation (KD) constant in different buffers was determined. This study demonstrated that divalent cations increased the affinity of [3H]epibatidine. Since quaternary ammonium molecules are known to inhibit AChE, the obtained affinity constants of the tested bispyridinium compounds were compared with the inhibition of human AChE. In competition experiments, bispyridinium derivatives of longer linker length displaced [3H]epibatidine and inhibited AChE strongly. Bispyridinium compounds with short linkers, at most, have an allosteric interaction with the [3H]epibatidine binding sites and barely inhibited AChE. In dependence on alkane linker length, the bispyridinium compounds seemed to interact at different binding sites. However, the exact binding sites of the bispyridinium compounds responsible for the positive pharmacological effects have still not been identified, making predictive drug design difficult.