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The acetylcholine binding protein (AChBP) is considered an analogue for the ligand-binding domain of neuronal nicotinic acetylcholine receptors (nAChRs). Its stability and solubility in aqueous buffer allowed the development of an online bioaffinity analysis system. For this, a tracer ligand which displays enhanced fluorescence in the binding pocket of AChBP was identified from a concise series of synthetic benzylidene anabaseines. Evaluation and optimization of the bioaffinity assay was performed in a convenient microplate reader format and subsequently transferred to the online format. The high reproducibility has the prospect of estimating the affinities of ligands from an in-house drug discovery library injected in one known concentration. Furthermore, the online bioaffinity analysis system could also be applied to mixture analysis by using gradient HPLC. This led to the possibility of affinity ranking of ligands in mixtures with parallel high-resolution mass spectrometry for compound identification.

Acetylcholine-binding protein is a water-soluble homologue of the extracellular ligand-binding domain of cys-loop receptors. It is used as a structurally accessible prototype for studying ligand binding to these pharmaceutically important pentameric ion channels, in particular to nicotinic acetylcholine receptors, due to conserved binding site residues present at the interface between two subunits. Here we report that an aromatic conjugated small molecule binds acetylcholine-binding protein in an ordered π–π stack of three identical molecules per binding site, two parallel and one antiparallel. Acetylcholine-binding protein stabilizes the assembly of the stack by aromatic contacts. Thanks to the plasticity of its ligand-binding site, acetylcholine-binding protein can accommodate the formation of aromatic stacks of different size by simple loop repositioning and minimal adjustment of the interactions. This type of supramolecular binding provides a novel paradigm in drug design.

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