Online Fluorescence Enhancement Assay for the Acetylcholine Binding Protein with Parallel Mass Spectrometric Identification

Abstract

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.

At-Line Cellular Screening Methodology for Bioactives in Mixtures Targeting the α7-Nicotinic Acetylcholine Receptor

“…This phenomenon of excessive band broadening observed for high concentrations of ligand injected is common when working with on-line and at-line reconstructed bioassay chromatograms. 13 ACh showed a strong and repeatable signal in the Ca 2+ -flux assay when relatively high concentrations were injected. ACh also elicited a Ca 2+ -signal in the nanomolar range (500 nM injected concentration, about 45 nM in the assay), which indicated that the signals might be caused not only by signaling via the α7-nAChR (pKi of ACh is 5.1 ± 0.1 for the α7-nAChR 30 ).…”

“…[11][12][13][14] Most importantly, functional bioassays can also be implemented in these screening methodologies. 9 A functional bioassay gives the advantage of not only measuring binding affinity of a ligand, but also measuring functional activity and allowing distinction between, for example, agonism, antagonism, and allosteric modulation.…”

High-Resolution Bioactivity Profiling of Mixtures toward the Acetylcholine Binding Protein Using a Nanofractionation Spotter Technology

“…Expression and purification from the medium is described by Celie et al 15 the assay is based on the competition of ligands with the tracer ligand daHBa, which has an increased fluorescence yield in the active site of aChBP. 10 the following triS/PBS buffer was used: 1 mM KH 2 Po 4 , 3 mM na 2 HPo 4 , 0.16 mM naCl, and 20 mM trizma-base at pH 7.5 with 400 mg/l EliSa Br. For operation of the superloops (10 ml in-house-built superloops) delivering the biochemical assay constituents (aChBP and tracer ligand daHBa), two Shimadzu lC10advp pumps (50 µl/min) were used, resulting in continuous delivery of 6.6 nM aChBP (lower superloop) and 150 nM daHBa (upper superloop) to an inverted y-shaped mixing piece prior to coupling to the spotter heart.…”

“…Evaluation, validation, and actual high-resolution bioactivity profiling of pure compounds and mixtures on the AChBP aided by the nanofractionation spotter technology the reason for using the aChBP biochemical assay format in this offline mode rather than the recently described online format 10 is the capability of the offline format to identify compounds with slow binding kinetics, which require long incubation times for binding equilibrium to be reached. Efficient measurement of these types of compounds, such as α-bungarotoxin, 17 a peptidic antagonist of the α7 naChr with a molecular weight of ˜8000 da, is not possible with the online format.…”

Online magnetic bead based dynamic protein affinity selection coupled to LC–MS for the screening of acetylcholine binding protein ligands

“…After its crystal structure was published [19], it has become a model for nAChRs to efficiently screen compound libraries for potential ligands and for structure based synthetic approaches in medicinal chemistry [20]. The nAChR family is intensively studied in relation to its potential as pharmaceutical target against epilepsy, Alzheimer's disease, pain relief, Parkinson's disease, anxiety and cognitive and attention deficits [17,18,[21][22][23].…”