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

Pochet, Lionel; Heus, Ferry; Jonker, Niels; Lingeman, H.; Smit, August B.; Niessen, W.M.A.; Kool, Jeroen

doi:10.1016/j.jchromb.2011.04.023

Cited by 28 publications

(9 citation statements)

References 25 publications

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“…To characterize the binding specificity of toxins from various neurotoxic elapid venoms to nAChRs, we used a ligand-fishing approach adapted from a previously published method (Pochet et al, 2011). Elapid venoms ( N. haje , N. naja , N. kaouthia , D. viridis , B. caeruleus, M. fulvius , and O. s. scutellatus ) were incubated with recombinant His-tagged nAChR mimics (α7-AChBP or Ls-AChBP), after which the toxin-bound fraction was purified using magnetic beads.…”

Section: Resultsmentioning

confidence: 99%

“…Ligand-fishing using AChBP was adapted from a previously published method (Pochet et al, 2011). Briefly, 20 µg of venom and 20 µg of recombinant α7-AChBP were brought up in binding buffer (1 mM KH 2 PO 4 , 3 mM Na 2 HPO 4 , 0.16 mM NaCl, 20 mM Tris–Cl, pH 7.5) to a final volume of 200 µl and incubated for 2 h at room temperature with gentle agitation.…”

Section: Methodsmentioning

confidence: 99%

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A Decoy-Receptor Approach Using Nicotinic Acetylcholine Receptor Mimics Reveals Their Potential as Novel Therapeutics Against Neurotoxic Snakebite

et al. 2019

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Snakebite is a neglected tropical disease that causes 138,000 deaths each year. Neurotoxic snake venoms contain small neurotoxins, including three-finger toxins (3FTxs), which can cause rapid paralysis in snakebite victims by blocking postsynaptic transmission via nicotinic acetylcholine receptors (nAChRs). These toxins are typically weakly immunogenic and thus are often not effectively targeted by current polyclonal antivenom therapies. We investigated whether nAChR mimics, also known as acetylcholine binding proteins (AChBPs), could effectively capture 3FTxs and therefore be developed as a novel class of snake-generic therapeutics for combatting neurotoxic envenoming. First, we identified the binding specificities of 3FTx from various medically important elapid snake venoms to nAChR using two recombinant nAChR mimics: the AChBP from Lymnaea stagnalis and a humanized neuronal α7 version (α7-AChBP). We next characterized these AChBP-bound and unbound fractions using SDS-PAGE and mass spectrometry. Interestingly, both mimics effectively captured long-chain 3FTxs from multiple snake species but largely failed to capture the highly related short-chain 3FTxs, suggesting a high level of binding specificity. We next investigated whether nAChR mimics could be used as snakebite therapeutics. We showed that while α7-AChBP alone did not protect against Naja haje (Egyptian cobra) venom lethality in vivo , it significantly prolonged survival times when coadministered with a nonprotective dose of antivenom. Thus, nAChR mimics are capable of neutralizing specific venom toxins and may be useful adjunct therapeutics for improving the safety and affordability of existing snakebite treatments by reducing therapeutic doses. Our findings justify exploring the future development of AChBPs as potential snakebite treatments.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A Decoy-Receptor Approach Using Nicotinic Acetylcholine Receptor Mimics Reveals Their Potential as Novel Therapeutics Against Neurotoxic Snakebite

et al. 2019

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“…173,180,185 An example of yet another precolumn bioactivity profiling methodology used in nAChR drug discovery is a magnetic bead-based affinity-selection methodology for the identification of AChBP ligands in mixtures. 135 A summary and comparison of complex-mixture screening techniques used in drug discovery approaches is given in Table 3 .…”

Section: Approachesmentioning

confidence: 99%

“…Nonradioactive binding assays can be performed in both homogeneous and heterogeneous assay formats, based on the different types of assay component interactions. There are various types of instrumentation for measuring binding assays, such as plate reader formats, flow cytometry, 132,133 chromatographic separation formats (frontal chromatography, quantitative affinity chromatography), 134–136 and surface plasmon resonance (SPR) analysis. 137,138 Nonradioactive binding assays developed for screening α7-nAChR and 5-HT 3 R ligands include SPR-based AChBP ligand binding assays, 138 TIRF assays for studying the 5-HT 3 R, 128,129 and fluorescence enhancement assays using AChBP as a soluble binding protein that mimics the extracellular domain of the α7-nAChR.…”

Section: Approachesmentioning

confidence: 99%

Drug Discovery on Natural Products: From Ion Channels to nAChRs, from Nature to Libraries, from Analytics to Assays

et al. 2019

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Natural extracts are complex mixtures that may be rich in useful bioactive compounds and therefore are attractive sources for new leads in drug discovery. This review describes drug discovery from natural products and in explaining this process puts the focus on ion-channel drug discovery. In particular, the identification of bioactives from natural products targeting nicotinic acetylcholine receptors (nAChRs) and serotonin type 3 receptors (5-HT3Rs) is discussed. The review is divided into three parts: “Targets,” “Sources,” and “Approaches.” The “Targets” part will discuss the importance of ion-channel drug targets in general, and the α7-nAChR and 5-HT3Rs in particular. The “Sources” part will discuss the relevance for drug discovery of finding bioactive compounds from various natural sources such as venoms and plant extracts. The “Approaches” part will give an overview of classical and new analytical approaches that are used for the identification of new bioactive compounds with the focus on targeting ion channels. In addition, a selected overview is given of traditional venom-based drug discovery approaches and of diverse hyphenated analytical systems used for screening complex bioactive mixtures including venoms.

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“…A number of studies have demonstrated that magnetic bead-based approaches can quickly identify active compounds from diverse kinds of mixtures and its integration with liquid chromatography and mass spectrometry makes it possible to accurately elucidate the structures of these bioactives [23,24]. Compared with traditional methods, magnetic beads exhibit several distinct characteristics.…”

Section: Classification Of Ligand Fishing Strategiesmentioning

confidence: 99%

Ligand Fishing: A Remarkable Strategy for Discovering Bioactive Compounds from Complex Mixture of Natural Products

et al. 2016

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Identification of active compounds from natural products is a critical and challenging task in drug discovery pipelines. Besides commonly used bio-guided screening approaches, affinity selection strategy coupled with liquid chromatography or mass spectrometry, known as ligand fishing, has been gaining increasing interest from researchers. In this review, we summarized this emerging strategy and categorized those methods as off-line or on-line mode according to their features. The separation principles of ligand fishing were introduced based on distinct analytical techniques, including biochromatography, capillary electrophoresis, ultrafiltration, equilibrium dialysis, microdialysis, and magnetic beads. The applications of ligand fishing approaches in the discovery of lead compounds were reviewed. Most of ligand fishing methods display specificity, high efficiency, and require less sample pretreatment, which makes them especially suitable for screening active compounds from complex mixtures of natural products. We also summarized the applications of ligand fishing in the modernization of Traditional Chinese Medicine (TCM), and propose some perspectives of this remarkable technique.

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

Cited by 28 publications

References 25 publications

A Decoy-Receptor Approach Using Nicotinic Acetylcholine Receptor Mimics Reveals Their Potential as Novel Therapeutics Against Neurotoxic Snakebite

A Decoy-Receptor Approach Using Nicotinic Acetylcholine Receptor Mimics Reveals Their Potential as Novel Therapeutics Against Neurotoxic Snakebite

Drug Discovery on Natural Products: From Ion Channels to nAChRs, from Nature to Libraries, from Analytics to Assays

Ligand Fishing: A Remarkable Strategy for Discovering Bioactive Compounds from Complex Mixture of Natural Products

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