Radiosynthesis and in vitro validation of 3H-NS14492 as a novel high affinity alpha7 nicotinic receptor radioligand

Janus H. Magnussen, Anders Ettrup, Cornelius K. Donat, Dan Peters, Martin Holst Friborg Pedersen, Gitte M. Knudsen, Jens D. Mikkelsen

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    Abstract

    The neuronal alpha 7 nicotinic acetylcholine receptor is a homo-pentameric ligand-gated ion channel that is a promising drug target for cognitive deficits in Alzheimer's disease and schizophrenia. We have previously described 11C-NS14492 as a suitable agonist radioligand for in vivo positron emission tomography (PET) occupancy studies of the alpha 7 nicotinic receptor in the pig brain. In order to investigate the utility of the same compound for in vitro studies, 3H-NS14492 was synthesized and its binding properties were characterized using in vitro autoradiography and homogenate binding assays in pig frontal cortex. 3H-NS14492 showed specific binding to alpha 7 nicotinic receptors in autoradiography, revealing a dissociation constant (Kd) of 2.1 ± 0.7 nM and a maximum number of binding sites (Bmax) of 15.7±2.0 fmol/mg tissue equivalent. Binding distribution was similar to that of another selective ligand 125I-alpha-bungarotoxin (125I-BTX) in autoradiography, and unlabeled NS14492 displaced 125I-BTX with an inhibition constant (Ki) of 23 nM. 3H-NS14492 bound to alpha 7 nicotinic receptors in homogenized pig frontal cortex with a Kd of 0.8±0.3 nM and a Bmax of 30.2±11.6 fmol/mg protein. This binding assay further revealed the Ki rank order for a number of alpha 7 nicotinic receptor agonists, and positive allosteric modulators (PAMs). Further, we saw increased binding of 3H-NS14492 to pig frontal cortex membranes when co-incubated with PNU-120596, a type II PAM. Taken together, these findings show that 3H-NS14492 is a useful new in vitro radioligand for the pig alpha 7 nicotinic receptor. © 2015 Elsevier B.V. All rights reserved.
    Original languageEnglish
    JournalEuropean Journal of Pharmacology
    Volume762
    Pages (from-to)35-41
    Number of pages7
    ISSN0014-2999
    DOIs
    Publication statusPublished - 2015

    Keywords

    • Alpha7
    • Radioligand
    • Autoradiography
    • Binding assay
    • PET ligand

    Cite this

    Magnussen, J. H., Ettrup, A., Donat, C. K., Peters, D., Pedersen, M. H. F., Knudsen, G. M., & Mikkelsen, J. D. (2015). Radiosynthesis and in vitro validation of 3H-NS14492 as a novel high affinity alpha7 nicotinic receptor radioligand. European Journal of Pharmacology, 762, 35-41. https://doi.org/10.1016/j.ejphar.2015.04.036