TY - JOUR
T1 - Radiosynthesis and in vitro validation of (3)H-NS14492 as a novel high affinity alpha7 nicotinic receptor radioligand
AU - Magnussen, Janus H
AU - Ettrup, Anders
AU - Donat, Cornelius K
AU - Peters, Dan
AU - Pedersen, Martin H F
AU - Knudsen, Gitte M
AU - Mikkelsen, Jens D
N1 - Copyright © 2015 Elsevier B.V. All rights reserved.
PY - 2015/5/29
Y1 - 2015/5/29
N2 - The neuronal α7 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 α7 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 α7 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-α-bungarotoxin (125I-BTX) in autoradiography, and unlabeled NS14492 displaced 125I-BTX with an inhibition constant (Ki) of 23 nM. 3H-NS14492 bound to α7 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 α7 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 α7 nicotinic receptor.
AB - The neuronal α7 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 α7 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 α7 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-α-bungarotoxin (125I-BTX) in autoradiography, and unlabeled NS14492 displaced 125I-BTX with an inhibition constant (Ki) of 23 nM. 3H-NS14492 bound to α7 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 α7 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 α7 nicotinic receptor.
U2 - 10.1016/j.ejphar.2015.04.036
DO - 10.1016/j.ejphar.2015.04.036
M3 - Journal article
C2 - 25941084
SN - 0014-2999
VL - 762
SP - 35
EP - 41
JO - European Journal of Pharmacology
JF - European Journal of Pharmacology
ER -