Radiosynthesis and in vitro validation of (3)H-NS14492 as a novel high affinity alpha7 nicotinic receptor radioligand.
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 (11)C-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, (3)H-NS14492 was synthesized and its binding properties were characterized using in vitro autoradiography and homogenate binding assays in pig frontal cortex. (3)H-NS14492 showed specific binding to α7 nicotinic receptors in autoradiography, revealing a dissociation constant (Kd) of 2.1±0.7nM and a maximum number of binding sites (Bmax) of 15.7±2.0fmol/mg tissue equivalent. Binding distribution was similar to that of another selective ligand (125)I-α-bungarotoxin ((125)I-BTX) in autoradiography, and unlabeled NS14492 displaced (125)I-BTX with an inhibition constant (Ki) of 23nM. (3)H-NS14492 bound to α7 nicotinic receptors in homogenized pig frontal cortex with a Kd of 0.8±0.3nM and a Bmax of 30.2±11.6fmol/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 (3)H-NS14492 to pig frontal cortex membranes when co-incubated with PNU-120596, a type II PAM. Taken together, these findings show that (3)H-NS14492 is a useful new in vitro radioligand for the pig α7 nicotinic receptor.
Neurobiology Research Unit, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.
Janus H
JH
Magnussen
Neurobiology Research Unit, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.
Anders
A
Ettrup
Neurobiology Research Unit, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.
Cornelius K
CK
Donat
DanPET AB, Malmö, Sweden.
Dan
D
Peters
The Hevesy Laboratory, DTU Nutech, The Technical University of Denmark, Denmark.
Martin H F
MH
Pedersen
Neurobiology Research Unit, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.
Gitte M
GM
Knudsen
Neurobiology Research Unit, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark. Electronic address: jens_mikkelsen@dadlnet.dk.
Jens D
JD
Mikkelsen
D000818:Animals; D053961:Azabicyclo Compounds / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D060326:Chemistry Techniques, Synthetic; D008024:Ligands; D010069:Oxadiazoles / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011854:Radiochemistry; D013552:Swine; D064569:alpha7 Nicotinic Acetylcholine Receptor / Q000378:metabolism*
C562073:4-(5-(1-methyl-1H-pyrrol-2-yl)-1,3,4-oxadiazol-2-yl)-1,4-diazabicyclo(3.2.2)nonane; D053961:Azabicyclo Compounds; D008024:Ligands; D010069:Oxadiazoles; D064569:alpha7 Nicotinic Acetylcholine Receptor
Autoradiography; Binding assay; PET ligand; Radioligand; alpha7