Synthesis and characterization of [³H]-SN56, a novel radioligand for the σ₁ receptor.
The study of the binding characteristics of σ ligands in vivo and in vitro requires radiolabeled probes with high affinity and selectivity. The radioligand presently used for in vitro studies of the σ₁ receptor, [³H](+)-pentazocine, has significant limitations; it is difficult to synthesize, has limited chemical stability, and can be problematic to obtain. Evaluation of a series of novel 2(3H)-benzothiazolone compounds revealed SN56 to have sub-nanomolar and preferential affinity for the σ₁ subtype, relative to σ₂ and non-sigma, binding sites. The goal of this study was to characterize the binding of [³H]-SN56 to σ₁ receptors isolated from rat brain. Standard in vitro binding techniques were utilized to 1) determine the specificity and affinity of binding to σ₁ receptors, 2) confirm that[³H]-SN56 labels sites previously identified as σ₁ by comparing binding to sites labeled by [³H](+)-pentazocine, and 3) characterize the kinetics of binding. The results indicate that [³H]-SN56 exhibits 1) specific, saturable, and reversible binding to the σ₁ receptor, with B(max)=340±10 fmol/mg and K(d)=0.069±0.0074 nM, 2) competitive displacement by classical sigma compounds, yielding σ₁ K(i) values consistent with those reported in the literature, and 3) binding kinetics compatible with a 90 min incubation, and filtration for separation of free and bound radioligand. The results of these studies suggest that [(3)H]-SN56 may serve as a viable alternative to [³H](+)-pentazocine in radioligand binding assays.
Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, WV 26506, USA.
James A
JA
Fishback
Christophe
C
Mesangeau
Jacques H
JH
Poupaert
Christopher R
CR
McCurdy
Rae R
RR
Matsumoto
D000818:Animals; D001381:Azepines / Q000138:chemical synthesis / Q000378:metabolism*; D052160:Benzothiazoles / Q000138:chemical synthesis / Q000378:metabolism*; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D008297:Male; D010423:Pentazocine / Q000378:metabolism; D011485:Protein Binding; D011869:Radioligand Assay / Q000379:methods*; D019275:Radiopharmaceuticals / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017480:Receptors, sigma / Q000378:metabolism*; D000097605:Sigma-1 Receptor
D001381:Azepines; D052160:Benzothiazoles; D010423:Pentazocine; D019275:Radiopharmaceuticals; D017480:Receptors, sigma; D000097605:Sigma-1 Receptor; C557889:3-(2-(azepan-1-yl)ethyl)-6-propylbenzo(d)thiazol-2(3H)-one