Synthesis and characterization of [3H]-SN56, a novel radioligand for the σ1 receptor

The study of the binding characteristics of a ligands in vivo and in vitro requires radiolabeled probes with high affinity and selectivity. The radioligand presently used for in vitro studies of the sigma(1) receptor, [H-3](+)-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 sigma(1) subtype, relative to sigma(2) and non-sigma, binding sites. The goal of this study was to characterize the binding of [H-3]-SN56 to sigma(1) receptors isolated from rat brain. Standard in vitro binding techniques were utilized to 1) determine the specificity and affinity of binding to sigma(1) receptors, 2) confirm that [H-3]-SN56 labels sites previously identified as sigma(1) by comparing binding to sites labeled by [H-3](+)-pentazocine, and 3) characterize the kinetics of binding. The results indicate that [H-3]-SN56 exhibits 1) specific, saturable, and reversible binding to the sigma(1) receptor, with B-max = 340 +/- 10 fmol/mg and K-d = 0.069 +/- 0.0074 nM, 2) competitive displacement by classical sigma compounds, yielding sigma K-1(i) values consistent with those reported in the literature, and 3) binding kinetics compatible with a 90 mm incubation, and filtration for separation of free and bound radioligand. The results of these studies suggest that [H-3]-SN56 may serve as a viable alternative to [H-3](+)-pentazocine in radioligand binding assays. (C) 2010 Elsevier B.V. All rights reserved.