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.