A 96-well filtration method for radioligand binding analysis of σ receptor ligands

Abstract

σ Receptors represent a potential drug target for numerous therapeutic indications including cancer, depression, psychostimulant abuse, and stroke. Most published radioligand binding studies for σ receptors utilize a low throughput method employing a “cell harvester.” Higher throughput methods are required to facilitate efficient screening of large numbers of novel compounds. In this study, a series of reference compounds was analyzed with a new medium-throughput 96-well filtration method and the results were compared to those obtained using the conventional cell harvester-based method. The 96-well assay utilized rat liver membranes for the determination of both known σ receptor subtypes (σ1 and σ2) because this tissue contains high densities of both subtypes and fulfills criteria required for reliable use with the 96-well format. The new method gave comparable Ki values for reference ligands analyzed in parallel with samples prepared in rat brain membranes and processed on the traditional cell harvester. For σ1 receptors, equivalent affinity values were observed for both methods/tissues. For σ2 receptors, approximately 2-fold higher affinities were observed for most compounds in liver, as compared to brain membranes, but excellent correlation with brain-derived values was maintained. To further demonstrate the utility of the new method it was used to screen a novel series of 2(3H)-benzothiazolone compounds, resulting in the identification of several analogues with nanomolar affinity and greater than 50-fold specificity for σ1 versus σ2 receptors.

Involvement of Sigma-1 Receptors in the Antidepressant-like Effects of Dextromethorphan

“…This interpretation would be consistent with other reports of multiple regions for ligand interactions on the σ 1 receptor, some of which have functional ramifications for agonist vs. antagonist activity [35], [36], [37]. The affinity differences of dextromethorphan for its two putative binding sites appear to be similar (<100-fold difference) since competition binding assays of dextromethorphan at σ 1 receptors are consistent with a one-site fit [27]. The antidepressant-like effects of dextromethorphan are thought to be mediated through the competitive binding site since i) there appears to be a rightward shift in its dose response curve in the forced swim test with no apparent change in maximal effect, and ii) (+)-pentazocine, the σ 1 agonist used to label the receptor, has previously also been reported to produce similar antidepressant-like effects [10], [17].…”

“…The concentration of dextromethorphan used in these assays was based on the reported K i of the drug for σ 1 receptors [27]. Briefly, 15 concentrations of [ 3 H](+)-pentazocine (0.1–100 nM) were used to label σ 1 receptors in P 2 rat brain homogenates (400–500 µg/sample).…”

Evaluation of the antidepressant potential of dextromethorphan

“…This interpretation would be consistent with other reports of multiple regions for ligand interactions on the sigma-1 receptor, some of which have functional ramifications for agonist vs. antagonist activity (Cobos et al, 2005;Wu and Bowen, 2008;Yamamoto et al, 1999). The affinity differences of DM for its two putative binding sites appear to be similar (<100-fold difference) since competition binding assays of DM at sigma-1 receptors are consistent with a one-site fit (Fishback et al, 2012). The antidepressant-like effects of DM are thought to be mediated through the competitive binding site since i) there appears to be a rightward shift in its dose response curve in the FST with no apparent change in maximal effect, and ii) (+)-pentazocine, the sigma-1 receptor agonist used to label the receptor, has previously also been reported to produce similar antidepressant-like effects (Fishback et al, 2010;Matsuno et al, 1996).…”

“…Earlier competition binding studies showed that DM has significant affinity for sigma-1 receptors (138-652 nM) (Chou et al, 1999;Fishback et al, 2012;Nam et al, 2012;Werling et al, 2007b), and thus further characterization of the interaction of DM with sigma-1 receptors was undertaken in the current study. The saturation binding studies indicate that the interaction of DM with sigma-1 receptors is complex, with both a change in Bmax and Kd in the binding of Together, the data support the presence of at least two distinct sites or modes of interaction with which DM binds to the sigma-1 receptor, one with which it has competitive interactions, and another with which it has non-competitive interactions.…”

Structure activity relationship study of benzo[d]thiazol-2(3H)one based σ receptor ligands

“…A library of benzo[ d ]thiazol-2( 3H )one analogues were synthesized and their receptor binding affinities were evaluated in rat liver membranes using a 96 well format high throughput methodology. 25 The −1 receptors were labeled with 5nM [ 3 H](+)-pentazocine. The −2 receptors were labeled with 5 nM [ 3 H]DTG in the presence of 300 nM (+)-pentazocine.…”