Hazards of Using Masking Protocols When Performing Ligand Binding Assays: Lessons From the Sigma-1 and Sigma-2 Receptors

Abstract

Sigma-1 and sigma-2 receptors are emerging therapeutic targets. Although the molecular identity of the sigma-2 receptor has recently been determined, receptor quantitation has used, and continues to use, the sigma-1 selective agents (+) pentazocine or dextrallorphan to mask the sigma-1 receptor in radioligand binding assays. Here, we have assessed the suitability of currently established saturation and competition binding isotherm assays that are used to quantify parameters of the sigma-2 receptor. We show that whilst the sigma-1 receptor mask (+) pentazocine has low affinity for the sigma-2 receptor (K i 406 nM), it can effectively compete at this site with [³H] di-O-tolyl guanidine (DTG) at the concentrations frequently used to mask the sigma-1 receptor (100 nM and 1 μM). This competition influences the apparent affinity of DTG and other ligands tested in this system. A more troublesome issue is that DTG can displace (+) pentazocine from the sigma-1 receptor, rendering it partly unmasked. Indeed, commonly used concentrations of (+) pentazocine, 100 nM and 1 μM, allowed 37 and 11% respectively of sigma-1 receptors to be bound by DTG (300 nM), which could result in an overestimation of sigma-2 receptor numbers in assays where sigma-1 receptors are also present. Similarly, modelled data for 1 μM dextrallorphan show that only 71-86% of sigma-1 receptors would be masked in the presence of 300 nM DTG. Therefore, the use of dextrallorphan as a masking agent would also lead to the overestimation of sigma-2 receptors in systems where sigma-1 receptors are present. These data highlight the dangers of using masking agents in radioligand binding studies and we strongly recommend that currently used masking protocols are not used in the study of sigma-2 receptors. In order to overcome these problems, we recommend the use of a cell line apparently devoid of sigma-1 receptors [e.g., MCF7 (ATCC HTB-22)] in the absence of any masking agent when determining the affinity of agents for the sigma-2 receptor. In addition, assessing the relative levels of sigma-1 and sigma-2 receptors can be achieved using [³H] DTG saturation binding followed by two-site analysis of (+) pentazocine competition binding with [³H] DTG.

Simple ammonium salts acting on sigma-1 receptors yield potential treatments for cancer and depression

“…The binding of [ 3 H] DTG to MCF7 cells showed a pK d ± SEM of [ 3 H] DTG 7.92 ± 0.04, K d = 12 nM, B max 1700 ± 400 fmol.mg −1 . We have recently shown that [ 3 H] DTG binding is reduced by (+) pentazocine with low affinity, further confirming the absence of sigma-1 receptors from MCF7 cells 20 .…”

“…Nonspecific binding was determined using 100 μM reduced haloperidol. No sigma-1 receptor "masking agent" (e.g., (+) pentazocine) was used, as this adversely affects the determination of K d and K i values at the sigma-2 receptor 20 . Assays were terminated by addition of ice-cold sigma-2 wash buffer (150 mM NaCl, 10 mM Tris-HCl; pH 7.2) and filtration, using a cell harvester, through glass fibre filters (GF/C, Sigma-Aldrich, Poole, UK).…”

Adamantane-derived scaffolds targeting the sigma-2 receptor; an in vitro and in silico study

“…The compiled data on cell lines reported to express σ2 receptors is shown below ( Table 4 ). These cell lines are HT29, A549/ATCC, MAD-MB-468 ( Abbas et al, 2020 ), MAD-MB-435 ( Zeng et al, 2007 ), OVCAR-3 ( Peluso et al, 2008 ), DU-145 ( Christy et al, 1999 ), and MCF7 ( Vilner et al, 1995 ). Our compounds showed varying degrees of growth inihibiton.…”

Pharmacokinetics and brain σ1 receptor occupancy of MR309, a selective σ1 receptor antagonist

“…SA4503 has been reported to have σ1R/σ2R ratio of approximately 100‐fold, with no significant affinity to 36 other receptors, transporters or channels 35,36 . However, using masking protocols with selective ligands, the σ1R/σ2R ratio was reported to be 14‐fold in guinea pig brain membranes, although such masking protocols were shown to carry their own challenges and these data should be treated with caution 37,38 . The magnitude of the PET signal is proportional to the ratio of target density and affinity (B max /K d ).…”

“…35,36 However, using masking protocols with selective ligands, the σ1R/σ2R ratio was reported to be 14-fold in guinea pig brain membranes, although such masking protocols were shown to carry their own challenges and these data should be treated with caution. 37,38 The magnitude of the PET signal is proportional to the ratio of target density and affinity (B max /K d ). The σ2R, a transmembrane ER-resident regulator of the Niemann-Pick carrier protein, 39 contributed to the PET signals obtained in this study.…”