Functional assays to define agonists and antagonists of the sigma-2 receptor

doi:10.1016/j.ab.2013.12.008

Analytical Biochemistry

Volume 448, 1 March 2014, Pages 68-74

https://doi.org/10.1016/j.ab.2013.12.008 Get rights and content

Abstract

The sigma-2 receptor has been identified as a biomarker in proliferating tumors. To date there is no well-established functional assay for defining sigma-2 agonists and antagonists. Many sigma-2 ligands with diverse structures have been shown to induce cell death in a variety of cancer cells by triggering caspase-dependent and independent apoptosis. Therefore, in the current study, we used the cell viability assay and the caspase-3 activity assay to determine sigma-2 agonists and antagonists. Three classes of sigma-2 ligands developed in our laboratory were evaluated for their potency to induce cell death in two tumor cell lines, mouse breast cancer cell line EMT-6 and human melanoma cell line MDA-MB-435. The data showed that the EC₅₀ values of the sigma-2 ligands using the cell viability assay ranged from 11.4 μM to >200 μM, which were comparable with the EC₅₀ values obtained using the caspase-3 assay. Based on the cytotoxicity of a sigma-2 ligand relative to that of siramesine, a commonly accepted sigma-2 agonist, we have categorized our sigma-2 ligands into agonists, partial agonists, and antagonists. The establishment of functional assays for defining sigma-2 agonists and antagonists will facilitate functional characterization of sigma-2 receptor ligands and sigma-2 receptors.

Section snippets

Receptor binding assays

The sigma-1 and sigma-2 receptor binding affinities of sigma-2 ligands were determined as previously described [26]. Briefly, guinea pig brain (sigma-1 assay) or rat liver (sigma-2 assay) membrane homogenates (∼300 μg protein) were diluted with 50 mM Tris–HCl, pH 8.0 and incubated with either ∼5 nM [³H](+)-pentazocine (34.9 Ci/mmol; sigma-1 assay) or 1 nM [³H]RHM-1 (80 Ci/mmol; sigma-2 assay) in a total volume of 150 μl in 96 well plates at 25 °C. The concentrations of each compound ranged from 0.1 nM

Chemical structures and the sigma receptor binding affinities of the sigma-2 ligands

In the current study, 11 sigma-2 ligands developed in our laboratory including 6 azabicylononane (ABN) analogs, 4 benzamide analogs, and 1 tropane analog, were evaluated for the potency to induce cell death in the cell viability assay and the caspase-3 assay. Siramesine, commonly accepted as a sigma-2 agonist, was also included in these two assays as a control. The structures of these sigma-2 ligands and their affinities for sigma-1 and sigma-2 receptors are published previously and are shown

Discussion

Determination of sigma-2 agonists/antagonists is important for characterization of sigma-2 ligands for their biological effects. Well-characterized sigma-2 agonists and antagonists will facilitate their investigation as agents for imaging cell proliferation of solid tumors, for chemotherapy, and for studying the biological functions of sigma-2 receptors in cancer and in normal tissues. In the current study, we have proposed to use a cell viability assay and caspase-3 assay to define the

Acknowledgment

This study was supported by CA 102869 and U.S. Army Medical Research and Material Command under DAMD 17-01-1-0446.

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And fortunately, the orientation of σ2 receptor ligands within the binding pocket were highly similar in both molecular docking and isolated receptor crystals, lending further credence to computational methods. Functional assessment of σ2 receptor ligands was previously attempted in literature, contingent upon the ability to induce caspase-3 activation and apoptosis, and it was found that not all ligands with high σ2 receptor affinity cause apoptotic cell death, thus giving a basic functional characterization (agonist/partial agonist/antagonist) to σ2 receptor ligands (Zeng et al., 2014). However, pharmacodynamic uncertainty did not prevent investigators from targeting this receptor with all manner of ligands ranging from radioactive, fluorescent, and covalently-bond anticancer conjugates with promising results (Lee et al., 2016; Makvandi et al., 2016; Alamri et al., 2020a).
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¹: Current address: Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA

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Functional assays to define agonists and antagonists of the sigma-2 receptor

Abstract

Section snippets

Receptor binding assays

Chemical structures and the sigma receptor binding affinities of the sigma-2 ligands

Discussion

Acknowledgment

Biochem. Pharmacol.

Pharmacol. Ther.

Nat. Commun.

Eur. J. Pharmacol.

Life Sci.

Bioorg. Med. Chem.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Biochem. Pharmacol.

J. Steroid Biochem. Mol. Biol.

J. Biol. Chem.

Cell Metab.

Bioorg. Med. Chem.

Sigma Receptors: Historical Perspective and Background

The effects of morphine- and nalorphine- like drugs in the nondependent and morphine-dependent chronic spinal dog

J. Pharmacol. Exp. Ther.

Cloning and functional characterization of a sigma receptor from rat brain

J. Neurochem.