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

Abstract

Sigma-1 and sigma-2 receptors are emerging therapeutic targets. We have identified that simple ammonium salts bind to these receptors and are effective in vivo. Radioligand binding assays were used to obtain structure-activity relationships of these salts. MTS assays were performed to determine their effect on growth in MCF7 and MDA-MB-486 cells. Anticancer properties were tested in NMRI mice transplanted with a fragment of mouse adenocarcinoma (MAC13). Antidepressant activity was tested using the forced-swim test and tail suspension tests. Dipentylammonium (K_i 43 nM), tripentylammonium (K_i 15 nM) and trihexylammonium (K_i 9 nM) showed high affinity for the sigma-1 receptor. Dioctanoylammonium had the highest affinity (K₅₀ 0.05 nM); this also showed the highest affinity for sigma-2 receptors (K_i 13 nM). Dipentylammonium was found to have antidepressant activity in vivo. Branched-chain ammonium salts showed lower affinity. Bis(2-ethylhexyl)ammonium (K₅₀ 29 µM), triisopentylammonium (K₅₀ 196 µM) and dioctanoylammonium showed a low Hill slope, and fitted a 2-site binding model for the sigma-1 receptor. We propose this two-site binding can be used to biochemically define a sigma-1 receptor antagonist. Bis(2-ethylhexyl)ammonium and triisopentylammonium were able to inhibit the growth of tumours in vivo. Cheap, simple ammonium salts act as sigma-1 receptor agonists and antagonists in vivo and require further investigation.

Figure 1

Certain ammonium salt sigma-1 receptor ligands reduce cellular metabolism in MDA-MB-468 cells. Dioctylammonium (n = 4, filled circles), dicyclohexylammonium (n = 4, open circles), bis(2-ethylhexyl)ammonium (n = 6, filled squares) and triisopentylammonium (n = 6, open squares) reduced cellular activity in a concentration-dependent manner. Error bars show SEM. This figure is modified from the PhD thesis by J.M. Brimson.

Figure 2

Sigma-1 receptor siRNA hinders the ability of (a) bis(2-ethylhexyl)ammonium and (b) triisopentylammonium to reduce cellular metabolism in MDA-MB-468 cells. Levels of binding of 30 nM [³H] (+) pentazocine (c) are also shown. SiRNA targeting the sigma-1 receptor caused a shift in the pIC₅₀ for bis(2-ethylhexyl)ammonium from 3.37 to 2.7 (P = 0.044, unpaired t-test), and for triisopentylammonium from 2.9 to 2.3 (P = 0.0095, unpaired t-test). Error bars show SEM, n = 3. SiRNA also caused a reduction in the sigma-1 receptor number, as monitored using a fixed concentration (30 nM) of [³H] (+) pentazocine (P < 0.0001, unpaired t-test, n = 8).This figure is reproduced from the PhD thesis by J.M. Brimson. Panel (c) has been previously presented.

Figure 3

The effect of dipentylammonium (DPA, 5 mg.kg⁻¹) and sigma-1 receptor antagonist BD1047 (4 mg.kg⁻¹) on despair behaviour in mice subjected to the forced-swim test. The control consisted of 10 ml.kg⁻¹ saline; fluoxetine (10 mg.kg⁻¹) was used as a positive control. Five mice were used for each data point. Error bars show SEM. ANOVA followed by Bonferroni post hoc multiple comparison gave: *control vs fluoxetine, P 0.0022; **control vs dipentylammonium, P < 0.0001; control vs BD1047, P = 0.13; ^##dipentylammonium vs BD1047 + dipentylammonium, P = 0.0001. This figure is reproduced from the PhD thesis by J.M. Brimson.

Figure 4

The effect of dipentylammonium (DPA, 5 mg.kg⁻¹) and sigma-1 receptor antagonist BD1047 (4 mg.kg⁻¹) on despair behaviour in mice subjected to the tail suspension test. The control consisted of an injection of 10 ml.kg⁻¹ saline; fluoxetine (10 mg.kg⁻¹) was used as a positive control. Ten mice were used for each data point, except BD1047 + dipentylammonium (n = 5). Error bars represent SEM. ANOVA followed by Bonferroni’s post hoc multiple comparison gave: **control vs fluoxetine P = 0.005, **control vs dipentylammonium, P = 0.0001; control vs BD1047 + dipentylammonium, P = 0.358; dipentylammonium vs BD1047 + dipentylammonium, P = 0.15. This figure is reproduced from the PhD thesis by J.M. Brimson.

Figure 5

The effects of dipentylammonium and triisopentylammonium on serotonin, dopamine and norepinepherine reuptake. (a) fluoxetine (open squares) dose-dependently prevents serotonin reuptake (IC₅₀ 83 nM), whereas dipentylammonium (open circles) and triisopentylammonium (closed squares) required a far higher concentration to prevent any serotonin uptake (IC₅₀ 1.1 mM and IC₅₀ 7.8 mM respectively). (b) GBR-12935 (open squares) inhibited dopamine transport with IC₅₀ of 76 nM whereas dipentylammonium (open circles) and triisopentylammonium (closed squares) required a far higher concentration to prevent any dopamine uptake (IC₅₀ 0.6 mM and IC₅₀ 1.6 mM respectively). (c) Desipramine (open squares), potently inhibited norepinepherine (NE) reuptake with IC₅₀ 9 nM, whereas dipentylammonium (open circles) and triisopentylammonium (closed squares) required a far higher concentration to prevent any norepinepherine uptake (IC₅₀ 0.2 mM and IC₅₀ 7.8 mM respectively).

Figure 6

The effects of bis(2-ethylhexyl)ammonium and triisopentylammonium on metabolism of MAC13 cells in the MTS assay. Bis(2-ethylhexyl)ammonium (open circles) had a mean pIC₅₀ of 5.3 ± 0.2, and triisopentylammonium (filled squares) had a mean pIC₅₀ of 4.4 ± 0.2 from 6 independent assays. This figure is modified from the PhD thesis by J.M. Brimson.

Figure 7

The prevention of MAC13 tumour growth in vivo by bis(2-ethylhexyl)ammonium (open circles) and triisopentylammonium (filled squares) compared to vehicle treated mice (filled circles). Drug treatment commenced 12 days after implantation of the tumour. Error bars represent SEM from 6 mice. ^*Statistically significant tumour growth from day 12, determined using 2-way repeated measures ANOVA with Tukey’s post hoc test, P < 0.001. ^#Statistically significant reduction in tumour growth compared to the equivalent day of in the control, P < 0.001. This figure is modified from the PhD thesis by J.M. Brimson.