SERM isomers reduce basal G-protein activity via CB1 and CB2Rs.

<p>The ability of SERMs to modulate basal G-protein activity via [A] CB1R, [B] CB2R and [C] MORs was evaluated by examining [³⁵S]-GTPγS binding in the presence or absence of a receptor-saturating concentration (10 μM) of all compounds. G-protein modulation by full agonists CP-55,940 (10 μM) and DAMGO (10 μM) was examined to serve as positive controls for activation of [A-B] CBRs and [C] MORs, respectively. G-protein modulation by the inverse agonists AM-281 and AM-630 was examined to serve as positive controls for regulation of [A] CB1 and [B] CB2R signaling. The mean ± SEM of [³⁵S]GTPγS binding is presented as percent of G-protein activity in the presence of vehicle. ^a,b[³⁵S]GTPγS binding produced by individual SERMs acting at hCB1 [A], hCB2 [B] or hMOR [C] receptors designated by different letters above bars, is significantly different (P<0.05, one-way ANOVA; Tukey <i>Post-hoc</i> test). *,**Bar graphs comparing E and Z isomers of individual SERMs that are designated by asterisks, are significantly different from activity at respective receptors (P<0.05, 0.01; student’s t-test).</p

Modulation of adenylyl cyclase activity by SERM isomers in CHO-hCB2 cells.

<p>Modulation of adenylyl cyclase activity by SERM isomers in CHO-hCB2 cells.</p

SERM isomer antagonism of CP-55,940 inhibition of AC-activity in intact CHO-hCB1 cells.

<p>SERM isomer antagonism of CP-55,940 inhibition of AC-activity in intact CHO-hCB1 cells.</p

SERM isomers modulate forskolin-stimulated AC activation via G_i/o proteins and CB2Rs.

<p>[A-C] Modulation of forskolin-stimulated cAMP production by SERMs (10 μM) in intact CHO-hCB2 and CHO-hMOR cells was evaluated. Drugs were examined in CHO-hCB2 cells (+/- 100 ng PTX pretreatment) and in CHO-hMOR cells not expressing CBRs. Intracellular cAMP values (mean ± SEM) are presented as percent response compared to levels observed in the presence of vehicle. Statistics revealed that no drug altered basal cAMP levels in CHO-hCB2 cells treated with PTX (P<0.01; student’s <i>t</i>-test) or in CHO-hMOR cells (P<0.01; one-sample <i>t</i>-test).</p

Antagonism of CP-55,940 inhibition of forskolin-stimulated AC activity by SERM isomers in intact CHO-hCB1 cells.

<p>CHO-hCB1 cells were pre-incubated for 30 min with receptor saturating concentrations of individual SERMs and were subsequently co-incubated for 7 min with increasing concentrations of CP-55,940. Measurements of CP-55,940 effects alone on potency (IC₅₀) and efficacy (E_MAX) of intracellular cAMP were obtained and were compared to the shifts in IC₅₀ and E_MAX values observed in [A-D]. All IC₅₀, EC₅₀, and K_B values (mean ± SEM) were derived from non-linear regression analysis of the curves shown in [A-D] and are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167240#pone.0167240.t003" target="_blank">Table 3</a> with statistical analysis. Open squares represent the concentration-effect curve for CP-55,940 alone, while filled symbols represent the action of CP-55,940 in the presence of the SERM indicated [A-C] or the selective CB1R inverse agonist AM-281 [D].</p

Structure of tamoxifen stereoisomers and subsequent cytochrome P450 metabolites.

<p>E- and Z-Tamoxifen (Tam) are metabolized by several CYP450 enzymes, the primary being CYP2D6 and CYP3A4/5, which yields stereoisomers of 4-hydroxytamoxifen (4OHT) and 4-hydroxy-N-desmethyl tamoxifen (End), respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167240#pone.0167240.ref033" target="_blank">33</a>].</p

Antagonism of CP-55,940 inhibition of forskolin-stimulated AC activity by SERM isomers in intact CHO-hCB2 cells.

<p>CHO-hCB2 cells were pre-incubated for 30 min with receptor saturating concentrations of individual SERMs and were co-incubated for 7 min with increasing concentrations of the agonist CP-55,940. Measurements of CP-55,940 effects alone on potency (IC₅₀) and efficacy (E_MAX) of intracellular cAMP were obtained and were compared to the shifts in IC₅₀ and E_MAX values observed in [A-D]. All IC₅₀, EC₅₀, and K_B values (mean ± SEM) were derived from non-linear regression analysis of the curves shown in [A-D] and are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167240#pone.0167240.t004" target="_blank">Table 4</a> with statistical analysis. Open squares represent the concentration-effect curve for CP-55,940 alone, while filled symbols represent the action of CP-55,940 in the presence of the SERM indicated [A-C] or the selective CB2R inverse agonist AM-630 [D].</p

Competition binding of SERM isomers employing CHO-hCB1 and CHO-hCB2 membranes.

<p>Competition binding of SERM isomers employing CHO-hCB1 and CHO-hCB2 membranes.</p

SERM isomers exhibit mid-nanomolar to low-micromolar affinities for CB1 and CB2Rs.

<p>A measure of affinity (K_i) of E and Z isomers of Tam, 4OHT, and End for respective CB1 and CB2Rs was obtained by conducting competition binding studies, employing 0.2 nM [³H]-CP-55,940 and increasing concentrations of test compounds. K_i values (mean ± SEM) were derived from non-linear regression analysis of the curves shown in [A-C]. Individual K_i values and statistical analysis of pK_i values are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167240#pone.0167240.t001" target="_blank">Table 1</a>. Filled squares and circles represent binding of respective E and Z isomers to CB1Rs, open squares and circles represent binding of respective E and Z isomers to CB2Rs.</p

Modulation of forskolin-stimulated cAMP production by SERM isomers in intact CHO-hCB1 and CHO-hCB2 cells.

<p>The potency (IC₅₀) and efficacy (E_MAX) for modulation of forskolin-stimulated AC was evaluated by analyzing concentration-effect curves for SERMs in intact CHO-hCB1 and CHO-hCB2 cells. All IC₅₀ and E_MAX values (mean ± SEM) were derived from non-linear regression analysis of the curves shown in [A-D] and are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167240#pone.0167240.t002" target="_blank">Table 2</a> with statistical analysis. For panels [A-C], filled squares and circles represent modulation of adenylyl cyclase activity by E- and Z-isomers acting at hCB1Rs, respectively, while open squares and circles demonstrate modulation by E- and Z-isomers at CB2Rs. In panel [D], modulation of adenylyl cyclase activity by the selective CB1R inverse agonist AM-281 (filled circles) and CB2R inverse agonist AM-630 (open circles) is depicted.</p