PGRMC1 has been reported to interact with multiple proteins including Insig-1, SCAP, P450 proteins, PAIR-BP1, and epidermal growth factor (EGFR)[29C33]. of siramesine, a generally accepted MMV008138 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. for sigma-1 receptors (nM)for sigma-2 receptors (nM) /th th valign=”top” align=”right” rowspan=”1″ colspan=”1″ Recommendations /th /thead Azabicyclononane AnalogsSV1191417.05.2aSV1666292.028.3aWC-261436.02.6b2b262.05.9cYUN2452250.05.0cSV9592.53.1cBenzamide AnalogsRHM-13078.010.3dISO-1330.07.0eYUN2345484.012.4dYUN25212900.08.2dTropane AnalogRHM-138544.012.3fSiramesine Analogsiramesine17.00.12g Open in a separate windows a b c d e fThe chemical structure of RHM-138 is usually published previously . The binding affinities of RHM-138 for sigma-1 and sigma-2 affinities are not published previously. g The determination of the potency of sigma-2 ligands to induce cell death in the cell viability assay and the caspase-3 assay The EC50 values of the sigma-2 ligands were decided in mouse breast cancer cell collection EMT-6 and human melanoma MMV008138 cell collection MDA-MB-435 using the cell viability assay. The data are shown in Table 2. The dose-response curves for the representative sigma-2 ligands (SV119, WC-26, RHM-1 and siramesine) are shown in Fig. 2A and C. Siramesine exhibited the most potent cytotoxicity with the EC50 values of 5.3 M and 9.3 M in EMT-6 and MDA-MB-435, respectively, after 48 hour treatment. The data are consistent with the EC50 values of siramesine in other cell lines reported previously [23, 27]. Open in a separate window Fig. 2 The dose response curves of the sigma-2 ligands in cell viability assay and caspase-3 activation assay. EMT-6 cells (A and B) or MDA-MB-435 cells (C and D) were treated with increasing concentrations of SV119 (), siramesine (), WC-26 (), or RHM-1 () for 24 h. The cell viability and caspase-3 activation were then determined by MTS assay (A and LEPREL2 antibody C) and caspase-3 assay (B and D), respectively. The bars represent mean SD in the representative experiment performed in triplicates. Table 2 MMV008138 EC50 of sigma-2 ligands in EMT-6 and MDA-MB-435 cell lines using MTS assay thead th valign=”top” align=”left” rowspan=”1″ colspan=”1″ /th th colspan=”2″ valign=”top” align=”center” rowspan=”1″ EMT-6 /th th colspan=”2″ valign=”top” align=”center” rowspan=”1″ MDA-MB-435 /th th colspan=”5″ valign=”bottom” align=”left” rowspan=”1″ hr / /th th valign=”top” align=”left” rowspan=”1″ colspan=”1″ Compound /th th valign=”top” align=”right” rowspan=”1″ colspan=”1″ EC50 (M, 24h) /th th valign=”top” align=”right” rowspan=”1″ colspan=”1″ EC50 (M, 48h) /th th valign=”top” align=”right” rowspan=”1″ colspan=”1″ EC50 (M, 24h) /th th valign=”top” align=”right” rowspan=”1″ colspan=”1″ EC50 (M, 48h) /th /thead Azabicyclononane AnalogsSV11916.0 1.411.4 1.736.7 3.318.5 1.9SV16630.0 1.018.5 2.134.1 4.425.0 5.57WC-2642.5 3.512.3 1.649.7 2.544.9 5.32b65.5 7.129 1.463.1 5.833.3 3.26YUN245 200154.5 30.4136.9 4.6101.0 15.1SV95 200 200 200 200Benzamide AnalogsRHM-1 200 200 200 200ISO-1 200 200 200 200YUN234 20091 12.7 200 200YUN252 200137.5 3.5 200 200Tropane AnalogSiramesine Analogsiramesine14.9 4.05.3 1.09.4 0.49.3 0.9 Open in a separate window Azabiocyclonane analogs exhibited different potency of cytotoxicity with EC50 values ranging from 11.4 M to 200 M. Benzamide analogs, YUN234, YUN252, MMV008138 RHM-1 and ISO-1, showed little cytotoxicity (Table 2 and Fig. 3). Tropane analog RHM-138 exhibited potent cytotoxicity. Open in a separate windows Fig. 3 Cytotoxicity of sigma-2 ligands using MTS assay. EMT-6 and MDA-MB-435 cells were treated with 50 M of sigma-2 ligands for 24h. MTS assay was then performed and cytotoxicity of the sigma-2 ligand was decided using the formula 1 and 2 explained in Material and Methods. The bars represent mean SD in three impartial experiments performed in triplicates. The EC50 values of the sigma-2 ligands were also decided using the caspase-3 assay in both EMT-6 and MDA-MB-435 cell lines. The data are shown in Table 3. The dose response curves for the.