Sigma-2 receptor (Kd = 0.12 nM)
Sigma-1 receptor (Kd = 17 nM) [1]

IC50 (sigma 1) = 17 nM, IC50 (sigma 2) = 0.12 nM, IC50 (5-HT1A) = 21000 nM, IC50 (5-HT1A) = 2000 nM, IC50 (D2) = 800 nM, and IC50 (alpha 1) = 330 nM are the binding affinities displayed by simeimesine hydrochloride[1]. In different cell lines (HaCaT, Hsc-4, HeLa and MCF-7, neuroblastoma cell line SH-SY5Y, and glioblastoma cell line U-87MG), siramesine hydrochloride (0-50μM; 8 hours) induces cell death[2]. In HaCaT and U-87MG cells, siramesine hydrochloride (0–40 μM; 2-48 hours) activates caspases[2].

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Siramesine induced cell death in various cell lines (HaCaT, Hsc-4, HeLa, MCF-7, SH-SY5Y, U-87MG) at concentrations above 20 μM within 8 hours, with 90% cell death at 40-50 μM. The pancaspase inhibitor Z-VAD-FMK largely reduced cell death in HaCaT and HeLa cells but not in MCF-7 cells (which lack caspase-3). Cysteine cathepsin inhibitor E-64d had no effect in any cell line. The lipophilic antioxidant α-tocopherol (0.3 mM) prevented cell death efficiently in all cell lines [1].
At concentrations below 20 μM, Siramesine did not affect viability within 8 hours but decreased viability after prolonged incubation (48 hours) in HaCaT cells [1].
Caspase activity (DEVDase) increased at all Siramesine concentrations tested. At ≥25 μM, DEVDase activity was detected much earlier (4 hours) compared to lower concentrations (48 hours). α-Tocopherol largely prevented caspase activation at 25 μM but had less effect at 40 μM. In U-87MG cells, caspase activation was considerably lower than in HaCaT cells [1].
Siramesine at ≥25 μM decreased mitochondrial membrane potential (MMP) in both HaCaT and U-87MG cells within 15 minutes, which was not prevented by Z-VAD-FMK or E-64d but was largely prevented by α-tocopherol. At ≤10 μM, most cells retained MMP until 48 hours [1].
Siramesine induced cardiolipin peroxidation (assessed by NAO staining) and major structural changes in mitochondria, largely prevented by α-tocopherol. In HaCaT cells, ≥25 μM induced cytochrome c release into cytosol within 15 minutes, prevented by α-tocopherol [1].
Siramesine generated significant ROS within 15 minutes, especially at high concentrations, efficiently blocked by α-tocopherol. At 10 μM, ROS peak occurred at 8 hours. The hydrophilic antioxidant N-acetyl-cysteine (8 mM) did not block viability or loss of MMP [1].
Transmission electron microscopy revealed apoptotic morphology (chromatin condensation, apoptotic bodies) in HaCaT cells treated with 25-40 μM Siramesine for 6 hours, prevented by α-tocopherol. U-87MG cells showed rounded shape and vacuolization at 40 μM but minimal apoptosis. In both cell lines, Golgi cisternae became enlarged and vacuolated, especially at higher concentrations; this was reduced but not prevented by α-tocopherol [1].
Siramesine caused rapid loss of LysoTracker Green accumulation at all concentrations tested (5-40 μM) within 15 minutes, not prevented by Z-VAD-FMK, E-64d, or α-tocopherol. However, no lysosomal membrane permeabilization (LMP) was detected by digitonin-based cathepsin activity assay or by cathepsin L immunodetection in cytosolic fractions, even after prolonged incubation. N-acetyl-β-D-glucosaminidase activity confirmed absence of LMP up to 7 hours at concentrations below 20 μM [1].
Siramesine caused accumulation of proforms of cathepsins L, B, and C without processing to mature forms, indicating impaired lysosomal degradation. The autophagic marker LC3-II accumulated with time; immunocytochemistry showed that LC3-positive vesicles did not colocalize with LAMP-2-positive vesicles, suggesting impaired fusion of autophagosomes with lysosomes [1].
Siramesine did not trigger ER membrane permeabilization (assessed by Fluo-4 intracellular calcium measurement) even at 40 μM within 30 minutes. Some ER swelling was observed in U-87MG cells at 40 μM after 6 hours, likely an indirect effect [1].

SA4503 or siramesine given jointly with MEM (as well as with AMA) decreased the immobility time in rats. Siramesine was previously reported to exhibit potent anticancer activity in a mouse cancer model (referenced as Ostenfeld et al., 2005), but no new in vivo experiments are presented in this manuscript [1].

Cell viability assay: Cells were plated in 24-well plates at 0.5×10^5 cells/well, treated with increasing Siramesine concentrations for 8-48 hours, then stained with annexin V and propidium iodide and analyzed by flow cytometry. Live cells defined as annexin V/PI double-negative. Each experiment performed in duplicate, at least two independent experiments [1].
Caspase activity measurement: Total cell extracts prepared in caspase buffer with detergents (50 mM HEPES, 200 mM NaCl, 10% sucrose, 0.1% CHAPS, 5 mM MgCl2, 0.02% BSA, 1% NP-40, 0.5% Triton X-100, pH 7.5), sonicated, centrifuged. 75 μg protein incubated with 10 μM fluorogenic substrate Ac-DEVD-AFC. Initial velocities measured at 37°C using microplate reader (excitation 400 nm, emission 505 nm). Three independent experiments in triplicate [1].
Mitochondrial membrane potential: Cells stained with JC-1 (3.5 μg/mL) for 15 minutes, analyzed by flow cytometry. Red cells considered as cells with intact mitochondria. Also NAO (50 nM) staining for cardiolipin integrity [1].
Lysosomal pH: Cells stained with LysoTracker Green DND-26 (40 nM) for 10 minutes, analyzed by flow cytometry [1].
Lysosomal membrane permeabilization assay: Cells treated with Siramesine, then incubated with digitonin (15-200 μg/mL) in acetate buffer (50 mM Na-acetate, 150 mM NaCl, 0.5 mM EDTA, pH 5.6) for 12 minutes on ice. Cysteine cathepsin activity measured fluorimetrically using Z-FR-AMC (30 μM) with 5 mM DTT (excitation 380 nm, emission 460 nm). Cytosolic activity (plasma membrane lysis only) vs total activity (all membranes lysed) compared [1].
Cytochrome c release: Cytosolic extracts prepared using digitonin (15-30 μg/mL), then Western blot with anti-cytochrome c antibody [1].
ROS measurement: Cells incubated with 5 μM CM-H2DCFDA for 40 minutes, then analyzed by flow cytometry [1].
Western blot: Total extracts in RIPA buffer, resolved on 12.5% or 15% SDS-PAGE, transferred to nitrocellulose, probed with antibodies against cathepsin L, LC3, β-actin [1].
Immunocytochemistry: Cells grown on poly-L-lysine-coated coverslips, fixed in cold methanol, blocked with 3% BSA, incubated with anti-LC3 (rabbit) and anti-LAMP-2A (mouse) primary antibodies, then with fluorescent secondary antibodies (green and red), mounted with ProLong Gold antifade, observed under fluorescence microscope [1].
Transmission electron microscopy: Cells fixed with 2% glutaraldehyde, postfixed with 2% OsO4 containing 1.5% potassium ferricyanide, stained en bloc with 1.5% uranyl acetate, dehydrated in ethanol series, embedded in epoxy resin. Ultrathin sections (60-70 nm) cut and examined with Philips CM100 TEM. Stereological analysis of Golgi cisternae using test grid: intersections of Golgi cisternal membranes counted, relative surface density calculated as ratio of intersection length to cytoplasmic area [1].

Rats

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Cell Death Dis.2013 Oct 3;4:e818.

Siramesine was originally developed by H. Lundbeck A/S. It was shown to be a potent inducer of cell death with anticancer activity. The compound has been reported to act as a lysosomotropic detergent in some studies (e.g., Ostenfeld et al., 2005, 2008), but the present study demonstrates that cell death is primarily mediated through mitochondrial destabilization rather than lysosomal membrane permeabilization. The compound also affects Golgi morphology and autophagic flux. It is unlikely that Siramesine acts exclusively through sigma-2 receptors; rather, it exhibits multiple molecular targets inside the cell [1].

C30H32CLFN2O

491.05

490.218

224177-60-0

Siramesine;147817-50-3;Siramesine fumarate;163630-79-3

9891778

White to off-white solid powder

225 °C

7.353

1

3

6

35

656

0

ILSRGIFRZZSGPN-UHFFFAOYSA-N

InChI=1S/C30H31FN2O.ClH/c31-25-12-14-26(15-13-25)33-21-23(27-9-2-4-11-29(27)33)7-5-6-18-32-19-16-30(17-20-32)28-10-3-1-8-24(28)22-34-30;/h1-4,8-15,21H,5-7,16-20,22H2;1H

1'-[4-[1-(4-fluorophenyl)indol-3-yl]butyl]spiro[1H-2-benzofuran-3,4'-piperidine];hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.09 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.09 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.09 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)