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Several recent advances are leading to a better understanding of sigma receptors. Here we focus on our recent findings regarding cellular functions of sigma-2 receptors and discuss their possible clinical implications. Agonists at sigma-2 receptors induced changes in cell morphology and apoptosis in various cell types. Sigma-2 receptor activation produced both transient and sustained increases in [Ca++]i, derived from different intracellular stores. These changes in [Ca++]i and cytotoxic effects are mediated by intracellular sigma-2 receptors. Sigma-2 agonists induced apoptosis in drug-resistant cancer cells, enhanced the potency of DNA damaging agents, and down-regulated expression of p-glycoprotein mRNA. Thus, sigma-2 receptor agonists may be useful in treatment of drug-resistant cancers. Sigma radioligands have been used in tumor imaging. We also discuss how sigma-2 antagonists might prevent the irreversible motor side effects of typical neuroleptics. Sigma-2 receptors may subserve a novel signalling pathway to apoptosis, involved in regulation of cell proliferation and/or viability.

Recently, we demonstrated that sigma-2 receptors may have the potential to be a biomarker of tumour cell proliferation (Mach et al (1997) Cancer Res57: 156–161). If sigma-2 receptors were a biomarker of tumour cell proliferation, they would be amenable to detection by non-invasive imaging procedures, thus eliminating many of the problems associated with the flow cytometric measures of tumour cell proliferation presently used in the clinic. To be a good biomarker of tumour cell proliferation, the expression of sigma-2 receptors must be essentially independent of many of the biological, physiological, and/or environmental properties that are found in solid tumours. In the investigation reported here, the mouse mammary adenocarcinoma lines, 66 (diploid) and 67 (aneuploid), 9L rat brain tumour cells, and MCF-7 human breast tumour cells were used to study the extent and kinetics of expression of sigma-2 receptors in proliferative (P) and quiescent (Q) tumour cells as a function of species, cell type, ploidy, pH, nutrient depletion, metabolic state, recruitment from the Q-cell compartment to the P-cell compartment, and treatment with tamoxifen. In these experiments, the expression of sigma-2 receptors solely reflected the proliferative status of the tumour cells. None of the biological, physiological, or environmental properties that were investigated had a measurable effect on the expression of sigma-2 receptors in these model systems. Consequently, these data suggest that the proliferative status of tumours and normal tissues can be non-invasively assessed using radiolabelled ligands that selectively bind sigma-2 receptors. © 1999 Cancer Research Campaign

1-Benzyl-4-(N-2-pyrrolidinylethoxy)benzene (PBPE) is a cytotoxic derivative of the antitumoral drug tamoxifen. PBPE binds with high-affinity and specificity to the microsomal antiestrogen-binding site (AEBS). PBPE, as well as some other high-affinity AEBS ligands, shares structural features with high-affinity and selective sigma receptor ligands in the N-(arylethyl)-N-alkyl-2-(1-pyrrolidinyl)ethylamine class, such as BD1008, which are cytotoxic against tumoral cells. Based on these structural and pharmacological similitudes, we set out to examine whether AEBS and sigma receptors could be related binding sites. We showed that BD1008 had a high affinity for AEBS. However, prototypical sigma receptor ligands were very low-affinity competitors on AEBS. Surprisingly, AEBS ligands displayed a high affinity for sigma-1 and sigma-2 receptor subtypes, showing that AEBS and sigma receptor-binding sites were not mutually exchangeable. Moreover, phenytoin, which is an allosteric modulator of sigma-1 receptor, was a competitive inhibitor of [3H]tamoxifen on AEBS. These results suggest that the tamoxifen-binding site on AEBS and the sigma ligand-binding site on sigma receptors were not identical but related entities. We also showed here that the high-affinity and specific AEBS ligands also bound sigma receptors with high affinity. Moreover, the compounds that were capable of displacing tamoxifen from AEBS were cytotoxic against tumoral cells but not against the AEBS-deficient cell line Rtx-6. These results confirm that AEBS and sigma receptors might belong to the same family of proteins, and that the tamoxifen-binding site might be involved in the cytotoxicity of AEBS ligands and some classes of sigma compounds.

We have isolated a cDNA clone from a human placental choriocarcinoma cell (JAR) cDNA library which codes for a functional type 1 sigma receptor. An RT-PCR product obtained from guinea pig kidney mRNA using primers specific for the guinea pig sigma receptor cDNA was used to screen the cDNA library. The hSigmaR1 cDNA predicts a protein of 223 amino acids with a single putative transmembrane domain. The amino acid sequence exhibits 93% identity with the guinea pig sigma receptor. When functionally expressed in HeLa cells, the hSigmaR1 cDNA enhances the binding of [3H]-haloperidol, a sigma receptor ligand, to the HeLa cell membranes. The inhibitor specificity of the cloned hSigmaR1 indicates that it is the type 1 sigma receptor. Several human tissues, including placenta, liver, and brain, and several human cell lines express the SigmaR1 mRNA (1.7 kb) to a variable extent.

The determination of the structure and function of the sigma receptor subtypes and their physiological role(s) has been impeded by the unavailability of selective ligands. We have developed a new class of sigma subtype selective receptor ligands that are (E)-8-benzylidene derivatives of the synthetic opioid (+/-)-, (+)-, and (-)-2-methyl-5-(3-hydroxyphenyl)morphan-7-one (1). The derivatives can be prepared by reaction of 1, (+)-1, and (-)-1 with the appropriate benzaldehyde under Claisen-Schmidt conditions. Incorporation of substituted (E)-8-benzylidene moieties onto the 7-keto precursor of (+)-2-methyl-5-(3-hydroxyphenyl)morphan, (+)-1, produces compounds (-)-2 through (-)-7 (5.8-32.0 nM, sigma 1), which have between a 25- and 131-fold increase in affinity for the sigma 1 receptor subtype relative to the keto precursor (+)-1 (Ki = 762 nM, sigma 1). Compound (-)-2 is the most selective of this group (16-fold) for the sigma 1 subtype versus sigma 2. Substitution of an (E)-8-benzylidene moiety onto the 7-keto precursor of (-)-2-methyl-5-(3-hydroxyphenyl)morphan, (-)-1, produces compounds (+)-2-(+)-9 (6.4-52.6 nM, sigma 2), which have at least a 475-3906-fold increase in affinity for the sigma 2 receptor subtype relative to the keto precursor (-)-1 (Ki = 25 x 10(3) nM). This enhancement of sigma 2 receptor affinity is accompanied by substantial selectivity of all of these dextrorotatory products for the sigma 2 relative to the sigma 1 subtype (32-238-fold), and thus, they are among the most sigma 2 selective compounds currently known. Furthermore, the sigma 1 subtype is highly enantioselective for the levorotatory isomers, (-)-2-(-)-7 (41-1034-fold), whereas the sigma 2 subtype is only somewhat enantioselective for the dextrorotatory isomers, (+)-2-(+)-7 (2.6-9.3-fold). All of these derivatives retain substantial affinity for the mu opioid receptor. Despite the high affinity of the dextrorotatory derivatives for the mu opioid receptor, the high affinity and selectivity for sigma 2 over sigma 1 sites will surely prove beneficial as tools for the delineation of the function and physiological role of sigma 2 receptors.

A series of 4-(1H-indol-3-yl)-1-butyl-substituted 4-phenylpiperidines, 4-phenyl-1,2,3,6-tetrahydropyridines, and 4-phenylpiperazines was synthesized. The phenyl group was optionally substituted with 4-fluoro or 2-methoxy substituents. High affinity for both sigma 1 and sigma 2 binding sites was achieved with these compounds. Additionally, these compounds had relatively high affinity for serotonin 5-HT1A and 5-HT2A, dopamine D2, and adrenergic alpha 1 receptors. Introduction of a 4-fluorophenyl substituent at the indole nitrogen atom rendered very selective sigma 2 ligands with subnanomolar affinity for the sigma 2 binding site. The prototype of such a compound was 1-(4-fluorophenyl)-3-[4-[4-(4-fluorophenyl)-1-piperidinyl]-1-butyl]-1H- indole, 11a (code no. Lu 29-253). This compound had the following binding affinities: IC50 (sigma 1) = 16 nM, IC50 (sigma 2) = 0.27 nM, IC50 (5-HT1A) = 22,000 nM, IC50 (5-HT2A) = 270 nM, IC50 (D2) = 4200 nM, IC50 (alpha 1) = 220 nM. Spiro-joining of the phenyl and the piperidine rings into a spiro[isobenzofuran-1(3H),4'-piperdine] ring system resulted in even more selective compounds. Variations of the 1-substituent at the indole and of the chain length of the alkylene spacer group were studied. The optimal compound was the spiro analogue of compound 11a. This compound is 1'-[4-[1-(4-fluorophenyl)-1H-indol-3-yl]-1-butyl]spiro[isobenzofuran- 1(3H),4'-piperidine], 14f (code no. Lu 28-179), with the binding affinities: IC50 (sigma 1) = 17 nM, IC50 (sigma 2) = 0.12 nM, IC50 (5-HT1A) = 21,000 nM, IC50 (5-HT2A) = 2000 nM, IC50 (D2) = 800 nM, IC50 (alpha 1) = 330 nM. However, the most selective sigma 2 versus sigma 1 ligand was the tropane derivative 1-(4-fluorophenyl)-3-[4-[3-(4-fluorophenyl)-8-azabicyclo[3.2.1]oct-2- en-8-yl]-1-butyl]-1H-indole, 15a. This compound had the following binding affinities: IC50 (sigma 1) = 1200 nM, IC50 (sigma 2) = 2.5 nM. Potent anxiolytic activity in the black/white box exploration test in rats was found with the two most prominent sigma 2 ligands Lu 29-253 and Lu 28-179. Good penetration into the CNS was documented both after subcutaneous and peroral administration of Lu 28-179 by ex vivo binding studies. Long duration of action was demonstrated both in ex vivo binding (T1/2 approximately 20 h) and in the black/white box exploration test.

Four members of a novel class of sigma (σ) ligands were investigated for σ subtype selectivity. (−)-1 S ,5 S - and (+)-1 R ,5 R -( E )-8-Benzylidene-5-(3-hydroxyphenyl)-2-methylmorphan-7-one (CB-64L and CB-64D, respectively) exhibited σ 1 K 1 = 10.5 nM and 3063 nM; σ 2 K i = 154 nM and 16.5 nM, respectively. The corresponding 3,4-dichloro derivatives, (−)-1 S ,5 S - and (+)-1 R ,5 R -( E )-8-(3,4-dichlorobenzylidene)-5-(3-hydroxyphenyl)-2-methylmorphan-7-one (CB-182 and CB-184, respectively) were also examined. CB-182 ((−)-isomer) showed σ 1 and σ 2 K i = 27.3 nM and 35.5 nM, respectively, whereas CB-184 ((+)-isomer) exhibited σ 1 and σ 2 K i = 7436 nM and 13.4 nM, respectively. Thus, the two σ subtypes showed opposite enantioselectivity for these compounds, with (−) > (+) at σ 1 and (+) > (−) at σ 2 . Importantly, CB-64D and CB-184 showed high σ 2 affinity and, respectively, 185-fold and 554-fold selectivity for σ 2 receptors over σ 1 . While high σ 2 selectivity relative to σ 1 was achieved with these compounds, they both exhibited high affinity at mu (μ) opioid receptors ( K i = 37.6 nM and 4.5 nM, respectively). Despite this, CB-64D and CB-184 will be useful tools for further characterization of σ 2 receptors.

Cancers of the lungs and head and neck are one of the leading causes of death, particularly in Kentucky, which leads the nation in multiple categories related to smoking. Thus, there is an urgent need for new biomarkers and therapeutic targets for airway cancers. The sigma-2 receptor is an intracellular receptor for numerous drugs and metabolites and was recently found to be identical to Pgrmc1 (progesterone receptor membrane component 1), a cytochrome-related protein implicated in metabolism and hormone signaling. S2RPgrmc1 is up-regulated in multiple types of cancer and is required for tumor cell proliferation and motility in vitro and tumor formation and metastasis in vivo. Furthermore, small molecule inhibitors of S2RPgrmc1 suppressed the growth of lung, breast and cervical cancer cell lines. In the present study, we have analyzed S2RPgrmc1 levels in clinical tumor samples from squamous cell lung cancers (SqCLC), lung adenocarcinomas, head and neck cancers and others. S2RPgrmc1 levels were increased in tumors throughout and correlated with survival in lung cancer (n=43, p=0.02) and with grade in head and neck cancer (higher in grade 2 versus 1, p=0.007). One of the cancer types with highest S2RPgrmc1 expression was squamous cell lung cancer (SqCLC), and treatment options for SqCLC are limited. S2RPgrmc1 was highly expressed in SqCLC cell lines, and SqCLC cell survival was inhibited by siRNA knockdown of S2RPgrmc1 or the S2RPgrmc1 inhibitor AG-205. S2RPgrmc1 is part of a family of secreted cytochromes, and we found that S2RPgrmc1 was significantly elevated in the plasma of 42 lung cancer patients compared to non-cancer patients and was significantly higher in the plasma of stage I lung cancer patients compared to stage 2-4 patients. Purified, recombinant S2RPgrmc1 increased the proliferation of NSCLC cell lines, suggesting that secreted S2RPgrmc1 may contribute to lung tumor growth. Together, the results demonstrate that S2RPgrmc1 correlates with key clinical endpoints in tumors and is a therapeutic target in some cancers with few therapeutic options, particularly squamous cell lung cancer. Furthermore, S2RPgrmc1 is a potential plasma biomarker for early stage lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3604. doi:1538-7445.AM2012-3604

Sigma-2 receptor binding sites, recently identified as progesterone receptor membrane component 1 (PGRMC1) (Nature Communications, 2011; 2:380), are highly expressed in tumor cells. We have previously reported that measuring the sigma-2 receptor expression is a novel strategy for evaluating the proliferative status of tumors in vivo using the non-invasive imaging technique, positron emission tomography (PET). Our group has developed a series of sigma-2 receptor radioligands as PET imaging probes, one sigma-2 receptor radiotracer [18F]ISO-1 is under clinical evaluation for imaging the proliferation status in the patients with different types of cancers. PGRMC1 depleted human ovarian SKOV-3 cancer cells have been reported to lack tumor formation in mice and PGRMC1 depleted tumors displayed poor microvasculature system (Endocrinology, 2009; 150:4846-4854). To further understand the regulation of PGRMC1 in cell cycle and cancer proliferation, we have stably transfected PGRMC1-eYFP, enhanced yellow fluorescent protein conjugated PGRMC1, into human pancreatic BxPC3 cancer cells and those cells were transplanted into immunodeficient nude mice to grow into solid tumors; subsequently, a traditional fluorescence imaging system and an optical-resolution photoacoustic microscopy (OR-PAM) were utilized for in vivo monitoring of BxPC3 cancer cells growth and observing the surrounding microvasculature development for those tumors under conditions of sigma-2 receptor/PGRMC1 overexpression. Tumor tissue sections were prepared and in vitro fluorescence microscopy analysis of PGRMC1-eYFP cells revealed that PGRMC1-eYFP overexpressed cells form cluster patterns which may represent the proliferation priority created by PGRMC1 overexpression. The subcellular localization of PGRMC1-eYFP appeared to be similar to that of sigma-2 receptors reported previously by our group using the sigma-2 receptor fluorescent probes (Cancer Res 2007; 67, 6708-6716). PGRMC1-eYFP BxPC3 cells were surrounded by fully developed microvasculature system. Further application of this in vivo imaging platform for evaluating the sigma-2 receptor pharmacology and imaging proliferation is ongoing in our group. (Supported by CA 102869 and CA136398). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 356. doi:1538-7445.AM2012-356

The sigma-2 receptor is overexpressed in various human tumors. Sigma-2 receptor selective radiotracers have been shown to target various solid tumors in rodents and in human patients using positron emission tomography. Sigma-2 receptor ligands can be internalized into tumor cells by the endocytotic pathway. Therefore, sigma-2 receptor ligands are excellent candidates as tumor-targeted delivery agents when covalently attached to drugs. Smac is a protein released from mitochondria into the cytosol in response to apoptotic stimuli. Small-molecule Smac mimetic compounds (SMC) have been developed by several laboratories as anticancer drugs. In this study we validate sigma-2 ligand as a tumor-targeting drug delivery agent for treating ovarian cancer. We have synthesized sigma-2 ligand-conjugated SMC (SSMC), SW III-123. Our data show that SW III-123 has adequate sigma-2 receptor binding affinities (Kiα2 =190 nM and Kiα1 =2046 nM). SW III-123 exhibits potent antitumor activities with EC50 values at 4.0 μM, 2.3 μM and 2.4 μM in human ovarian cancer cell lines SKOV-3, CaOV-3, and BG-1, respectively. In contrast, unconjugated SMC, SW IV-52s, shows little cytotoxicity in these cell lines (EC50 > 200 μM). These results suggest that the sigma-2 ligand has successfully delivered SMC into ovarian cancer cells. Western blot results show that SW III-123 degrades inhibitor of apoptosis proteins (IAPs), XIAP and cIAP-1, in SKOV-3 cells, possibly by interacting with the baculovirus IAP repeat (BIR) domains of IAPs. SW III-123 cleaves caspase-3, 8, and 9 dose-dependently in SKOV-3 cells, indicating that SW III-123 kills ovarian cancer cells by activating both intrinsic and extrinsic apoptotic pathways. In conclusion, sigma-2 ligand is a promising tumor-targeting drug delivery agent. SSMC will likely offer a novel class of therapeutic drugs for treating ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5690. doi:1538-7445.AM2012-5690

The interactions of a new compound SR 31747 with sigma sites were examined in rat spleen membranes and in human peripheral blood leukocytes (PBL). Nanomolar concentrations of SR 31747 selectively inhibited in a non-competitive manner the binding of the prototypic sigma ligands [3H](+)-pentazocine, [3H]-3PPP and [3H]DTG on rat spleen membranes. Characterization of SR 31747 binding sites using [3H]SR 31747 as a ligand showed that this compound binds reversibly, with high affinity to one class of sites on rat spleen membranes (Kd 0.66 nM, Bmax 5646 fmol/mg protein). The pharmacological profile of [3H]SR 31747 binding sites was consistent with the presence of specific sites distinct from classical sigma 1 and sigma 2 receptor subtypes strongly suggesting an allosteric modulation of sigma sites by SR 31747. Similarly, [3H]SR 31747 binding sites were demonstrated on human PBL and also on purified subpopulations of human mononuclear cells (granulocytes, NK cells, T4, T8 and B lymphocytes). Administered to mice by i.p. or oral route 30 min before sacrifice, SR 31747 strongly inhibited the binding of [3H](+)-3PPP to mice spleen membranes with ED50 values of 0.18 and 1.43 mg/kg, respectively. Taken together these results could suggest a potential immunological activity of SR 31747 either directly or through allosteric modulation of peripheral sigma sites.

Rat liver and kidney were investigated for the presence of sigma (σ) receptor subtypes by radioligand binding with three highly selective σ probes and by photoaffinity labeling using [H]azido-di-o-tolyguanidine ([3H]azido-DTG). [3(+)- Pentazocine, a highly selective σ1 probe, bound to sites in liver membranes with Kd = 7.5 nM and Bmax = 2929 fmol/mg protein. [3H](+)-Pentazocine binding sites in kidney had Kd = 23.3 nM and Bmax = 229fmol/mg protein. [3H1,3- Di-o-tolylguanidine ([3H]DTG) and [3H](+)-3-(3-hydroxyphenyl-N-(1-propyl)piperidine ([3H](+)-3-PPP) label both σ1 and σ2 receptors. Parameters for [3H]DTG in the liver were Kd = 17.9 nM and Bmax - 11 895 fmol.mg protein. Similar parameters were observed for [3H](+)-3-PPP, Kd = 51.9 nM and Bmax = 11 070 fmol/mg protein. [3H]DTG bound to rat kidney with Kd = 45.8 nM and Bmax = 1190 fmol/mg protein. The observation that either [3H]DTG or [3H](+)-3-PPP labeled a higher number of sites relative to [3H](+)-pentazocine suggested that liver and kidney contain both subtypes of σ receptor. This was confirmed by competition studies vs. [3H](+)-pentazocine and [3H]DTG (in the presence of dextrallorphan to mask σ1 sites). In both tissues, [3H](+)-pentazocine labeled sites with high affinity for haloperidol and enantioselectivity for (+)-benzomorphans over (−)-benzomorphans. [3H]DTG + dextrallorphan labeled sites in both tissues which also high affinity for haloperidol, but which had the characteristic σ2 property of low affinity for (+)-benzomorphans and enantioselectivity for (−)-benzomorphans over the corresponding (+)-isomer. Similar results were obtained with [3H](+)-3-PPP + dextrallorphan. Several novel aryl diamines, such as 1S, 2R-cis-N-[2-(3,4-dichlorophenylethyl]-N-methyl-2-(1-pyrrolidinyl)cyclohexylamine BD737) and N-2- (3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine (BD1008), bound to both sites with high affinity. Photoaffinity labeling with 10 nM [3H]azido-DTG resulted in specific labeling of polypeptides of 25 kDa and 21.5 kDa and 21.5 kDa. Dextrallorphan (100 nM or 500 nM) completely blocked labeling of the 25 kDa polypeptide, but had no effect on labeling of the lower molecular weight protein. (+)-10,11-Dihydro-5-methyl-5H-dibenzol[a,d]cyclohepten-5,10-imine((+)_-MK-801) had no effect on labeling of either polypeptide. These data are consistent with the notion that the 25 kDa and 21.5 proteins represent σ2 and σ2 receptors, respectively. Thus, rat liver and kidney contain high densities of σ1 and σ2 receptors, with σ2 sites comprising 75–80% of the total σ population. The high density of σ receptor subtypes may indicate important functions in hepatic and renal tissues.

A series of polyamines based on the high affinity sigma receptor ligand N-[2-(3,4-dichlorophenyl)-ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine (3) were developed and evaluated for their binding characteristics at sigma-1 and sigma-2 receptor subtypes. The data indicated that a considerable degree of structural variation is possible while still retaining nanomolar affinity at sigma receptors. As the structure of the polyamines was varied, their binding at sigma-1 and sigma-2 subtypes showed quite different and in some cases opposite trends, supporting the belief that these are pharmacologically distinct entities. Polyamines containing two nitrogen atoms showed optimal binding at both sigma-1 and sigma-2 receptor subtypes. Although additional nitrogen atoms resulted in decreased affinity at sigma-1 and sigma-2 subtypes, an increase in selectivity for sigma-2 subtypes was evident; the parent 3 showed greater selectivity for sigma-1 subtypes. Internitrogen spacings had a large effect on binding affinity and subtype selectivity. For example, the difference between N-[3-(1-pyrrolidinyl)propyl]-N'-(3,4-dichlorobenzyl)-N,N'- dimethylethylenediamine (8) [K(i) = 29.9 nM at sigma-1 receptor and 18.3 nM at sigma-2 receptor] to N-[3-(1-pyrrolidinyl)propyl]-N'-(3,4-dichlorobenzyl)- N,N'-dimethylethylenediamine (10) [K(i) = 1.49 nM at sigma-1 receptor and 12.1 nM at sigma-2 receptor] illustrates the importance of internitrogen spacing. Triamines 11 and 13 [Ki(sigma-2)/K(i)(sigma-1) = 0.19 and 0.10, respectively] containing the N-N-N-Ar spacings 3-3-2 and 4-4-2, proved to be the most sigma-2 subtype selective of the 15 polyamines examined in this study. The N-N-N spacings appear to be an important factor in their sigma-2 subtype selectivity. These compounds will serve as templates in the design of still further sigma-2 subtype selective ligands. The pyrrolidine ring (present in most of the polyamines tested in this series) proved to be an important recognition site for sigma receptor binding activity. Furthermore, alkyl substitution also appears to be important since the stripped down polyamines N-[2-(3,4-dichlorophenyl)ethyl]ethylenediamine (15) and N1-[2-(3,4-dichlorophenyl)ethyl]diethylenetriamine (16) exhibited relatively low binding affinity.

Sigma binding sites represent a unique class of receptors and are highly expressed in tumor cells. Two sigma binding site subtypes, known as sigma-1 and sigma-2 receptors, were distinguished based on differences in their pharmacological profiles and molecular weights. The sigma-1 receptor gene has been cloned and well characterized in the CNS and cancer biology. However, the sigma-2 receptor has not yet been cloned. The purpose of the current study is to identify and clone sigma-2 receptor. We have synthesized an irreversible sigma-2 receptor selective photoaffinity probe, WC-II-21, containing a fluorescein isothiocyanate (FITC) group. This ligand has high binding affinity for sigma-2 receptor (Ki = 13 nM) and relatively low binding affinity for sigma-1 receptor (Ki > 1,000 nM). This ligand irreversibly labeled sigma-2 receptors in the rat liver membrane fractions after UV irradiation. The ligand-labeled proteins were solubilized, enriched and then separated through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The position of a predominant band of the ligand-labeled protein in the gel was determined by western blot analysis using FITC antibody. MALDI-mass spectrometry analysis for the ligand-labeled proteins indentified the protein progesterone receptor membrane component-1 (PGRMC-1) with a convincingly high score of 81. Based on the similarities of the reported biological characteristics and functions between PGRMC1 and sigma-2 receptor, PGRMC1 was investigated in the current study. Immunocytochemistry revealed that PGRMC1 colocalized with molecular markers of endoplasmic reticulum and mitochondria in human melanoma cell line MDA-MB435. The subcellular localization of sigma-2 receptor also colocalized with fluorescent markers of endoplasmic reticulum and mitochondria as reported previously from our group (Cancer Res 2007; 67, 6708-6716). Thus, PGRMC1 and sigma-2 receptor appear to localize in the same subcellular organelles in the cell. PGRMC1 was knocked down with PGRMC1 siRNA in MDA-MB435 cells. Sigma-2 radioligand binding activity was significantly reduced in the PGRMC1 knocked down cells relative to the nontargeting siRNA transfected cells. These data suggest that PGRMC1 is the putative sigma-2 receptor. Further validation of PGRMC1 as the sigma-2 receptor is ongoing in our group. (Supported by CA 102869). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1054.

Introduction Amyloid beta (Aβ) oligomers are one of the most toxic structural forms of the Aβ protein and are hypothesized to cause synaptotoxicity and memory failure as they build up in Alzheimer's disease (AD) patients' brain tissue. We previously demonstrated that antagonists of the sigma-2 receptor complex effectively block Aβ oligomer toxicity. CT1812 is an orally bioavailable, brain penetrant small molecule antagonist of the sigma-2 receptor complex that appears safe and well tolerated in healthy elderly volunteers. We tested CT1812's effect on Aβ oligomer pathobiology in preclinical AD models and evaluated CT1812's impact on cerebrospinal fluid (CSF) protein biomarkers in mild to moderate AD patients in a clinical trial (ClinicalTrials.gov NCT02907567). Methods Experiments were performed to measure the impact of CT1812 versus vehicle on Aβ oligomer binding to synapses in vitro, to human AD patient post mortem brain tissue ex vivo, and in living APPSwe /PS1dE9 transgenic mice in vivo. Additional experiments were performed to measure the impact of CT1812 versus vehicle on Aβ oligomer-induced deficits in membrane trafficking rate, synapse number, and protein expression in mature hippocampal/cortical neurons in vitro. The impact of CT1812 on cognitive function was measured in transgenic Thy1 huAPPSwe/Lnd+ and wild-type littermates. A multicenter, double-blind, placebo-controlled parallel group trial was performed to evaluate the safety, tolerability, and impact on protein biomarker expression of CT1812 or placebo given once daily for 28 days to AD patients (Mini-Mental State Examination 18-26). CSF protein expression was measured by liquid chromatography with tandem mass spectrometry or enzyme-linked immunosorbent assay in samples drawn prior to dosing (Day 0) and at end of dosing (Day 28) and compared within each patient and between pooled treated versus placebo-treated dosing groups. Results CT1812 significantly and dose-dependently displaced Aβ oligomers bound to synaptic receptors in three independent preclinical models of AD, facilitated oligomer clearance into the CSF, increased synaptic number and protein expression in neurons, and improved cognitive performance in transgenic mice. CT1812 significantly increased CSF concentrations of Aβ oligomers in AD patient CSF, reduced concentrations of synaptic proteins and phosphorylated tau fragments, and reversed expression of many AD-related proteins dysregulated in CSF. Discussion These preclinical studies demonstrate the novel disease-modifying mechanism of action of CT1812 against AD and Aβ oligomers. The clinical results are consistent with preclinical data and provide evidence of target engagement and impact on fundamental disease-related signaling pathways in AD patients, supporting further development of CT1812.

Sigma σ1 and σ2 receptors are targets of cocaine. Despite sharing a similar name, the two receptors are structurally unrelated and their physiological role is unknown. Cocaine increases the level of dopamine, a key neurotransmitter in CNS motor control and reward areas. While the drug also affects dopaminergic signaling by allosteric modulations exerted by σ1R interacting with dopamine D1 and D2 receptors, the potential regulation of dopaminergic transmission by σ2R is also unknown. We here demonstrate that σ2R may form heteroreceptor complexes with D1 but not with D2 receptors. Remarkably σ1, σ2, and D1 receptors may form heterotrimers with particular signaling properties. Determination of cAMP levels, MAP kinase activation and label-free assays demonstrate allosteric interactions within the trimer. Importantly, the presence of σ2R induces bias in signal transduction as σ2R ligands increase cAMP signaling whereas reduce MAP kinase activation. These effects, which are opposite to those exerted via σ1R, suggest that the D1 receptor-mediated signaling depends on the degree of trimer formation and the differential balance of sigma receptor and heteroreceptor expression in acute versus chronic cocaine consumption. Although the physiological role is unknown, the heteroreceptor complex formed by σ1, σ2, and D1 receptors arise as relevant to convey the cocaine actions on motor control and reward circuits and as a key factor in acquisition of the addictive habit.

Drug addiction is a chronic, debilitating disease that affects millions of people around the world causing a substantial societal burden. Despite decades of research efforts, treatment possibilities remain limited and relapse represents the most treatment-resistant element. Neurosteroid sigma-1 receptors have been meticulously studied in psychostimulant reinforced Pavlovian learning, while the sigma-2 receptor subtype has remained unexplored. Recent development of selective sigma-2 receptor ligands have now made it possible to investigate if the sigma-2 receptor system is a potential target to treat drug addiction. We examined the effect of the sigma-2 receptor agonist Siramesine (Lu 28-179) on cocaine-associated locomotion, Pavlovian learning, and reward neurocircuitry using electrophysiology recordings and in vivo microdialysis. We found that Siramesine significantly attenuated conditioned place preference acquisition and expression, as well as it completely blocked cocaine-primed reinstatement. Siramesine, in a similar manner as the selective sigma-1 receptor antagonist BD 1063, decreased acute locomotor responses to cocaine. Immunohistochemistry suggests co-expression of progesterone receptor membrane component 1/sigma-2 receptors and vesicular glutamate transporter 1 in presynaptic boutons of the nucleus accumbens (NAc). Whole-cell voltage clamp recordings of neurons in the NAc indicated that Siramesine decreases the presynaptic release probability of glutamate. Further, we demonstrated, via in vivo microdialysis, that Siramesine significantly decreased cocaine-evoked dopamine release in the striatum of freely moving mice. Collectively, these findings demonstrate that sigma-2 receptors regulate neurocircuitry responsible for positive reinforcement and thereby play a role in cocaine-reinforced Pavlovian behaviors.

Background Sigma-2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. This mechanism of apoptosis is poorly understood, with varying reports of caspase-3 dependence. We evaluated multiple sigma-2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of human pancreatic cancer. Results Fluorescently labeled sigma-2 receptor ligands of two classes (derivatives of SW43 and PB282) localize to cell membrane components in Bxpc3 and Aspc1 pancreatic cancer cells and accumulate in lysosomes. We found that interactions in the lysosome are critical for cell death following sigma-2 ligand treatment because selective inhibition of a protective lysosomal membrane glycoprotein, LAMP1, with shRNA greatly reduced the viability of cells following treatment. Sigma-2 ligands induced lysosomal membrane permeabilization (LMP) and protease translocation triggering downstream effectors of apoptosis. Subsequently, cellular oxidative stress was greatly increased following treatment with SW43, and the hydrophilic antioxidant N-acetylcysteine (NAC) gave greater protection against this than a lipophilic antioxidant, α-tocopherol (α-toco). Conversely, PB282-mediated cytotoxicity relied less on cellular oxidation, even though α-toco did provide protection from this ligand. In addition, we found that caspase-3 induction was not as significantly inhibited by cathepsin inhibitors as by antioxidants. Both NAC and α-toco protected against caspase-3 induction following PB282 treatment, while only NAC offered protection following SW43 treatment. The caspase-3 inhibitor DEVD-FMK offered significant protection from PB282, but not SW43. Conclusions Sigma-2 ligand SW43 commits pancreatic cancer cells to death by a caspase-independent process involving LMP and oxidative stress which is protected from by NAC. PB282 however undergoes a caspase-dependent death following LMP protected by DEVD-FMK and α-toco, which is also known to stabilize the mitochondrial membrane during apoptotic stimuli. These differences in mechanism are likely dependent on the structural class of the compounds versus the inherent sigma-2 binding affinity. As resistance of pancreatic cancers to specific apoptotic stimuli from chemotherapy is better appreciated, and patient-tailored treatments become more available, ligands with high sigma-2 receptor affinity should be chosen based on sensitivities to apoptotic pathways.