Sigma Ligands Research Articles

Allosteric Modulation of Opioid G-Protein Coupled Receptors by Sigma1 Receptors.

Since their proposal in 1976, the concept of sigma1 receptors has been continually evolving. Initially thought to be a member of the opioid receptor family, molecular studies have now identified its genes and established its structure crystallographically. Much effort has now revealed its importance as a chaperone in the endoplasmic reticulum, but its functions extend beyond this. Sigma1 receptors have been associated with a host of signaling systems. Evidence over the past 20years has established the modulatory effects of sigma1 ligands on opioid systems. Despite their inability to bind directly to opioid receptors, sigma1 ligands can modulate opioid analgesia in vivo and signal transduction mechanisms in vitro. Furthermore, sigma1 receptors can physically associate with GPCRs. Together, these findings show that sigma1 ligands can function as allosteric modulators of GPCR function through their association with the sigma1 receptors, which are in direct physical association with opioid receptors, members of the G-protein coupled family of receptors.

Sigma1 Pharmacology in the Context of Cancer.

Sigma1 (also known as sigma-1 receptor, Sig1R, σ1 receptor) is a unique pharmacologically regulated integral membrane chaperone or scaffolding protein. The majority of publications on the subject have focused on the neuropharmacology of Sigma1. However, a number of publications have also suggested a role for Sigma1 in cancer. Although there is currently no clinically used anti-cancer drug that targets Sigma1, a growing body of evidence supports the potential of Sigma1 ligands as therapeutic agents to treat cancer. In preclinical models, compounds with affinity for Sigma1 have been reported to inhibit cancer cell proliferation and survival, cell adhesion and migration, tumor growth, to alleviate cancer-associated pain, and to have immunomodulatory properties. This review will highlight that although the literature supports a role for Sigma1 in cancer, several fundamental questions regarding drug mechanism of action and the physiological relevance of aberrant SIGMAR1 transcript and Sigma1 protein expression in certain cancers remain unanswered or only partially answered. However, emerging lines of evidence suggest that Sigma1 is a component of the cancer cell support machinery, that it facilitates protein interaction networks, that it allosterically modulates the activity of its associated proteins, and that Sigma1 is a selectively multifunctional drug target.

Sigma-2 receptor and progesterone receptor membrane component 1 (PGRMC1) are two different proteins: Proofs by fluorescent labeling and binding of sigma-2 receptor ligands to PGRMC1

A controversial relationship between sigma-2 and progesterone receptor membrane component 1 (PGRMC1) proteins, both representing promising targets for the therapy and diagnosis of tumors, exists since 2011, when the sigma-2 receptor was reported to be identical to PGRMC1. Because a misidentification of these proteins will lead to biased future research hampering the possible diagnostic and therapeutic exploitation of the two targets, there is the need to solve the debate on their identity. With this aim, we have herein investigated uptake and distribution of structurally different fluorescent sigma-2 receptor ligands by flow cytometry and confocal microscopy in MCF7 cells, where together with intrinsic sigma-2 receptors, PGRMC1 was constitutively present or alternatively silenced or overexpressed. HCT116 cells, with constitutive or silenced PGRMC1, were also studied. These experiments showed that the fluorescent sigma-2 ligands bind to their receptor irrespective of PGRMC1 expression. Furthermore, isothermal titration calorimetry was conducted to examine if DTG and PB28, two structurally distinct nanomolar affinity sigma-2 ligands, bind to purified PGRMC1 proteins that have recently been revealed to form both apo-monomeric and heme-mediated dimeric forms. While no binding to apo-PGRMC1 monomer was detected, a micromolar affinity to heme-mediated dimerized PGRMC1 was demonstrated in DTG but not in PB28. The current data provide evidence that sigma-2 receptor and PGRMC1 are not identical, paving the pathway for future unbiased research in which these two attractive targets are treated as different proteins while the identification of the true sigma-2 protein further needs to be pursued.

Abstract B58: Combination therapy with the novel small molecule drug conjugate SW V-49 and gemcitabine is a potent pancreatic cancer therapeutic

Abstract Cancer-selective drug delivery is an important concept in improving treatment while minimizing off-site toxicities, and sigma-2 receptors, which are overexpressed in solid tumors, represent attractive pharmacologic targets. Select sigma-2 ligands have been shown to be rapidly internalized selectively into cancer cells while retaining the ability to deliver small molecules as drug cargoes. We previously conjugated a derivative of Erastin, a small molecule with cytotoxic activity in cells bearing KRAS mutations, to sigma-2 ligand SV119, forming the novel small molecule drug conjugate SW V-49. SW V-49 demonstrated a reduction in tumor burden and improved median survival in multiple murine models of pancreatic cancer compared to either sigma-2 ligand (SV119), Erastin, or vehicle treatment alone. In the present work, we report on the selective and targeted delivery of SW V-49 to pancreatic tumors as well as the enhanced efficacy of treatment in combination with gemcitabine, a standard chemotherapeutic for pancreatic cancer. To determine delivery and uptake of SW V-49 in vivo, C57BL/6 mice bearing orthotopic KP-2 tumors were treated with SW V-49, and mass spectrometry was utilized to assess drug concentration within tissue. We observed increased delivery of SW V-49 to the tumor-bearing pancreas compared to liver, kidney, and muscle over 24 hours. To validate drug delivery in a genetically-engineered murine model, mass spectrometry was utilized to assess tissue uptake of SW V-49 in KPPC (p48-CRE/LSL-KRas/p53flox/flox) mice bearing palpable pancreatic tumors. Further evidence of SW V-49 tumor cell specificity is demonstrated by the significant reduction in EpCAM-positive neoplastic cells in the pancreatic tumors of C57BL/6 mice bearing orthotopic syngeneic KCKO tumors after treatment with SW V-49. In addition, we performed in vivo experimentation to demonstrate SW V-49 and gemcitabine combination therapy was superior in both subcutaneous and orthotopic murine models of pancreatic cancer. The KP-2 syngeneic subcutaneous tumor model demonstrated dense stroma similar to that of the human disease, and combination therapy with SW V-49 and gemcitabine resulted in significantly superior tumor reduction relative to all controls. We validated the utility of combination therapy in the KCKO syngeneic orthotopic model and again demonstrated the smallest tumor burden in the combination therapy group compared to control-treated animals. Together, these findings suggest that combination therapy utilizing the targeted therapeutic SW V-49 with systemic gemcitabine has potential translational utility. SW V-49 was successfully delivered in vivo to pancreatic tumors and reduced EpCAM-positive neoplastic cells, and combination therapy was superior to either agent alone and reduced tumor burden in multiple murine models of pancreatic cancer. Thus, SW V-49 is a promising novel therapeutic for pancreatic cancer and warrants further investigation. Citation Format: Kerri A. Ohman, Timothy M. Nywening, Suwanna Vangveravong, Dirk Spitzer, William G. Hawkins.{Authors}. Combination therapy with the novel small molecule drug conjugate SW V-49 and gemcitabine is a potent pancreatic cancer therapeutic. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B58.

N-phenylpropyl-N′-substituted piperazines occupy sigma receptors and alter methamphetamine-induced hyperactivity in mice

This study examined the effect of the N-phenylpropyl-N′-substituted piperazine ligands SA4503 (3.4-dimethoxyphenethyl), YZ-067 (4-methoxyphenethyl), YZ-185 (3-methoxyphenethyl) and Nahas-3h (4-methoxybenzyl) on methamphetamine-induced hyperactivity in mice. In a previous study in rats, SA4503 increased methamphetamine-induced hyperactivity at a lower ligand dose and enhanced it at a higher dose. The other ligands have not been investigated in this assay. Presently, mice were administered sigma ligands, and specific [125I]E-IA-DM-PE-PIPZE and [125I]RTI-121 binding was measured to determine σ1 sigma receptor and dopamine transporter occupancy, respectively. Mice were also administered sigma ligands followed by methamphetamine, and locomotor activity was measured. Each of the ligands occupied σ1 sigma receptors (ED50=0.2–0.6μmol/kg) with similar potency, but none occupied the transporter (ED50>10μmol/kg). At the highest dose tested (31.6μmol/kg) all four sigma ligands significantly attenuated methamphetamine-induced hyperactivity. Interestingly, SA4503, YZ-067 and Nahas-3h, but not YZ-185, enhanced methamphetamine-induced hyperactivity at lower ligand doses (1–3.16μmol/kg). These results suggest that these ligands function as stimulant agonists at lower doses and as antagonists at higher does, with subtle changes in the substitution pattern at the 3- and 4-positions of the phenethyl group contributing to the nature of the interactions. Overall, these data indicate a complex role for σ1 sigma receptor ligands in methamphetamine's behavioral effects.

σ1 receptor ligands control a switch between passive and active threat responses.

Humans and many animals exhibit freezing behavior in response to threatening stimuli. In humans, inappropriate threat responses are fundamental characteristics of several mental illnesses. To identify small molecules that modulate threat responses, we developed a high-throughput behavioral assay in zebrafish (Danio rerio) and characterized the effects of 10,000 compounds on freezing behavior. We found three classes of compounds that switch the threat response from freezing to escape-like behavior. We then screened these for binding activity across 45 candidate targets. Using target profile clustering we implicated the sigma-1 receptor in the mechanism of behavioral switching and confirmed that known sigma-1 ligands also disrupt freezing behavior. Furthermore, mutation of the sigma-1 gene prevented the behavioral effect of escape-inducing compounds. The compound ‘finazine’ potently bound mammalian sigma-1 and altered rodent threat response behavior. Thus, pharmacological and genetic interrogation of the freezing response revealed sigma-1 as a mediator of vertebrate threat responses.

Conjugation to the sigma-2 ligand SV119 overcomes uptake blockade and converts dm-Erastin into a potent pancreatic cancer therapeutic.

Cancer-selective drug delivery is an important concept in improving treatment while minimizing off-site toxicities, and sigma-2 receptors, which are overexpressed in solid tumors, represent attractive pharmacologic targets. Select sigma-2 ligands have been shown to be rapidly internalized selectively into cancer cells while retaining the capacity to deliver small molecules as drug cargoes. We utilized the sigma-2-based drug delivery concept to convert Erastin, a clinically underperforming drug, into a potent pancreatic cancer therapeutic. The Erastin derivative des-methyl Erastin (dm-Erastin) was chemically linked to sigma-2 ligand SV119 to create SW V-49. Conjugation increased the killing capacity of dm-Erastin by nearly 35-fold in vitro and reduced the size of established tumors and doubled the median survival in syngeneic and patient-derived xenograft models when compared to non-targeted dm-Erastin. Mechanistic analyses demonstrated that cell death was associated with robust reactive oxygen species production and could be efficiently antagonized with antioxidants. Mass spectrometry was employed to demonstrate selective uptake into pancreatic cancer cells. Thus, targeted delivery of dm-Erastin via conjugation to the sigma-2 ligand SV119 produced efficient tumor control and prolonged animal survival with minimal off-target toxicities, and SW V-49 represents a promising new therapeutic with the potential to advance the fight against pancreatic cancer.

New bifunctional antioxidant/σ1 agonist ligands: Preliminary chemico-physical and biological evaluation

We previously reported bifunctional sigma-1 (σ1) ligands endowed with antioxidant activity (1 and 2). In the present paper, pure enantiomers (R)-1 and (R)-2 along with the corresponding p-methoxy (6, 11), p-fluoro derivatives (7, 12) were synthesized. σ1 and σ2 affinities, antioxidant properties, and chemico-physical profiles were evaluated. Para derivatives, while maintaining strong σ1 affinity, displayed improved σ1 selectivity compared to the parent compounds 1 and 2. In vivo evaluation of compounds 1, 2, (R)-1, 7, and 12 showed σ1 agonist pharmacological profile. Chemico-physical studies revealed that amides 2, 11 and 12 were more stable than corresponding esters 1, 6 and 7 under our experimental conditions. Antioxidant properties were exhibited by fluoro derivatives 7 and 12 being able to increase total antioxidant capacity (TAC). Our results underline that p-substituents have an important role on σ1 selectivity, TAC, chemical and enzymatic stabilities. In particular, our data suggest that new very selective compounds 7 and 12 could be promising tools to investigate the disorders in which σ1 receptor dysfunction and oxidative stress are contemporarily involved.

Structural Insights into Sigma1 Function.

Sigma1 (also known as this sigma-1 receptor) is an unusual and enigmatic transmembrane protein implicated in a diverse array of biological processes ranging from neurodegenerative disease to cancer. Despite decades of research, the molecular architecture of Sigma1 is only beginning to become clear. Recent work has established that Sigma1 is an oligomer, and crystallographic studies have now offered the first high-resolution views of its molecular structure. For the first time, these results provide a detailed framework to understand mutagenesis data and the molecular pharmacology of Sigma1 ligands. Structural data also raise new questions surrounding the mechanisms of ligand activity and the molecular basis for interactions between Sigma1 and other proteins. As Sigma1 research enters the structural era, the field is poised for new discoveries and reevaluation of old data and old models.

Sigma Receptor Binding Assays.

Sigma receptors, both Sigma-1(S1R) and Sigma-2 (S2R), are small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated sites. A number of drugs bind to sigma receptors, including the antipsychotic haloperidol and (+)-pentazocine, an opioid analgesic. Sigma receptors are implicated in many central nervous system disorders, in particular Alzheimer's disease and conditions associated with motor control, such as Amyotrophic Lateral Sclerosis (ALS). Described in this unit are radioligand binding assays used for the pharmacological characterization of S1R and S2R. Methods detailed include a radioligand saturation binding assay for defining receptor densities and a competitive inhibition binding assay employing [³H]-(+)-pentazocine for identifying and characterizing novel ligands that interact with S1R. Procedures using [³H]-1,3-di(2-tolyl)guanidine ([³H]-DTG), a nonselective sigma receptor ligand, are described for conducting a saturation binding and competitive inhibition assays for the S2R site. These protocols are of value in drug discovery in identifying new sigma ligands and in the characterization of these receptors.

Kinetics modeling and occupancy studies of a novel C-11 PET tracer for VAChT in nonhuman primates

IntroductionDeficits in cholinergic function have been found in the aged brain and in neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (PD). The vesicular acetylcholine transporter (VAChT) is a reliable biomarker for the cholinergic system. We previously reported the initial in vitro and ex vivo characterization of (−)-[11C]TZ659 as a VAChT specific ligand. Here, we report the in vivo specificity, tracer kinetics, and dose-occupancy studies in the nonhuman primate brain. MethodsMicroPET brain imaging of (−)-[11C]TZ659 was performed under baseline conditions in two male macaques. Tracer kinetic modeling was carried out using a two-tissue compartment model (2TCM) and Logan plot with arterial blood input function and using a simplified reference tissue model (SRTM) and Logan plot (LoganREF) without blood input. Specificity for VAChT was demonstrated by pretreatment with (+)-pentazocine, (−)-vesamicol, or S-(−)-eticlopride. Target occupancy (Occ) was calculated following pretreatment with escalating doses of (−)-vesamicol. ResultsBaseline PET imaging revealed selective retention in the striatum with rapid clearance from the cerebellar hemispheres as a reference region. Total volume of distribution (VT) values derived from both 2TCM and Logan analysis with blood input revealed ~3-fold higher levels of (−)-[11C]TZ659 in the striatum than the cerebellar hemispheres. Injection of (−)-vesamicol either as a blocking or displacing agent significantly reduced striatal uptake of (−)-[11C]TZ659. In contrast, pretreatment with the sigma-1 ligand (+)-pentazocine had no impact. Pretreatment with the S-(−)-eticlopride, a dopamine D2–like receptor antagonist, increased striatal uptake of (−)-[11C]TZ659. Striatal binding potential (BPND, range of 0.33–1.6 with cerebellar hemispheres as the reference region) showed good correlation (r2=0.97) between SRTM and LoganREF. Occupancy studies found that ~0.0057mg/kg of (−)-vesamicol produced 50% VAChT occupancy in the striatum. Conclusion(−)-[11C]TZ659 demonstrated specific and reversible VAChT binding and favorable pharmacokinetic properties for assessing the density of VAChT in the living brain.

The pre-clinical characterization of an alpha-emitting sigma-2 receptor targeted radiotherapeutic

RationaleThe sigma-2 receptor is a protein with a Heme binding region and is capable of receptor-mediated endocytosis. It is overexpressed in many cancers making it a potential vector for therapeutic drug delivery. Our objective was to introduce an alpha-emitting radionuclide, astatine-211, into a selective sigma-2 ligand moiety to provide cytotoxic capabilities without adversely altering the pharmacological characteristics. In this study we investigated the in vitro/in vivo tumor targeting and estimated dosimetry of alpha-emitting sigma-2 ligand, 5-(astato-211At)-N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2,3-dimethoxybenzamide (211At-MM3), in a pre-clinical human breast cancer model. MethodsAstatine-211 was produced in a cyclotron and isolated by dry distillation. Radiosynthesis of 211At-MM3 was performed using a tin precursor through radioastatodestannylation. In vitro sigma-2 binding experiments using 211At-MM3 were carried out in live EMT6 and MDA-MB-231 breast cancer cells and liver homogenate tissue. In vivo biodistribution experiments were performed using EMT6 mouse breast cancer cells in BALB/c female mice. Approximately 370kBq of 211At-MM3 was administered intravenously and at time points of 5min, 1, 2, 4, 8, and 24h organs/tissue were harvested. Estimated human dosimetry was extrapolated from biodistribution data using OLINDA/EXM (VU e-Innovations). ResultsAstatine-211 was successfully produced and isolated in quantities suitable for in vitro and small animal in vivo experiments. Radiosynthesis of 211At-MM3 was reproducible with high radiochemical purity. Astatine-211-MM3 exhibited picomolar affinity to the sigma-2 receptor in contrast to the iodinated analog that had nanomolar affinity. Prolonged tumor targeting was measured through biodistribution studies with a maximal tumor to muscle ratio of 9.02 at 4h. Estimated human dosimetry revealed doses of up to 370MBq in an adult female patient were below organ radiation limits with the potential to provide a high therapeutic dose to tumors. ConclusionThe sigma-2 receptor could serve as a suitable targeting platform for designing radiotherapeutics. 211At-MM3 showed tumor targeting properties in vitro/in vivo and favorable estimated human dosimetry establishing the proof of concept for future development as a radiotherapeutic for the treatment of breast cancer.

Knockdown of interleukin-10 induces the redistribution of sigma1-receptor and increases the glutamate-dependent NADPH-oxidase activity in mouse brain neurons.

BackgroundIn the central nervous system, interleukin-10 (IL-10) provides trophic and survival effects directly on neurons, modulates neurite plasticity, and has a pivotal importance in the neuronal regeneration in neurodegenerative and neuroinflammatory conditions. This cytokine is primarily produced by glial cells and has beneficial effects on the neuronal viability. However, the mechanisms of IL-10-elicited neuroprotection are not clear.ResultsMembrane preparations, isolated from wild-type (Wt) and IL-10 knockout (KO) mice brain were used in this study. It has been shown that compared to wild-type mice, in IL-10 KO mice brain, the amount of immunoglobulin binding protein (BiP) is greatly increased, whereas the content of sigma receptor-1 (SigR1) is not changed significantly. Co-immunoprecipitation experiments have shown that the association of SigR1 with small GTPase Rac1 (Ras-related C3 botulinum toxin substrate 1), NR2B subunit of NMDA-receptor (NMDAR) and inositol-3-phosphate receptor (IP3R) is higher in the IL-10 KO mice brain than in the Wt mice brain. Besides, we have found that either glutamate or sigma ligands, separately or together, do not change glutamate-induced NADPH-oxidase (NOX) activity in Wt-type mice brain membrane preparations, whereas in IL-10 KO mice high concentration of glutamate markedly increases the NOX-dependent production of reactive oxygen species (ROS). Glutamate-dependent ROS production was decreased to the normal levels by the action of sigma-agonists.ConclusionsIt has been concluded that IL-10 deprivation, at least in part, can lead to the induction of ER-stress, which causes BiP expression and SigR1 redistribution between components of endoplasmic reticulum (ER) and plasma membrane. Moreover, IL-10 deficiency can change the specific organization of NMDAR, increasing the surface expression of SigR1-sensitive NR2B-containing NMDAR. In these conditions, glutamate-dependent ROS production is greatly increased leading to the initiation of apoptosis. In this circumstances, sigma-ligands could play a preventive role against NMDA receptor-mediated excitotoxicity.

Abstract 3481: Sigma-2 ligands sensitize triple negative breast cancer cells to PARP inhibitor treatment

Abstract Triple-negative breast cancer (TNBC) accounts for 15-25% of breast cancer cases. Effective targeted treatment for TNBC is lacking. Poly (ADP-ribose) polymerase (PARP) is a key enzyme involved in single-strand DNA break repair. PARP inhibitors have been used in combination with various chemotherapeutic agents in TNBC with limited success. The sigma-2 receptor is associated with cellular proliferation and quiescence and has been used as the target for imaging solid tumors by positron emission tomography. The sigma-2 receptor has been identified as progesterone receptor membrane component 1 (PGRMC1) protein complex. Recent work has suggested that the sigma-2 receptor may also be an effective therapeutic target. In this study we looked for potential synergy between PARP inhibitors and sigma-2 ligands using a triple negative breast cancer cell line, MDA-MB231. We showed that the sigma-2 ligand, SW43, and a PGRMC1 ligand, AG205, induced cell toxicity and caspase 3 activation in a dose-dependent manner. The combination of SW43 and PARP inhibitor, Olaparib, resulted in additive cytotoxicity by cell viability assay and colony forming assay. Olaparib inhibited PARP activity in MDA-MB231 cell line, suggesting that the single-strand DNA break repair may be inhibited. SW43 and AG205 treatment decreased PGRMC1 and EGFR protein levels, suggesting that sigma-2/PGRMC1 ligands reduce growth factor signaling. In conclusion, SW43 and Olaparib induce cell death by separate signaling pathways. Sigma-2 ligands may selectively sensitize tumors to PARP inhibitor treatment in triple negative breast cancer patients. Citation Format: Chenbo Zeng, Julia Mankoff, Robert H. Mach. Sigma-2 ligands sensitize triple negative breast cancer cells to PARP inhibitor treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3481. doi:10.1158/1538-7445.AM2015-3481

Abstract 5428: Sigma-2 receptor-induced cell death: a novel approach to triple-negative breast cancer treatment

Abstract Triple negative breast cancers (TNBC) comprise 10-20% of all breast cancers. The 5-year survival rate of women with metastatic TNBC is less than 30%, and the mortality rate is nearly 100%, despite adjuvant chemotherapy (Lehmann, B.D. et al., 2011). TNBC lacks estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor (HER2) amplification, and is particularly aggressive and drug-resistant. There is a lack of targeted therapies available for TNBC, as current endocrine and drug therapies that target ER, PR, or HER2 are ineffective (Foulkes, W.D. et al., 2010). Sigma-2 receptors are highly upregulated in all tumor cell lines studied thus far, and sigma-2 agonists induce cell death in these cancer cells (Crawford and Bowen, 2002). We previously reported that CM572, an irreversible sigma-2 partial agonist, had cytotoxic effects in MCF-7 breast cancer cells (Nicholson et al., Proc. Amer. Assoc. Cancer Res. 74: #3237, 2014). Here, we present initial findings that validate the sigma-2 receptor as a potential chemotherapeutic target for the treatment of TNBC. In the MDA-MB-231 TNBC cell line, the estimated Bmax value for sigma-2 receptors (65.7 ± 17.0 fmol/mg protein) is approximately 20-fold higher than the Bmax for sigma-1 receptors (3.4 ± 2.4 fmol/mg protein). The sigma-2 selective ligands CM572, SV119, and siramesine (sigma-2 Ki values of 14.7 nM, 5.2 nM, and 0.12 nM, respectively) caused comparable cell death in a dose-dependent manner in TNBC cells and the much less aggressive MCF-7 cells (which have ER and PR) after 48 hour treatment. CM572 had an EC50 of 1.93 μM in MDA-MB-231 cells (95.0 ± 2.52% maximal cytotoxicity) and 1.04 μM in MCF-7 cells (89.1 ± 7.59% maximal cytotoxicity). SV119 had an EC50 of 23.3 μM in MDA-MB-231 cells (100 ± 0.49% maximal cytotoxicity) and 17.6 μM in MCF-7 cells (100 ± 1.11% maximal cytotoxicity). Siramesine had an EC50 of 5.14 μM in MDA-MB-231 cells (99.7 ± 0.47% maximal cytotoxicity) and 2.81 μM in MCF-7 cells (99.8 ± 0.26% maximal cytotoxicity). Therefore, sigma-2 ligands have similar potencies in TNBC and non-triple negative breast cancer cells, indicating that these drugs have cytotoxic effects that are independent of triple negative receptor status. Treatment with 30 μM of SV119 for 24-48 hours yielded decreased protein levels of full-length Bid, indicating activation of this pro-apoptotic member of the Bcl-2 family. Bid cleavage was previously found in sigma-2 ligand-mediated apoptosis in SK-N-SH neuroblastoma cells (Nicholson et al., Proc. Amer. Assoc. Cancer Res. 74: #3237, 2014), and it may be caused by the involvement of caspases or other proteases. In summary, the data show that the sigma-2 receptor is able to induce apoptotic cell death in TNBC cells. Sigma-2 selective ligands CM572, SV119, and siramesine produced significant cytotoxic effects on TNBC cells, suggesting that sigma-2 receptors are an exciting novel therapeutic target for TNBC. Citation Format: Zongyi Liu, Hilary E. Nicholson, Wayne D. Bowen. Sigma-2 receptor-induced cell death: a novel approach to triple-negative breast cancer treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5428. doi:10.1158/1538-7445.AM2015-5428

Novel indole-based sigma-2 receptor ligands: synthesis, structure–affinity relationship and antiproliferative activity

Novel sigma-2 ligand 1b induced G1 phase cell cycle arrest in DU145 cells and displayed antiproliferative activity.

A Direct Interaction between the Sigma-1 Receptor and the hERG Voltage-gated K+ Channel Revealed by Atomic Force Microscopy and Homogeneous Time-resolved Fluorescence (HTRF®)

The sigma-1 receptor is an endoplasmic reticulum chaperone protein, widely expressed in central and peripheral tissues, which can translocate to the plasma membrane and modulate the function of various ion channels. The human ether-à-go-go-related gene encodes hERG, a cardiac voltage-gated K(+) channel that is abnormally expressed in many human cancers and is known to interact functionally with the sigma-1 receptor. Our aim was to investigate the nature of the interaction between the sigma-1 receptor and hERG. We show that the two proteins can be co-isolated from a detergent extract of stably transfected HEK-293 cells, consistent with a direct interaction between them. Atomic force microscopy imaging of the isolated protein confirmed the direct binding of the sigma-1 receptor to hERG monomers, dimers, and tetramers. hERG dimers and tetramers became both singly and doubly decorated by sigma-1 receptors; however, hERG monomers were only singly decorated. The distribution of angles between pairs of sigma-1 receptors bound to hERG tetramers had two peaks, at ∼90 and ∼180° in a ratio of ∼2:1, indicating that the sigma-1 receptor interacts with hERG with 4-fold symmetry. Homogeneous time-resolved fluorescence (HTRF®) allowed the detection of the interaction between the sigma-1 receptor and hERG within the plane of the plasma membrane. This interaction was resistant to sigma ligands, but was decreased in response to cholesterol depletion of the membrane. We suggest that the sigma-1 receptor may bind to hERG in the endoplasmic reticulum, aiding its assembly and trafficking to the plasma membrane.

Abstract 3237: Characterization of CM-572 and CM-769: Novel irreversible modulators of sigma-2 receptor function

Abstract Sigma-2 receptors are highly upregulated in a wide variety of cancer cells compared to normal cells, and increase by at least ten-fold in rapidly proliferating cancer cells compared to quiescent. Activation of the sigma-2 receptor induces apoptosis. Thus, there has been increasing interest in sigma-2 receptors as targets for cancer therapeutic and diagnostic agents and a need for small molecule tools to study function. We previously reported analogs of the sigma-2 antagonist, SN-79, that incorporate an isothiocyanate (ITC) group in order to impart irreversible binding to the receptor (Nicholson et al., Proc. Amer. Assoc. Cancer Res. 54: #2242, 2013). Here we further characterize CM-572 (sigma-2 Ki = 320nM; ITC in heterocyclic ring) and CM-769 (sigma-2 Ki = 271nM; ITC in fluorophenyl ring) for their effects in SK-N-SH neuroblastoma, MCF-7 breast, and PANC-1 pancreatic cancer cell lines. Previous data showed that CM-572 potently and selectively acylated sigma-2 receptors over sigma-1, while CM-769 was less potent at irreversible binding, presumably due to non-optimal placement of the ITC group. CM-572 induced dose-dependent cell death in SK-N-SH neuroblastoma cells with ED50 about 5uM. CM-572 was also cytotoxic in MCF-7 breast and PANC-1 pancreatic cancer cells, however with reduced potency and efficacy (40% and 50% maximal cell death, respectively, at 30uM). The cytotoxic effect of CM-572 in SK-N-SH cells was retained after a 60min pretreatment of cells, washout of compound, and continued incubation for 24h, indicating irreversible agonist activity. Interestingly, CM-769 produced an apparent dose-dependent proliferative effect on SK-N-SH neuroblastoma cells. This effect was also retained after 60min pretreatment and washout, again indicating an irreversible affect. A subtoxic dose of CM-572 (300nM) was able to partially attenuate CM-769-induced cell proliferation, indicating that these two compounds act at the same receptor site. Cytosolic BID cleavage is a hallmark of sigma-2 receptor induced apoptosis in SK-N-SH cells. CM-572 (30uM) induced rapid BID cleavage beginning at 30min, with near completion at 6h. By contrast, CM-769 (30uM) did not induce BID cleavage, but rather appeared to increase cytosolic levels of full-length BID relative to control, consistent with the observed proliferative or protective effect. Together, the data indicate that CM-572 is an irreversible partial agonist at sigma-2 receptors. This is consistent with previously demonstrated effects of CM-572 on CB-64D (an agonist)-induced calcium release and cytotoxicity, where low-dose CM-572 attenuated CB-64D effects, and high-dose CM-572 mimicked CB-64D. CM-769 is an irreversible inverse agonist at the sigma-2 receptor, a novel effect not shown by any sigma-2 ligand to date. These two compounds represent novel tools for further study of sigma-2 receptor function. Citation Format: Hilary E. Nicholson, Pei Ling Chia, Anthony Comeau, Christophe Mesangeau, Christopher McCurdy, Wayne D. Bowen. Characterization of CM-572 and CM-769: Novel irreversible modulators of sigma-2 receptor function. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3237. doi:10.1158/1538-7445.AM2014-3237

Sigma receptors as potential therapeutic targets for neuroprotection

Sigma receptors comprise a unique family of proteins that have been implicated in the pathophysiology and treatment of many central nervous system disorders, consistent with their high level of expression in the brain and spinal cord. Mounting evidence indicate that targeting sigma receptors may be particularly beneficial in a number of neurodegenerative conditions including Alzheimer׳s disease, Parkinson׳s disease, stroke, methamphetamine neurotoxicity, Huntington׳s disease, amyotrophic lateral sclerosis, and retinal degeneration. In this perspective, a brief overview is given on sigma receptors, followed by a focus on common mechanisms of neurodegeneration that appear amenable to modulation by sigma receptor ligands to convey neuroprotective effects and/or restorative functions. Within each of the major mechanisms discussed herein, the neuroprotective effects of sigma ligands are summarized, and when known, the specific sigma receptor subtype(s) involved are identified. Together, the literature suggests sigma receptors may provide a novel target for combatting neurodegenerative diseases through both neuronal and glial mechanisms.

Conjugation to SMAC mimetic potentiates sigma-2 ligand induced tumor cell death in ovarian cancer

Background: Drug resistance is a significant problem in the treatment of ovarian cancer and can be caused by multiple mechanisms. Inhibition of apoptosis by the inhibitor of apoptosis proteins (IAPs) represents one such mechanism, and can be overcome by a mitochondrial protein called second mitochondria-derived activator of caspases (SMAC). We have previously shown that the ligands of sigma-2 receptors effectively induce tumor cell death. Additionally, because sigma-2 receptors are preferentially expressed in tumor cells, their ligands provide an effective mechanism for selective anti-cancer therapy. Methods: In the current work, we have improved upon the previously described sigma-2 ligand SW43 by conjugating it to a pro-apoptotic small molecule SMAC mimetic SW IV-52, thus generating the novel cancer therapeutic SW IV-134. The new cancer drug was tested for receptor selectivity and tumor cell killing activity in vitro and in vivo. Results: We have shown that SW IV-134 retained adequate sigma-2 receptor binding affinity in the context of the conjugate and potently induced cell death in ovarian cancer cells. The cell death induced by SW IV-134 was significantly greater than that observed with either SW43 or SW IV-52 alone and in combination. Furthermore, the intraperitoneal administration of SW IV-134 significantly reduced tumor burden and improved overall survival in a mouse xenograft model of ovarian cancer without causing significant adverse effects to normal tissues. Mechanistically, SW IV-134 induced degradation of cIAP-1 and cIAP-2 leading to NF-қB activation and TNFα-dependent cell death. Conclusions: Our findings suggest that coupling sigma-2 ligands to SMAC peptidomimetics enhances their effectiveness while maintaining the cancer selectivity. This encouraging proof-of-principle preclinical study supports further development of tumor-targeted small peptide mimetics via ligands to the sigma-2 receptor for future clinical applications.