Sigma Binding Research Articles

Genome-wide promoter assembly in E. coli measured at single-base resolution.

When detected at single-base-pair resolution, the genome-wide location, occupancy level, and structural organization of DNA-binding proteins provide mechanistic insights into genome regulation. Here we use ChIP-exo to provide a near-base-pair resolution view of the epigenomic organization of the Escherichia coli transcription machinery and nucleoid structural proteins at the time when cells are growing exponentially and upon rapid reprogramming (acute heat shock). We examined the site specificity of three sigma factors (RpoD/σ70, RpoH/σ32, and RpoN/σ54), RNA polymerase (RNAP or RpoA, -B, -C), and two nucleoid proteins (Fis and IHF). We suggest that DNA shape at the flanks of cognate motifs helps drive site specificity. We find that although RNAP and sigma factors occupy active cognate promoters, RpoH and RpoN can occupy quiescent promoters without the presence of RNAP. Thus, promoter-bound sigma factors can be triggered to recruit RNAP by a mechanism that is distinct from an obligatory cycle of free sigma binding RNAP followed by promoter binding. These findings add new dimensions to how sigma factors achieve promoter specificity through DNA sequence and shape, and further define mechanistic steps in regulated genome-wide assembly of RNAP at promoters in E. coli.

Sigma-1 Receptor and Binding Immunoglobulin Protein Interact with Ulinastatin Contributing to a Protective Effect in Rat Cerebral Ischemia/Reperfusion

To investigate impact of ulinastatin (UTI) on sigma-1 receptor (σ1R) and binding immunoglobulin protein (BiP) after cerebral ischemia/reperfusion injury. The middle cerebral artery occlusion (MCAO) model was used to induce cerebral ischemia/reperfusion injury. Eighty male Sprague Dawley rats were randomly divided into 6 groups: control, MCAO, MCAO+50,000 U/kg UTI, MCAO+100,000 U/kg UTI, MCAO+200,000 U/kg UTI, MCAO+300,000 U/kg UTI. At 24 and 48 hours after MCAO, infarct volume, neurological dysfunction, and grip strength test were measured, and level of σ1R and BiP proteins was further detected using Western blot. Molecular docking assays were carried out to verify interaction between σ1R, BiP, and UTI. The serum concentration of BiP and the binding assay between σ1R, BiP, and UTI were determined using enzyme-linked immunosorbent assay. UTI increased the modified neurological severity score and upregulated σ1R and BiP expression in the cerebral cortex after MCAO. The grip strength of forelimbs increased significantly in the MCAO+200,000 U/kg UTI and MCAO+300,000 U/kg UTI groups compared with the MCAO group, while BiP serum levels remained unchanged. The molecular docking assay indicated putative binding between σ1R, BiP, and UTI. The binding assay also revealed that both σ1R and BiP could be combined with UTI. UTI displays a neuroprotective effect via upregulation of σ1R and BiP during ischemia/reperfusion injury, suggesting that UTI modulates σ1R and BiP and their interaction may provide a novel insight into potential therapeutic mechanisms for stroke.

Abstract 1294: Differential sigma-2 receptor-mediated metabolic stimulative and apoptotic effects of monovalent CM571 and its homo-bivalent counterpart, MAM03055A

Abstract Sigma-2 receptors, recently identified as TMEM97, are highly expressed in various types of cancer cells. Sigma-2 receptor agonists have traditionally been characterized as ligands that induce programmed cell death in various cancer cell types. Sigma-2 receptor-mediated cell death involves a number of mechanisms including caspase activation, mitochondrial depolarization, and autophagy. Recently, we reported a novel metabolically stimulative function of the sigma-2 receptor with stimulation of glycolytic hallmarks, including increase in cellular ATP level, reduced ROS, and stabilization of HIF-1α. We have investigated a family of 6-substituted analogs of the canonical sigma-2 receptor antagonist, SN79, members of which can produce both the metabolic stimulative and the cytotoxic effects. Here we compare the activities of two closely related compounds. CM571, the 6-amino derivative, bound with high affinity to both sigma-1 and sigma-2 receptors (sigma-1 Ki = 15.5 nM; sigma-2 Ki = 21.7 nM). MAM03055A (MAM) is essentially a homo-bivalent dimer of CM571, linked by a thiourea moiety. MAM exhibited high affinity and selectivity for sigma-2 receptors (sigma-1 Ki = 3,371 nM; sigma-2 Ki = 55.9 nM). The functional effects of the two compounds were compared in human SK-N-SH neuroblastoma cells. CM571 caused a dose-dependent stimulation of MTT reduction, indicating the metabolic stimulative-effect, producing 40% stimulation at 30 µM. By contrast, MAM induced dose-dependent cell death with an EC50 = 4.24 µM. MAM induced time-dependent cleavage of proapoptotic Bid, a key component of the programmed cell death mechanism of sigma-2 ligands in these cells. Previous studies had shown that only irreversibly binding, isothiocyanate analogs of SN79 induced programmed cell death, while other analogs produced metabolic stimulation. Rat liver membranes were treated with compound, washed to remove unbound ligand, and then subjected to radioligand binding to determine recovery of sigma-1 receptors ([3H](+)-pentazocine) and sigma-2 receptors ([3H]DTG + (+)-pentazocine). Treatment with CM571 resulted in full recovery of both sigma-1 and sigma-2 binding. However, MAM treatment resulted in loss of sigma-2 binding and full recovery of sigma-1 activity, indicating irreversible binding to the sigma-2 receptor. Since there is no chemically reactive moiety in MAM to react covalently, MAM binds “pseudo-irreversibly”. Treatment of cells with MAM for 60 min, followed by washout produced nearly comparable levels of cell death 24 h later as continuous exposure of cells for 24 hours, consistent with pseudo-irreversible action at the sigma-2 receptor. The results show that mono-valent and bivalent sigma-2 ligands in this series show different modes of interaction with the receptor, resulting in divergent effects on cell viability. Citation Format: Cheri Z. Liu, Bridget M. McVeigh, Marco Mottinelli, Hilary E. Nicholson, Christopher R. McCurdy, Wayne D. Bowen. Differential sigma-2 receptor-mediated metabolic stimulative and apoptotic effects of monovalent CM571 and its homo-bivalent counterpart, MAM03055A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1294.

NKp46 Recognizes the Sigma1 Protein of Reovirus: Implications for Reovirus-Based Cancer Therapy

The recent approval of oncolytic virus for therapy of melanoma patients has increased the need for precise evaluation of the mechanisms by which oncolytic viruses affect tumor growth. Here we show that the human NK cell-activating receptor NKp46 and the orthologous mouse protein NCR1 recognize the reovirus sigma1 protein in a sialic-acid-dependent manner. We identify sites of NKp46/NCR1 binding to sigma1 and show that sigma1 binding by NKp46/NCR1 leads to NK cell activation in vitro Finally, we demonstrate that NCR1 activation is essential for reovirus-based therapy in vivo Collectively, we have identified sigma1 as a novel ligand for NKp46/NCR1 and demonstrated that NKp46/NCR1 is needed both for clearance of reovirus infection and for reovirus-based tumor therapy.IMPORTANCE Reovirus infects much of the population during childhood, causing mild disease, and hence is considered to be efficiently controlled by the immune system. Reovirus also specifically infects tumor cells, leading to tumor death, and is currently being tested in human clinical trials for cancer therapy. The mechanisms by which our immune system controls reovirus infection and tumor killing are not well understood. We report here that natural killer (NK) cells recognize a viral protein named sigma1 through the NK cell-activating receptor NKp46. Using several mouse tumor models, we demonstrate the importance of NK cells in protection from reovirus infection and in reovirus killing of tumors in vivo Collectively, we identify a new ligand for the NKp46 receptor and provide evidence for the importance of NKp46 in the control of reovirus infections and in reovirus-based cancer therapy.

4-aroylpiperidines and 4-(α-hydroxyphenyl)piperidines as selective sigma-1 receptor ligands: synthesis, preliminary pharmacological evaluation and computational studies.

BackgroundSigma (σ) receptors are membrane-bound proteins characterised by an unusual promiscuous ability to bind a wide variety of drugs and their high affinity for typical neuroleptic drugs, such as haloperidol, and their potential as alternative targets for antipsychotic agents. Sigma receptors display diverse biological activities and represent potential fruitful targets for therapeutic development in combating many human diseases. Therefore, they present an interesting avenue for further exploration. It was our goal to evaluate the potential of ring opened spipethiane (1) analogues as functional ligands (agonists) for σ receptors by chemical modification.ResultsChemical modification of the core structure of the lead compound, (1), by replacement of the sulphur atom with a carbonyl group, hydroxyl group and 3-bromobenzylamine with the simultaneous presence of 4-fluorobenzoyl replacing the spirofusion afforded novel potent sigma-1 receptor ligands 7a–f, 8a–f and 9d–e. The sigma-1 receptor affinities of 7e, 8a and 8f were slightly lower than that of 1 and their selectivities for this receptor two to threefold greater than that of 1.ConclusionsIt was found that these compounds have higher selectivities for sigma-1 receptors compared to 1. Quantitatitive structure–activity relationship studies revealed that sigma-1 binding is driven by hydrophobic interactions.Graphical abstractIdentified pharmacophore features for sigma binding.Electronic supplementary materialThe online version of this article (doi:10.1186/s13065-016-0200-1) contains supplementary material, which is available to authorized users.

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.

Sigma‐2 Receptor Ligands and Their Perspectives in Cancer Diagnosis and Therapy

The sigma-2 receptor is highly expressed in various rapidly proliferating cancer cells and regarded as a cancer cell biomarker. Selective sigma-2 ligands have been shown to specifically label the tumor sites, induce cancer cells to undergo apoptosis, and inhibit tumor growth. Sigma-2 ligands are potentially useful as cancer diagnostics, anticancer therapeutics, or adjuvant anticancer treatment agents. However, both the cloning of this receptor and the identification of its endogenous ligand have not been successful, and the lack of structural information has severely hindered the understanding of its physiological roles, its signaling pathways, and the development of more selective sigma-2 ligands. Recent data have implicated that sigma-2 binding sites are within the lipid rafts and that PGRMC1 (progesterone receptor membrane component 1) complex and sigma-2 receptor may be coupled with EGFR (epidermal growth factor receptor), mTOR (mammalian target of rapamycin), caspases, and ion channels. Due to its promising applications in cancer management, there are rapidly increasing research efforts that are being directed into this field. This review article updates the current understanding of sigma-2 receptor and its potential physiological roles, applications, interaction with other effectors, with special focuses on the development of sigma-2 ligands, their chemical structures, pharmacological profiles, applications in imaging and anticancer therapy.

Abstract 2242: Development of selective irreversible antagonists for sigma-2 receptors.

Abstract Sigma-1 and sigma-2 receptors are potential targets for therapeutic and diagnostic agents for various types of cancer. Sigma-1 receptors promote proliferation and survival, whereas activation of sigma-2 receptors induces apoptosis. Thus, there has been particular focus on sigma-2 receptors, which may be related to the PGRMC1 complex. While several selective ligands for sigma-2 receptors have been developed, there is a scarcity of tools for studying structure and function, particularly antagonists. SN-79 is a high affinity ligand for sigma-2 receptors, with 10-fold selectivity for sigma-2 over sigma-1 (Ki = 30 nM vs. 290 nM, respectively). Four isothiocyanate derivatives of SN-79 were characterized in rat liver membranes using [3H](+)-pentazocine to label sigma-1 and [3H]DTG (in presence of unlabeled (+)-pentazocine) for sigma-2. The isothiocyanate derivatives all exhibited lower affinity for sigma-2 receptors compared to SN-79. Sigma-2 Ki values were 320, 102, 395, and 271 nM for CM-572, CM-617, CM-621, and CM-769, respectively. Irreversible binding was examined by pretreating membranes for 60 min with the compound, extensively washing the membranes, and then determining recovery of radioligand binding. Loss of radioligand binding upon pretreatment results from “wash-resistant binding” of the compound and indicates covalent attachment to the receptor. Pretreatment with 100 nM CM-572 or CM-617 resulted in recovery of only 25% and 40% of sigma-2 binding, respectively. Treatment with 100 nM CM-621 or CM-769 had no effect. Therefore, while all four compounds bind to sigma-2 receptors with roughly comparable affinities, only CM-572 and CM-617 bind irreversibly. None of the compounds had an effect on sigma-1 receptor binding, indicating selective acylation of the sigma-2 receptor. In dose studies, CM-572 exhibited an IC50 ∼30 nM. Human SK-N-SH neuroblastoma cells were used to examine the biological activity of CM-572. Cells were pretreated for 1 h with CM-572 at concentrations up to 100 uM, then washed free of ligand, normal media added, and monitored by MTT assay after 24 h. There was no effect on cell viability up to a concentration of 10 uM, where ∼20% cytotoxicity was observed. About 80% cytotoxicity was observed at 100 uM. This suggests that CM-572 may have an irreversible partial agonist effect. However, pretreatment with CM-572 was unable to attenuate cell death induced by the sigma-2 agonist, CB-64D. A component of the signaling cascade of sigma-2 receptor activation is calcium release from the ER. In Fura-2 loaded cells, CM-572 produced no calcium signal at 3 uM and a small signal at 10 uM, though much smaller than the robust signal induced by CB-64D. Larger signals were produced at 30 uM and 100 uM CM-572. Interestingly, 3 uM and 10 uM CM-572 attenuated the calcium signal induced by 30 uM CB-64D. Taken together, these data indicate that CM-572 may be an irreversible partial agonist at sigma-2 receptors. Citation Format: Hilary Nicholson, Anthony Comeau, Christophe Mesangeau, Christopher R. McCurdy, Wayne D. Bowen. Development of selective irreversible antagonists for sigma-2 receptors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2242. doi:10.1158/1538-7445.AM2013-2242

Lysosomal Membrane Permeabilization is an Early Event in Sigma-2 Receptor Ligand Mediated Cell Death in Pancreatic Cancer

BackgroundSigma-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.ResultsFluorescently 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.ConclusionsSigma-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.

Role and regulation of plastid sigma factors and their functional interactors during chloroplast transcription – Recent lessons from Arabidopsis thaliana

Chloroplasts and other plastids within plant cells together are responsible for autotrophic growth and biosynthesis of metabolic key components. Genetically, the plastids are hybrid organelles composed of proteins that are either products of their own organellar genes or are nucleus-encoded and imported from the cytosol. This dual genetic principle is evident in the case of the multi-subunit RNA polymerase, i.e. a major enzyme of the plastid transcription apparatus, consisting of an organelle-encoded core surrounded by imported regulatory proteins. Representatives of the latter are the members of the plastid sigma factor family as well as a Ser/Thr-protein kinase (PTK/cpCK2) that functionally modifies these factors and controls transcription. The plant sigma factors contain regulatory phosphoacceptor sites within their unconserved (factor-specific) portion that precedes the conserved catalytic region. Phosphorylation state changes of these regulatory sites help establish the activity and promoter selectivity of individual members of this plant transcription factor family. The protein kinase itself responds to SH-group regulation by glutathione and transmits the redox signal via its phosphorylation activity to the plastid transcription apparatus. Other functional interactors include a set of sigma binding proteins that confer enhanced promoter binding in vitro and are thought to be involved in pathogenic stress responses of the chloroplast in vivo.

Abstract 1054: Identification of progesterone receptor membrane component-1 as the putative sigma-2 receptor

Abstract 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.

Sigma1 receptors and neuroblastoma differentiation

The sigma1 receptor is an integral membrane protein of ~26kD which shares no structural homology with any traditional receptor family. Sigma1 binding sites are highly conserved across mammalian species and are present in a broad range of tissue, with particularly high expression in distinct regions of the CNS. Given the CNS expression of sigma1 receptors and the neuro‐active properties of many sigma1 receptor ligands, we hypothesize that sigma1 receptors may play a role in neuronal differentiation. Differentiating neuroblastoma cells upregulates sigma1 receptors, with high levels in distinct vesicular structures throughout neurite outgrowths in contrast to an endoplasmic reticulum distribution in undifferentiated cells. These data suggest an association of sigma1 receptors with neuronal differentiation.Funded by grants to GWP (DA0641, DA02615 and DA00220) from the National Institute of Drug Abuse

A Medicinal‐Chemistry‐Guided Approach to Selective and Druglike Sigma 1 Ligands

Based on a medicinal-chemistry-guided approach, three novel series of druglike cycloalkyl-annelated pyrazoles were synthesized and display high affinity (pKi>8) for the sigma1 receptor. Structure-affinity relationships were established, and the different scaffolds were optimized with respect to sigma1 binding and selectivity versus the sigma2 receptor and the hERG channel, resulting in selective compounds that have Ki values (for sigma1) in the subnanomolar range. Selected compounds were screened for cytochrome P450 inhibition (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4), metabolic stability (rat and human liver microsomes), and cell-membrane permeability (Caco-2). They showed favorable in vitro ADME properties as well as favorable calculated druglike and experimental physicochemical properties. Furthermore, compounds 7 f and 17 a, for example, displayed high selectivity (affinity) for the sigma1 receptor against a wide range of other receptors (>60). With these valuable tool compounds in hand, we are further exploring the role of the sigma1 receptor in relevant animal models corresponding to such medicinal indications as drug abuse, pain, depression, anxiety, and psychosis.

σ1 and σ2 receptor binding affinity and selectivity of SA4503 and fluoroethyl SA4503

SA4503, a potent sigma(1) receptor agonist, is reported as having 103-fold higher affinity for sigma(1) (IC(50) = 17.4 nM) than sigma(2) (IC(50) = 1,784 nM) sites in guinea pig brain membranes. Modest structural changes appear to have major effects on sigma(1)/sigma(2) selectivity. The fluoroethyl analog, FE-SA4503, is described as having high primary affinity for sigma(2) sites (IC(50) = 2.11 nM) and a weaker interaction with sigma(1) sites (IC(50) = 6.48 nM). SA4503 and FE-SA4503 have been radiolabeled for PET studies, and both bind selectively to sigma(1) receptors in animal and human brain in vivo. We prepared SA4503 and FE-SA4503 as reference compounds for radioligand development efforts. In our hands, SA4503 is 14-fold selective for sigma(1) (K(i) = 4.6 nM) over sigma(2) (K(i) = 63.1 nM) sites in guinea pig brain homogenates. Further, FE-SA4503 exhibits the same 14-fold selectivity for sigma(1) (K(i) = 8.0 nM) over sigma(2) (K(i) = 113.2 nM) receptors. The main differences from previously reported values stem from sigma(2) affinity determinations. This protocol, displacement of [(3)H]DTG binding to sigma(2) sites using (+)-pentazocine (200 nM) to mask sigma(1) sites, was validated by the proper rank order of sigma(2) inhibitory potencies shown by a panel of additional ligands: ifenprodil > haloperidol > DTG >> (+)-pentazocine. Robust Pearson correlation (r = 1.0, P = 0.002; slope = 0.97) was observed for our pK(i) values against those from a prior study by others. The findings have bearing on structure-activity relationships for this active series, and on conclusions that might be drawn from experiments relying upon defined sigma(1)/sigma(2) binding selectivity.

Holding Back the Tide of Antibiotic Resistance

Another novel approach is being pursued by Wisconsin-based ConjuGon (http://www.conjugon.com/). This eight-person startup is using bacterial sexual reproduction to spread lethal genes in pathogen communities. When the lethal genes are expressed, the bacterial population dies out.“I'm a plasmid biologist,” says Marcin Filutowicz, professor of bacteriology at the University of Wisconsin-Madison, and one of ConjuGon's founders. “Bacterial plasmids are extra chromosomal elements that bacteria don't need to live. They transfer DNA molecules among bacteria. They carry genes which frequently encode for antibiotic resistance. Most of the DNA that is transferred between bacteria is done through plasmid exchange.” ConjuGon engineers specific plasmids with DNA that encodes toxins as well as benign bacteria that are immune to the toxins encoded by the plasmids. When immune bacteria transfer the plasmid that encodes for the toxins to the recipient bacteria, such as a pathogen like Pseudomonas aeruginosa, during conjugation the toxin is expressed, killing the pathogen.“It is a Trojan-horse-like approach where we introduce a DNA molecule from donor to recipient and then express in the recipients multiple toxins or single toxins,” says Filutowicz. “When we express multiple toxins, we reduce the probability for the recipient bacteria to acquire immunity.”ConjuGon is targeting genes that encode for antibiotics that block a certain RNA polymerase at several different locations in bacteria. The company has a patent on “Sigma Binding of RNA Polymerase and Uses Therof” for its method of high-throughput screening for small-molecule drugs that interfere with the binding of transcription factor sigma to the core RNA polymerase. ConjuGon chose this target because it is unlikely that bacteria would mutate in enough multiple ways to evade a compound that interferes with this interaction.Conjugon recently received a phase I SBIR grant from the Army to do research on a treatment for antibiotic-resistant Acineobacter baumannii infections contracted by wounded soldiers in Iraq and Afghanistan. Additionally, Ravi Shankar, associate professor, Department of Surgery and Cell Biology, Neurobiology & Anatomy Burn and Shock Trauma Unit at Loyola University Medical Center, is conducting the proof-of-concept studies on ConjuGon's approach on mice. Shankar is interested in ConjuGon's approach as a specialized or adjunct therapy for patients with burn or trauma wounds who have a long healing process. “Much of the time, especially with burn-wound cases, it is not about life or death. It is about achieving adequate wound coverage with grafts or synthetic material so the person can heal faster,” Shankar says. “But unfortunately, if you have an infection, many of the grafts fail. They lift off.” Conventional antibiotic therapy can be problematic as the antibiotic has to be delivered to the actual wound area of the patient. Thus, a systemic antibiotic is not enough. A majority of time, wound care is delivered via topical antibiotics and antimicrobials, such as topical silver emulsions.Shankar says that in the mouse studies that pathogens were totally eliminated within 30 min of administering ConjuGon treatment. “It actually works…. We have to see if it works on human beings,” Shankar says.

<![CDATA[Binding characteristics of sigma2 receptor ligands]]>

Sigma (sigma) receptors, once considered a type of opioid receptor, are now recognized as representing a unique receptive entity and at least two different types of sigma receptors have been identified: sigma1 and sigma2 receptors. Evidence suggests that these receptors might be targeted and exploited for the development of agents potentially useful for the treatment of several central disorders. This review primarily describes some of our efforts to understand those structural features that contribute to sigma2 receptor binding, and some recent work by other investigators is also included. Despite an inability to formulate a unified pharmacophore model for sigma2 binding due to the diversity of structure-types that bind at the receptor, and to the conformational flexibility of these ligands, significant progress has been made toward the development of some very high-affinity agents.

Editorial

The concept of sigma receptors or sigma binding sites emerged in the late seventies out of the field of opiate receptor research, when the dysphoric effects of several new opiate drugs could not sufficiently be explained by interaction with one of the known opiate receptors. Originally proposed as an additional subtype of opiate receptors it was soon recognized that the newly designed sigma site was distinct from the different subtypes of opiate receptors (typical G-protein coupled membrane receptors) but seemed to represent first candidates of a new class of drug binding sites with rather atypical localization (largely intracellularly), of unknown transduction mechanism and with rather obscure cellular functions. Similarly, their pharmacological relevance remained in dispute.

Neuropharmacology of the Anxiolytic Drug Opripramol, a Sigma Site Ligand

Although opipramol is structurally related to imipramine, it does not represent a tricyclic antidepressant drug as it does not inhibit the neuronal uptake of norepinephrine and/or serotonin. Unlike imipramine it is a rather potent sigma ligand with modest subclass selectivity which is similar in vitro as well as ex vivo. Opipramol is active in several behavioural paradigms indicative of anxiolytic properties at doses (1-10 mg/kg), which are also needed to occupy sigma binding sites. Somewhat higher doses (10-20 mg/kg) are needed for "antidepressant like" effects. The data allow the conclusion that interaction with sigma sites is involved in the anxiolytic and antidepressant effects of opipramol albeit a contribution of its weaker D (2)-antagonistic and 5-HT2-antagonistic properties cannot be totally be excluded.

Behavioral Pharmacology of σ-Ligands

Sigma (sigma) receptors, first defined as a subclass of opioid receptors, later confounded with the high affinity phencyclidine (PCP) binding sites, now are regarded as unique binding sites, distinct from opiate and PCP receptors, and related to higher brain function. The investigation of functional significance of sigma receptors in the brain has been hampered for many years by relative lack of specific tool drugs and by the unavailability of their coherent classification into postulated agonists and antagonists. However, a potential involvement of sigma receptors in psychotic disorders was first suggested soon after their discovery. The sigma receptors are classified into two subtypes, sigma (1) and sigma (2) receptors, of which the first was recently cloned from rodent and human tissues while the second has not yet been fully characterized. Although the precise mechanism of the functional response of these receptors is still uncertain, it is accepted that sigma receptors can modulate a number of central neurotransmitter systems, including noradrenergic, glutamatergic and dopaminergic ones. The sigma receptors have been postulated to be involved in numerous pharmacological and physiological functions, including motor disorders, psychotic disorders, neuroprotective mechanisms. In the last years, a number of compounds with a high affinity and selectivity for sigma binding sites have been discovered and investigated for their therapeutic potential. In this review, we try to summarize the behavioral effects of sigma receptor ligands that have been described, and their activity in animal models related to some brain disorders, especially schizophrenia and affective disorders.

Communication between Esigma(54) , promoter DNA and the conserved threonine residue in the GAFTGA motif of the PspF sigma-dependent activator during transcription activation.

Conversion of Esigma(54) closed promoter complexes to open promoter complexes requires specialized activators which are members of the AAA (ATPases Associated with various cellular Activities) protein family. The ATP binding and hydrolysis activity of Esigma(54) activators is used in an energy coupling reaction to remodel the Esigma(54) closed promoter complex and to overcome the sigma(54)-imposed block on open complex formation. The remodelling target for the AAA activator within the Esigma(54) closed complex includes a complex interface contributed to by Region I of sigma(54), core RNA polymerase and a promoter DNA fork junction structure, comprising the Esigma(54) regulatory centre. One sigma(54) binding surface on Esigma(54) activators is a conserved sequence known as the GAFTGA motif. Here, we present a detailed characterization of the interaction between Region I of sigma(54) and the Escherichia coli AAA sigma(54) activator Phage shock protein F. Using Esigma(54) promoter complexes that mimic different conformations adopted by the DNA during open complex formation, we investigated the contribution of the conserved threonine residue in the GAFTGA motif to transcription activation. Our results suggest that the organization of the Esigma(54) regulatory centre, and in particular the conformation adopted by the sigma(54) Region I and the DNA fork junction structure during open complex formation, is communicated to the AAA activator via the conserved T residue of the GAFTGA motif.