Structural determinants of the direct inhibition of GIRK channels by Sigma-1 receptor antagonist.

G-protein-gated inward rectifier K+ (GIRK) channels play a critical role in the regulation of the excitability of cardiomyocytes and neurons and include GIRK1, GIRK2, GIRK3 and GIRK4 subfamily members. BD1047 dihydrobromide (BD1047) is one of the representative antagonists of the multifunctional Sigma-1 receptor (S1R). In the analysis of the effect of BD1047 on the regulation of Gi-coupled receptors by S1R using GIRK channel as an effector, we observed that BD1047, as well as BD1063, directly inhibited GIRK currents even in the absence of S1R and in a voltage-independent manner. Thus, we aimed to clarify the effect of BD1047 on GIRK channels and identify the structural determinants. By electrophysiological recordings in Xenopus oocytes, we observed that BD1047 directly inhibited GIRK channel currents, producing a much stronger inhibition of GIRK4 compared to GIRK2. It also inhibited ACh-induced native GIRK current in isolated rat atrial myocytes. Chimeric and mutagenesis studies of GIRK2 and GIRK4 combined with molecular docking analysis demonstrated the importance of Leu77 and Leu84 within the cytoplasmic, proximal N-terminal region and Glu147 within the pore-forming region of GIRK4 for inhibition by BD1047. The activator of GIRK channels, ivermectin, competed with BD1047 at Leu77 on GIRK4. This study provides us with a novel inhibitor of GIRK channels and information for developing pharmacological treatments for GIRK4-associated diseases.

Dual DAT and sigma receptor inhibitors attenuate cocaine effects on nucleus accumbens dopamine dynamics in rats.

Psychostimulant use disorders (PSUD) are prevalent; however, no FDA-approved medications have been made available for treatment. Previous studies have shown that dual inhibitors of the dopamine transporter (DAT) and sigma receptors significantly reduce the behavioral/reinforcing effects of cocaine, which have been associated with stimulation of extracellular dopamine (DA) levels resulting from DAT inhibition. Here, we employ microdialysis and fast scan cyclic voltammetry (FSCV) procedures to investigate the effects of dual inhibitors of DAT and sigma receptors in combination with cocaine on nucleus accumbens shell (NAS) DA dynamics in naïve male Sprague Dawley rats. In microdialysis studies, administration of rimcazole (3, 10 mg/kg; i.p.) or its structural analog SH 3-24 (1, 3 mg/kg; i.p.), compounds that are dual inhibitors of DAT and sigma receptors, significantly reduced NAS DA efflux stimulated by increasing doses of cocaine (0.1, 0.3, 1.0 mg/kg; i.v.). Using the same experimental conditions, in FSCV tests, we show that rimcazole pretreatments attenuated cocaine-induced stimulation of evoked NAS DA release but produced no additional effect on DA clearance rate. Under the same conditions, JJC8-091, a modafinil analog and dual inhibitor of DAT and sigma receptors, similarly attenuated cocaine-induced stimulation of evoked NAS DA release but produced no additional effect on DA clearance rate. Our results provide the neurochemical groundwork towards understanding actions of dual inhibitors of DAT and sigma receptors on DA dynamics that likely mediate the behavioral effects of psychostimulants like cocaine.

Preclinical efficacy profiles of the sigma-1 modulator E1R and of fenfluramine in two chronic mouse epilepsy models.

OBJECTIVE: Given its key homeostatic role affecting mitochondria, ionotropic and metabotropic receptors, and voltage-gated ion channels, sigma-1 receptor (Sig1R) represents an interesting target for epilepsy management. Antiseizure effects of the positive allosteric modulator E1R have already been reported in acute seizure models. Although modulation of serotonergic neurotransmission is considered the main mechanism of action of fenfluramine, its interaction with Sig1R may be of additional relevance. METHODS: To further explore the potential of Sig1R as a target, we assessed the efficacy and tolerability of E1R and fenfluramine in two chronic mouse models, including an amygdala kindling paradigm and the intrahippocampal kainate model. The relative contribution of the interaction with Sig1R was analyzed using combination experiments with the Sig1R antagonist NE-100. RESULTS: Whereas E1R exerted pronounced dose-dependent antiseizure effects at well-tolerated doses in fully kindled mice, only limited effects were observed in response to fenfluramine, without a clear dose dependency. In the intrahippocampal kainate model, E1R failed to influence electrographic seizure activity. In contrast, fenfluramine significantly reduced the frequency of electrographic seizure events and their cumulative duration. Pretreatment with NE-100 reduced the effects of E1R and fenfluramine in the kindling model. Surprisingly, pre-exposure to NE-100 in the intrahippocampal kainate model rather enhanced and prolonged fenfluramine's antiseizure effects. SIGNIFICANCE: In conclusion, the kindling data further support Sig1R as an interesting target for novel antiseizure medications. However, it is necessary to further explore the preclinical profile of E1R in chronic epilepsy models with spontaneous seizures. Despite the rather limited effects in the kindling paradigm, the findings from the intrahippocampal kainate model suggest that it is of interest to further assess a possible broad-spectrum potential of fenfluramine.

The classical D1 dopamine receptor antagonist SCH23390 is a functional sigma-1 receptor allosteric modulator.

SCH23390 is a widely used D1 dopamine receptor (D1R) antagonist that also elicits some D1R-independent effects. We previously found that the benzazepine, SKF83959, an analog of SCH23390, produces positive allosteric modulation of the Sigma-1 receptor (Sig1R). SCH23390 does not bind to the orthodoxic site of Sig1R but enhances the binding of 3H (+)-pentazocine to Sig1R. In this study, we investigated whether SCH23390 functions as an allosteric modulator of Sig1R. We detected increased Sig1R dissociation from binding immunoglobulin protein (BiP) and translocation of Sig1R to the plasma membrane in response to SCH23390 in transfected HEK293T and SH-SY5Y cells, respectively. Activation of Sig1R by SCH23390 was further confirmed by inhibition of GSK3ß activity in a time- and dose-dependent manner; this effect was blocked by pretreatment with the Sig1R antagonist, BD1047, and by knockdown of Sig1R. SCH23390 also inhibited GSK3ß in wild-type mice but not in Sig1R knockout mice. Finally, we showed that SCH23390 allosterically modulated the effect of the Sig1R agonist SKF10047 on inhibition of GSK3ß. This positive allosteric effect of SCH23390 was further confirmed via promotion of neuronal protection afforded by SKF10047 in primary cortical neurons challenged with MPP+. These results provide the first evidence that SCH23390 elicits functional allosteric modulation of Sig1R. Our findings not only reveal novel pharmacological effects of SCH23390 but also indicate a potential mechanism for SCH23390-mediated D1R-independent effects. Therefore, attention should be paid to these Sig1R-mediated effects when explaining pharmacological responses to SCH23390.

Design, Synthesis, and Cytotoxic Assessment of New Haloperidol Analogues as Potential Anticancer Compounds Targeting Sigma Receptors.

Sigma receptors (SRs), including SR1 and SR2 subtypes, have attracted increasing interest in recent years due to their involvement in a wide range of activities, including the modulation of opioid analgesia, neuroprotection, and potential anticancer activity. In this context, haloperidol (HAL), a commonly used antipsychotic drug, also possesses SR activity and cytotoxic effects. Herein, we describe the identification of novel SR ligands, obtained by a chemical hybridization approach. There wereendowed with pan-affinity for both SR subtypes and evaluated their potential anticancer activity against SH-SY5Y and HUH-7 cancer cell lines. Through a chemical hybridization approach, we identified novel compounds (4d, 4e, 4g, and 4j) with dual affinity for SR1 and SR2 receptors. These compounds were subjected to cytotoxicity testing using a resazurin assay. The results revealed potent cytotoxic effects against both cancer cell lines, with IC50 values comparable to HAL. Interestingly, the cytotoxic potency of the novel compounds resembled that of the SR1 antagonist HAL rather than the SR2 agonist siramesine (SRM), indicating the potential role of SR1 antagonism in their mechanism of action. The further exploration of their structure-activity relationships and their evaluation in additional cancer cell lines will elucidate their therapeutic potential and may pave the way for the development of novel anticancer agents that target SRs.

Sigma-1 Receptor Signaling: In Search of New Therapeutic Alternatives for Cardiovascular and Renal Diseases.

Cardiovascular and renal diseases are among the leading causes of death worldwide, and regardless of current efforts, there is a demanding need for therapeutic alternatives to reduce their progression to advanced stages. The stress caused by diseases leads to the activation of protective mechanisms in the cell, including chaperone proteins. The Sigma-1 receptor (Sig-1R) is a ligand-operated chaperone protein that modulates signal transduction during cellular stress processes. Sig-1R interacts with various ligands and proteins to elicit distinct cellular responses, thus, making it a potential target for pharmacological modulation. Furthermore, Sig-1R ligands activate signaling pathways that promote cardioprotection, ameliorate ischemic injury, and drive myofibroblast activation and fibrosis. The role of Sig-1R in diseases has also made it a point of interest in developing clinical trials for pain, neurodegeneration, ischemic stroke, depression in patients with heart failure, and COVID-19. Sig-1R ligands in preclinical models have significantly beneficial effects associated with improved cardiac function, ventricular remodeling, hypertrophy reduction, and, in the kidney, reduced ischemic damage. These basic discoveries could inform clinical trials for heart failure (HF), myocardial hypertrophy, acute kidney injury (AKI), and chronic kidney disease (CKD). Here, we review Sig-1R signaling pathways and the evidence of Sig-1R modulation in preclinical cardiac and renal injury models to support the potential therapeutic use of Sig-1R agonists and antagonists in these diseases.

Pharmacokinetics and brain σ1 receptor occupancy of MR309, a selective σ1 receptor antagonist.

AIMS: Preclinical studies of MR309, a selective sigma-1 receptor (σ1R) antagonist, support a potential role in treating neuropathic pain. We report 2 studies that provide insight into the pharmacokinetics (PK) and brain σ1R binding of MR309. METHODS: Steady-state PK of MR309 (400 mg once daily and 200 mg twice-daily [BID] for 10 days; EudraCT 2015-001818-99 [PK study]) and the relationship between MR309 plasma exposure and brain σ1R occupancy (EudraCT 2017-000670-11 [positron emission tomography study]) were investigated in healthy volunteers. Positron emission tomography using the σ1R ligand [11 C]SA4503 was conducted at baseline, and 2 and 8 hours after a single dose of MR309 (200-800 mg). The relationship between brain σ1R occupancy and MR309 exposure was explored using data-driven model fitting. RESULTS: MR309 was well tolerated, brain σ1R occupancy ranged between 30.5 and 74.9% following single-dose MR309 (n = 7). MR309 BID provided a plasma PK profile with less fluctuation than once daily dosing (n = 16). MR309 200 mg BID yielded average steady state plasma concentrations between 2000 and 4000 ng/mL in the PK study, which corresponded to an estimated brain σ1R occupancy of 59-74%. CONCLUSION: MR309 200 mg BID dose was below the 75% σ1R occupancy threshold expected to elicit maximal antinociceptive effect as observed in neuropathic pain models. Further investigations of MR309 for neuropathic pain will require higher brain σ1R occupancy, and establish the optimal dose by elucidating the clinical impact of a broad range of brain σ1R occupancy across different neuropathic pain indications.

Long-Lasting Nociplastic Pain Modulation by Repeated Administration of Sigma-1 Receptor Antagonist BD1063 in Fibromyalgia-like Mouse Models.

Sigma-1 receptor (σ1R) ligands have been shown to be effective at relieving neuropathic and inflammatory pain, but have not yet been tested in experimental models of fibromyalgia. The objective of this study was to evaluate the effect of a σ1R antagonist (BD1063) compared to pregabalin. ICR-CD1 female mice were subjected to either six repeated injections of reserpine, to cause reserpine-induced myalgia (RIM6), or acidified saline intramuscular injections (ASI). In these two models, we evaluated the effect of BD1063 and pregabalin on thermal hypersensitivity, anxiety-like and depression-like behaviors, and on spinal cord gliosis. BD1063 exerted an antinociceptive effect on both reflexive (thermal hyperalgesia) and nonreflexive (anxiety- and depression-like) pain behaviors, and reduced spinal astroglial and microglial reactivity, following repeated treatment for 2 weeks. Interestingly, the effects of BD1063 were long-term, lasting several weeks after treatment discontinuation in both fibromyalgia-like models. Similar results were obtained with pregabalin, but the effects on pain behaviors lasted for a shorter length of time, and pregabalin did not significantly modulate spinal glial reactivity. The inhibitory and long-lasting effect of pharmacological blockade of σ1Rs on both sensory and affective dimensions of nociplastic-like pain and spinal cord gliosis in two experimental models of fibromyalgia support the application of this therapeutic strategy to treat fibromyalgia.

Examination of the Novel Sigma-1 Receptor Antagonist, SI 1/28, for Antinociceptive and Anti-allodynic Efficacy against Multiple Types of Nociception with Fewer Liabilities of Use.

Neuropathic pain is a significant problem with few effective treatments lacking adverse effects. The sigma-1 receptor (S1R) is a potential therapeutic target for neuropathic pain, as antagonists for this receptor effectively ameliorate pain in both preclinical and clinical studies. The current research examines the antinociceptive and anti-allodynic efficacy of SI 1/28, a recently reported benzylpiperazine derivative and analog of the S1R antagonist SI 1/13, that was 423-fold more selective for S1R over the sigma-2 receptor (S2R). In addition, possible liabilities of respiration, sedation, and drug reinforcement caused by SI 1/28 have been evaluated. Inflammatory and chemical nociception, chronic nerve constriction injury (CCI) induced mechanical allodynia, and adverse effects of sedation in a rotarod assay, conditioned place preference (CPP), and changes in breath rate and locomotor activity were assessed after i.p. administration of SI 1/28. Pretreatment with SI 1/28 produced dose-dependent antinociception in the formalin test, with an ED50 (and 95% C.I.) value of 13.2 (7.42-28.3) mg/kg, i.p. Likewise, SI 1/28 produced dose-dependent antinociception against visceral nociception and anti-allodynia against CCI-induced neuropathic pain. SI 1/28 demonstrated no impairment of locomotor activity, conditioned place preference, or respiratory depression. In summary, SI 1/28 proved efficacious in the treatment of acute inflammatory pain and chronic neuropathy without liabilities at therapeutic doses, supporting the development of S1R antagonists as therapeutics for chronic pain.

Sigma-2 receptor antagonists rescue neuronal dysfunction induced by Parkinson's patient brain-derived α-synuclein.

α-Synuclein oligomers are thought to have a pivotal role in sporadic and familial Parkinson's disease (PD) and related α-synucleinopathies, causing dysregulation of protein trafficking, autophagy/lysosomal function, and protein clearance, as well as synaptic function impairment underlying motor and cognitive symptoms of PD. Moreover, trans-synaptic spread of α-synuclein oligomers is hypothesized to mediate disease progression. Therapeutic approaches that effectively block α-synuclein oligomer-induced pathogenesis are urgently needed. Here, we show for the first time that α-synuclein species isolated from human PD patient brain and recombinant α-synuclein oligomers caused similar deficits in lipid vesicle trafficking rates in cultured rat neurons and glia, while α-synuclein species isolated from non-PD human control brain samples did not. Recombinant α-synuclein oligomers also increased neuronal expression of lysosomal-associated membrane protein-2A (LAMP-2A), the lysosomal receptor that has a critical role in chaperone-mediated autophagy. Unbiased screening of several small molecule libraries (including the NIH Clinical Collection) identified sigma-2 receptor antagonists as the most effective at blocking α-synuclein oligomer-induced trafficking deficits and LAMP-2A upregulation in a dose-dependent manner. These results indicate that antagonists of the sigma-2 receptor complex may alleviate α-synuclein oligomer-induced neurotoxicity and are a novel therapeutic approach for disease modification in PD and related α-synucleinopathies.

Sigma-1 receptor: A drug target for the modulation of neuroimmune and neuroglial interactions during chronic pain.

Immune and glial cells play a pivotal role in chronic pain. Therefore, it is possible that the pharmacological modulation of neurotransmission from an exclusively neuronal perspective may not be enough for adequate pain management, and the modulation of complex interactions between neurons and other cell types might be needed for successful pain relief. In this article, we review the current scientific evidence for the modulatory effects of sigma-1 receptors on communication between the immune and nervous systems during inflammation, as well as the influence of this receptor on peripheral and central neuroinflammation. Several experimental models of pathological pain are considered, including peripheral and central neuropathic pain, osteoarthritic, and cancer pain. Sigma-1 receptor inhibition prevents peripheral (macrophage infiltration into the dorsal root ganglion) and central (activation of microglia and astrocytes) neuroinflammation in several pain models, and enhances immune-driven peripheral opioid analgesia during painful inflammation, maximizing the analgesic potential of peripheral immune cells. Therefore, sigma-1 antagonists may constitute a new class of analgesics with an unprecedented mechanism of action and potential utility in several painful disorders.

Antagonism of Sigma-1 receptor blocks heavy alcohol drinking and associated hyperalgesia in male mice.

BACKGROUND: Alcohol use disorder (AUD) is a complex psychiatric disease characterized by high alcohol intake as well as hyperkatifeia and hyperalgesia during withdrawal. A role for Sigma-1 receptors (Sig-1Rs) in the rewarding and reinforcing effects of alcohol has started to emerge in recent years, as rat studies have indicated that Sig-1R hyperactivity may result in excessive alcohol drinking. Sig-1R studies in mice are very scarce, and its potential role in alcohol-induced hyperalgesia is also unknown. METHODS: In this study, we investigated the role of Sig-1R in alcohol drinking and associated hyperalgesia in male mice, using an intermittent access 2-bottle choice model of heavy drinking. RESULTS: The Sig-1R antagonist BD-1063 was found dose dependently to reduce both alcohol intake and preference, without affecting either water or sucrose intake, suggesting that the effects are specific for alcohol. Notably, the ability of BD-1063 to suppress ethanol intake correlated with the individual baseline levels of alcohol drinking, suggesting that the treatment was more efficacious in heavy drinking animals. In addition, BD-1063 reversed alcohol-induced hyperalgesia during withdrawal, assessed using an automatic Hargreaves test, without affecting thermal sensitivity in alcohol-naïve animals or locomotor activity in either group. CONCLUSIONS: These data show that Sig-1R antagonism dose-dependently reduced ethanol consumption in heavy drinking mice as well as its efficacy in reducing alcohol-induced hyperalgesia. These findings provide a foundation for the development of novel treatments for AUD and associated pain states.

Normalization of Calcium Balance in Striatal Neurons in Huntington's Disease: Sigma 1 Receptor as a Potential Target for Therapy.

Huntington's disease (HD) is a neurodegenerative, dominantly inherited genetic disease caused by expansion of the polyglutamine tract in the huntingtin gene. At the cellular level, HD is characterized by the accumulation of mutant huntingtin protein in brain cells, resulting in the development of the HD phenotype, which includes mental disorders, decreased cognitive abilities, and progressive motor impairments in the form of chorea. Despite numerous studies, no unambigous connection between the accumulation of mutant protein and selective death of striatal neurons has yet been established. Recent studies have shown impairments in the calcium homeostasis in striatal neurons in HD. These cells are extremely sensitive to changes in the cytoplasmic concentration of calcium and its excessive increase leads to their death. One of the possible ways to normalize the balance of calcium in striatal neurons is through the sigma 1 receptor (S1R), which act as a calcium sensor that also exhibits modulating chaperone activity upon the cell stress observed during the development of many neurodegenerative diseases. The fact that S1R is a ligand-operated protein makes it a new promising molecular target for the development of drug therapy of HD based on the agonists of this receptor.

Preclinical and clinical biomarker studies of CT1812: A novel approach to Alzheimer's disease modification.

INTRODUCTION: Amyloid beta (Aß) oligomers are one of the most toxic structural forms of the Aß 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 Aß 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 Aß 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 Aß 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 Aß 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 Aß 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 Aß 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 Aß 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-1 receptor antagonist (BD-1063) potentiates the antinociceptive effect of quercetin in neuropathic pain induced by chronic constriction injury.

Neuropathic pain is characterized by the presence of hyperalgesia and allodynia. Pharmacological treatments include the use of antiepileptics such as pregabalin or gabapentin, as well as antidepressants; however, given the role of the sigma-1 receptor in the generation and maintenance of pain, it has been suggested that sigma-1 receptor antagonists may be effective. There are also other alternatives that have been explored, such as the use of flavonoids such as quercetin. Due to the relevance of drug combinations in therapeutics, the objective of this work was to evaluate the effect of the combination of BD-1063 with quercetin in a chronic sciatic nerve constriction model using the "Surface of Synergistic Interaction" analysis method. The combination had preferable additive or synergistic effects, with BD-1063 (17.8 mg/kg) + QUER (5.6 mg/kg) showing the best antinociceptive effects. The required doses were also lower than those used individually to obtain the same level of effect. Our results provide the first evidence that the combination of a sigma-1 receptor antagonist and the flavonoid quercetin may be useful in the treatment of nociceptive behaviors associated with neuropathic pain, suggesting a new therapeutic alternative for this type of pain.

The Peripheral Role of CCL2 in the Anti-Nociceptive Effect of Sigma-1 Receptor Antagonist BD1047 on Inflammatory Hyperalgesia in Rats.

Our recent study demonstrated that the CC-chemokine ligand 2 (CCL2) present in primary afferent fibers (PAFs) plays an important role in the microglia-dependent neuronal activation associated with zymosan-induced inflammatory pain. The present study was aimed to evaluate whether BD1047 (a prototypical sigma-1 receptor (Sig-1R) antagonist) is capable of modifying elevated levels of inflammation-evoked CCL2 as a peripheral antinociceptive mechanism. In DRG primary culture, zymosan dose-dependently increased CCL2 release from isolectin B4 (IB4)-positive DRG neurons, a process that was inhibited by co-culture with BD1047. Single treatment of BD1047 before intraplantar injection of zymosan in rats significantly reduced thermal hyperalgesia and mechanical hyperalgesia, as well as CCL2 expression in DRG neurons and microglia activation in the spinal dorsal horn. In the Complete Freund's adjuvant (CFA)-induced inflammation model, repeated administration of BD1047 dramatically attenuated thermal hyperalgesia and mechanical hyperalgesia, and significantly diminished CCL2 immunoreactivity and microglia activation. Notably, CFA-induced inflammation significantly increased Sig-1R immunoreactivity in DRG neurons, which was co-localized with CCL2 and IB4, respectively. Taken together, our results suggest that BD1047's anti-nociceptive property was substantially mediated by the inhibition of CCL2 release in unmyelinated PAFs and that this may, in turn, have attenuated the spinal microglia activation that is associated with inflammatory pain.

Novel High Affinity Sigma-1 Receptor Ligands from Minimal Ensemble Docking-Based Virtual Screening.

Sigma-1 receptor (S1R) is an intracellular, multi-functional, ligand operated protein that also acts as a chaperone. It is considered as a pluripotent drug target in several pathologies. The publication of agonist and antagonist bound receptor structures has paved the way for receptor-based in silico drug design. However, recent studies on this subject payed no attention to the structural differences of agonist and antagonist binding. In this work, we have developed a new ensemble docking-based virtual screening protocol utilizing both agonist and antagonist bound S1R structures. This protocol was used to screen our in-house compound library. The S1R binding affinities of the 40 highest ranked compounds were measured in competitive radioligand binding assays and the sigma-2 receptor (S2R) affinities of the best S1R binders were also determined. This way three novel high affinity S1R ligands were identified and one of them exhibited a notable S1R/S2R selectivity.

Combination of Heme Oxygenase-1 Inhibition and Sigma Receptor Modulation for Anticancer Activity.

Cancer is a multifactorial disease that may be tackled by targeting different signaling pathways. Heme oxygenase-1 (HO-1) and sigma receptors (σRs) are both overexpressed in different human cancers, including prostate and brain, contributing to the cancer spreading. In the present study, we investigated whether HO-1 inhibitors and σR ligands, as well a combination of the two, may influence DU145 human prostate and U87MG human glioblastoma cancer cells proliferation. In addition, we synthesized, characterized, and tested a small series of novel hybrid compounds (HO-1/σRs) 1-4 containing the chemical features needed for HO-1 inhibition and σR modulation. Herein, we report for the first time that targeting simultaneously HO-1 and σR proteins may be a good strategy to achieve increased antiproliferative activity against DU145 and U87MG cells, with respect to the mono administration of the parent compounds. The obtained outcomes provide an initial proof of concept useful to further optimize the structure of HO-1/σRs hybrids to develop novel potential anticancer agents.

Effect of Dopamine D2 Receptor Antagonists on [18F]-FEOBV Binding.

The interaction of dopaminergic and cholinergic neurotransmission in, e.g., Parkinson's disease has been well established. Here, D2 receptor antagonists were used to assess changes in [18F]-FEOBV binding to the vesicular acetylcholine transporter (VAChT) in rodents using positron emission tomography (PET). After pretreatment with either 10 mg/kg haloperidol, 1 mg/kg raclopride, or vehicle, 90 min dynamic PET scans were performed with arterial blood sampling. The net influx rate (Ki) was obtained from Patlak graphical analysis, using a metabolite-corrected plasma input function and dynamic PET data. [18F]-FEOBV concentration in whole-blood or plasma and the metabolite-corrected plasma input function were not significantly changed by the pretreatments (adjusted p > 0.07, Cohen's d 0.28-1.89) while the area-under-the-curve (AUC) of the parent fraction of [18F]-FEOBV was significantly higher after haloperidol treatment (adjusted p = 0.022, Cohen's d = 2.51) than in controls. Compared to controls, the AUC of [18F]-FEOBV, normalized for injected dose and body weight, was nonsignificantly increased in the striatum after haloperidol (adjusted p = 0.4, Cohen's d = 1.77) and raclopride (adjusted p = 0.052, Cohen's d = 1.49) treatment, respectively. No changes in the AUC of [18F]-FEOBV were found in the cerebellum (Cohen's d 0.63-0.74). Raclopride treatment nonsignificantly increased Ki in the striatum 1.3-fold compared to control rats (adjusted p = 0.1, Cohen's d = 1.1) while it reduced Ki in the cerebellum by 28% (adjusted p = 0.0004, Cohen's d = 2.2) compared to control rats. Pretreatment with haloperidol led to a nonsignificant reduction in Ki in the striatum (10%, adjusted p = 1, Cohen's d = 0.44) and a 40-50% lower Ki than controls in all other brain regions (adjusted p < 0.0005, Cohen's d = 3.3-4.7). The changes in Ki induced by the selective D2 receptor antagonist raclopride can in part be quantified using [18F]-FEOBV PET imaging. Haloperidol, a nonselective D2/σ receptor antagonist, either paradoxically decreased cholinergic activity or blocked off-target [18F]-FEOBV binding to σ receptors. Hence, further studies evaluating the binding of [18F]-FEOBV to σ receptors using selective σ receptor ligands are necessary.

Urinary bladder sigma-1 receptors: A new target for cystitis treatment.

No adequate treatment is available for painful urinary bladder disorders such as interstitial cystitis/bladder pain syndrome, and the identification of new urological therapeutic targets is an unmet need. The sigma-1 receptor (σ1-R) modulates somatic pain, but its role in painful urological disorders is unexplored. The urothelium expresses many receptors typical of primary sensory neurons (e.g. TRPV1, TRPA1 and P2X3) and high levels of σ1-R have been found in these neurons; we therefore hypothesized that σ1-R may also be expressed in the urothelium and may have functional relevance in this tissue. With western blotting and immunohistochemical methods, we detected σ1-R in the urinary bladder in wild-type (WT) but not in σ1-R-knockout (σ1-KO) mice. Interestingly, σ1-R was located in the bladder urothelium not only in mouse, but also in human bladder sections. The severity of histopathological (edema, hemorrhage and urothelial desquamation) and biochemical alterations (enhanced myeloperoxidase activity and phosphorylation of extracellular regulated kinases 1/2 [pERK1/2]) that characterize cyclophosphamide-induced cystitis was lower in σ1-KO than in WT mice. Moreover, cyclophosphamide-induced pain behaviors and referred mechanical hyperalgesia were dose-dependently reduced by σ1-R antagonists (BD-1063, NE-100 and S1RA) in WT but not in σ1-KO mice. In contrast, the analgesic effect of morphine was greater in σ1-KO than in WT mice. Together these findings suggest that σ1-R plays a functional role in the mechanisms underlying cyclophosphamide-induced cystitis, and modulates morphine analgesia against urological pain. Therefore, σ1-R may represent a new drug target for urinary bladder disorders.