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Sigma receptor is a transmembrane non-GPCR protein expressed mainly in the endoplasmic reticulum membrane associated with mitochondria. It is classified into two types: Sigma-1 (S1R) and Sigma-2 (S2R) based on their biological functions. S1R has been implicated in many neurological disorders such as anxiety, schizophrenia, and depression. Therefore, S1R ligands possess a variety of potential clinical applications with a great interest in the treatment of neuropathic pain. In this study, we report the discovery of a novel lead compound for S1R binding, based on the thiazolidine-2,4-dione nucleus. We have explored hydrophobic groups of different sizes on both sides of the five-membered ring scaffold guided by the crystal structure of S1R. Six compounds showed more than 50% displacement of the radioligand at 10 µM concentration with compound 6c resulting in 100% displacement and a K

The σ(2) receptor has attracted intense interest in cancer imaging(1), psychiatric disease(2), neuropathic pain(3–5), and other areas of biology(6,7). We determined the crystal structure of this receptor in complex with the clinical candidate roluperidone(2) and the tool compound PB28(8). These structures templated a large-scale docking screen of 490 million virtual molecules, of which 484 compounds were synthesized and tested. 127 new chemotypes with affinities superior to 1 μM were identified, 31 of which had affinities superior to 50 nM. Hit rate fell smoothly and monotonically with docking score. We optimized three hits for potency and selectivity, achieving affinities ranging from 3 to 48 nM with up to 250-fold selectivity versus the σ(1) receptor. Crystal structures of two new ligands bound to the σ(2) receptor confirmed the docked poses. To investigate the contribution of the σ(2) receptor in pain, two potent σ(2)-selective ligands and one potent σ(1)/σ(2) non-selective ligand were tested for efficacy in a mouse neuropathic pain model. All three ligands demonstrated time-dependent decreases in mechanical hypersensitivity in the spared nerve injury model(9), supporting a role for the σ(2) receptor in nociception. This study illustrates the opportunities for rapid discovery of in vivo probes to study under-explored areas of biology using structure-based screens of diverse, ultra-large libraries.

Allosteric modulation represents an important approach in drug discovery because of its advantages in safety and selectivity. SOMCL-668 is the first selective and potent sigma-1 receptor allosteric modulator, discovered in our laboratory. The present work investigates the potential therapeutic effects of SOMCL-668 on phencyclidine (PCP)-induced schizophrenia-related behavior in mice and further elucidates underlying mechanisms for its antipsychotic-like effects. SOMCL-668 not only attenuated acute PCP-induced hyperactivity and PPI disruption, but also ameliorated social deficits and cognitive impairment induced by chronic PCP treatment. Pretreatment with the selective sigma-1 receptor antagonist BD1047 blocked the effects of SOMCL-668, indicating sigma-1 receptor-mediated responses. This was confirmed using sigma-1 receptor knockout mice, in which SOMCL-668 failed to ameliorate PPI disruption and hyperactivity induced by acute PCP and social deficits and cognitive impairment induced by chronic PCP treatment. Additionally, in vitro SOMCL-668 exerted positive modulation of sigma-1 receptor agonist-induced intrinsic plasticity in brain slices recorded by patch-clamp. Furthermore, in vivo lower dose of SOMCL-668 exerted positive modulation of improvement in social deficits and cognitive impairment induced by the selective sigma-1 agonist PRE084. Also, SOMCL-668 reversed chronic PCP-induced down-regulation in expression of frontal cortical p-AKT/AKT, p-CREB/CREB and BDNF in wide-type but not sigma-1 knockout mice. Moreover, administration of the PI3K/AKT inhibitor LY294002 abolished amelioration by SOMCL-668 of chronic PCP-induced schizophrenia-related behaviors by inhibition of BDNF expression. The present data provide initial, proof-of-concept evidence that allosteric modulation of the sigma-1 receptor may be a novel approach for the treatment of psychotic illness.

Sigma‐2 receptor/TMEM97 is overexpressed in many tumours, and sigma‐2 receptor ligands are under investigation for cancer therapy. We intended to evaluate the effect of PB28 on renal cancer in proliferation, migration and invasion in vitro and in vivo. Invasive renal cancer cell lines treated with PB28 (or sigma‐2 receptor antagonist 1) were subjected to cell proliferation, migration and invasion assays. The therapeutic effect of PB28 was performed on nude mice. Western blot for proteins in the PI3K‐AKT‐mTOR signalling pathway was conducted. A CCK‐8 assay was used to examine the effect of the combination of PB28 and cisplatin on renal cancer cells. Significant inhibitory effects were observed on proliferation, migration and invasion of 786‐O and ACHN cells after culturing with PB28. But, the outcomes of sigma‐2 receptor antagonist 1 presented the opposite tendency. PB28 significantly inhibited the proliferative and invasive ability of OS‐RC‐2 cells in vivo. Treatment resulted in decreased phosphorylation of constituents of the PI3K‐AKT‐mTOR pathway. The combination of PB28 and cisplatin showed enhanced efficacy in the inhibition of renal cancer cell proliferation. Taken together, PB28 inhibited the tumorigenic behaviours of renal cancer cells by regulating the PI3K‐AKT‐mTOR signalling pathway and was expected to be a sensitizer of cisplatin.

金沢大学疾患モデル総合研究センター, Background: This study chronologically evaluated the expression of the intensity and distribution of the sigma-1 receptor (σ1R) demonstrated by radiolabeled 2-[4-(2-iodophenyl)piperidino]cyclopentanol (OI5V) in a rat model of myocardial ischemia and reperfusion. Methods and Results: The left coronary artery was occluded for 30 min, followed by reperfusion. Dual-tracer autoradiography with 125I-OI5V and 99 mTc-MIBI was performed to assess the spatiotemporal changes in 125I-OI5V uptake (n=5-6). Significant and peaked 125I-OI5V uptake in the ischemic area was observed at 3 days after reperfusion, and the 125I-OI5V uptake ratio of ischemic area to normally perfused left ventricular area decreased gradually from 3 to 28 days (mean value ± SD; 0.90±0.12 at 1 day, 1.89±0.19 at 3 days, 1.52±0.17 at 7 days, 1.34±0.13 at 14 days, and 1.16±0.14 at 28 days, respectively). Triple-tracer autoradiography with 125I-OI5V, 99 mTc-MIBI, and 201TlCl was performed to evaluate 125I-OI5V uptake in the ischemic area in relation to the residual perfusion at 7 days (n=4). The 125I-OI5V uptake ratio of the non-salvaged area was higher compared to that of the salvaged area in the ischemic area. 123I-OI5V and 99 mTc-MIBI SPECT/CT was performed 3 days after reperfusion (n=3), and the in vivo images showed clear uptake of 123I-OI5V in the perfusion defect area. Conclusions: The present study confirmed the spatiotemporal expression pattern of σ1R expression. Non-invasive σ1R imaging with 123I or 125I-OI5V was feasible to monitor the expression of σ1R after myocardial ischemia and reperfusion. © 2021 Japanese Circulation Society. All rights reserved.

Purpose Association of the metal chelating portion of thiosemicarbazone with the cytotoxic activity of sigma-2 receptors appears a promising strategy for the treatment of pancreatic tumors. Here, we developed a novel sigma-2 receptor targeting thiosemicarbazone (FA4) that incorporates a moiety associated with lysosome destabilization and ROS increase in order to design more efficient antitumor agents. Methods The density of sigma receptors in pancreatic cancer cells was evaluated by flow cytometry. In these cells, cytotoxicity (MTT assay) and activation of ER- and mitochondria-dependent cell death pathways (mRNA expression of GRP78, ATF6, IRE1, PERK; ROS levels by MitoSOX and DCFDA-AM; JC-1 staining) induced by the thiosemicarbazones FA4, MLP44, PS3 and ACthio-1, were evaluated. The expression of autophagic proteins (ATG5, ATG7, ATG12, beclin, p62 and LC3-I) was also studied. In addition, the in vivo effect of FA4 in xenograft models with and without gemcitabine challenge was investigated. Results We found that FA4 exerted a more potent cytotoxicity than previously studied thiosemicarbazones (MLP44, PS3 and ACthio-1), which were found to display variable effects on the ER or the mitochondria-dependent pro-apoptotic axis. By contrast, FA4 activated pro-apoptotic pathways and decreased autophagy, except in MiaPaCa2 cells, in which autophagic proteins were expressed at lower levels and remained unmodified by FA4. FA4 treatment of PANC-1 xenografted mouse models, poorly responsive to conventional chemotherapy, significantly reduced tumor volumes and increased intratumor apoptosis compared to gemcitabine, with no signs of toxicity. Conclusions Our data indicate that FA4 exhibits encouraging activity in pancreatic cancer cells unresponsive to gemcitabine. These results warrant further investigation in patient-derived pancreatic cancers, and hold promise for the development of therapies that can more efficiently target the specific characteristics of individual tumor types.

International audience; Multiple Sclerosis (MS) is an autoimmune demyelinating and neurodegenerative disease of the central nervous system (CNS). Current management strategies suppress or modulate immune function, all with consequences and known side effects. They demonstrate a high level of success in limiting new relapses. However, the neurodegenerative process still affects both grey and white matter in the central nervous system. The sigma1 (S1R) ligand-regulated chaperone is implicated in many biological processes in various CNS-targeted diseases, acting on neural plasticity, myelination and neuroinflammation. Among the proteins involved in MS, S1R has therefore emerged as a promising new target. Standard and robust methods have been adopted to analyze the adsorption, distribution, metabolism, excretion (ADME) properties, safety pharmacology and toxicology of a previously synthetized simple benzamide-derived compound with nanomolar affinity for S1R, high selectivity, no cytotoxicity and good metabolic stability. The compound was also characterized as an agonist based on well-validated assays prior to in vivo investigations. Interestingly, we found that the oral administration of this compound resulted in an overall significant reduction in clinical progression in an MS experimental model. This effect is mediated through S1R action. Our results further suggest the potential use of this compound in the treatment of MS.

Obesity is increasing at epidemic rates across the US and worldwide, as are its co-morbidities, including type-2 diabetes and cardiovascular disease. Thus, targeted interventions to reduce the prevalence of obesity are of the utmost importance. The sigma-1 receptor (S1R) and sigma-2 receptor (S2R; encoded by Tmem97) belong to the same class of drug-binding sites, yet they are genetically distinct. There are multiple ongoing clinical trials focused on sigma receptors, targeting diseases ranging from Alzheimer’s disease through chronic pain to COVID-19. However, little is known regarding their gene-specific role in obesity. In this study, we measured body composition, used a comprehensive laboratory-animal monitoring system, and determined the glucose and insulin tolerance in mice fed a high-fat diet. Compared to Sigmar1+/+ mice of the same sex, the male and female Sigmar1−/− mice had lower fat mass (17% and 12% lower, respectively), and elevated lean mass (16% and 10% higher, respectively), but S1R ablation had no effect on their metabolism. The male Tmem97−/− mice exhibited 7% lower fat mass, 8% higher lean mass, increased volumes of O2 and CO2, a decreased respiratory exchange ratio indicating elevated fatty-acid oxidation, and improved insulin tolerance, compared to the male Tmem97+/+ mice. There were no changes in any of these parameters in the female Tmem97−/− mice. Together, these data indicate that the S1R ablation in male and female mice or the S2R ablation in male mice protects against diet-induced adiposity, and that S2R ablation, but not S1R deletion, improves insulin tolerance and enhances fatty-acid oxidation in male mice. Further mechanistic investigations may lead to translational strategies to target differential S1R/S2R regulations and sexual dimorphism for precision treatments of obesity.

Huntington’s disease (HD) is a genetic neurodegenerative disease caused by an expanded CAG repeat in the Huntingtin (HTT) gene that codes for an expanded polyglutamine (polyQ) repeat in exon-1 of the human mutant huntingtin (mHTT) protein. The presence of this polyQ repeat results in neuronal degeneration for which there is no cure nor treatment that modifies disease progression. In previous studies we have shown that small molecules that bind selectively to σ(2)R/TMEM97 can have significant neuroprotective effects in models of Alzheimer’s disease, traumatic brain injury and several other neurodegenerative diseases. In the present work we extend these investigations and show that certain σ(2)R/TMEM97-selective ligands decrease mHTT induced neuronal toxicity. We first synthesized a set of compounds designed to bind to σ(2)R/TMEM97 and determined their binding profiles (K(i) values) for σ(2)R/TMEM97 and other proteins in the central nervous system. Modulators with high affinity and selectivity for σ(2)R/TMEM97 were then tested in our HD cell model. Primary cortical neurons were cultured in vitro for seven days and then co-transfected with either a normal HTT construct (Htt N-586–22Q/GFP) or the mHTT construct Htt-N586–82Q/GFP. Transfected neurons were treated with either σ(2)R/TMEM97 or σ(1)R modulators for 48 h. After treatment, neurons were fixed and stained with Hoechst, and condensed nuclei were quantified to assess cell death in the transfected neurons. Significantly, σ(2)R/TMEM97 modulators reduce the neuronal toxicity induced by mHTT, and their neuroprotective effects are not blocked by NE-100, a selective σ(1)R antagonist known to block neuroprotection by σ(1)R ligands. These results indicate for the first time that σ(2)R/TMEM97 modulators can protect neurons from mHTT-induced neuronal toxicity, suggesting that targeting σ(2)R/TMEM97 may lead to a novel therapeutic approach to treat patients with HD.

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-naive 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.

Human salivary histatin 1 (Hst1) exhibits a series of cell-activating properties, such as promoting cell spreading, migration, and metabolic activity. We recently have shown that fluorescently labeled Hst1 (F-Hst1) targets and activates mitochondria, presenting an important molecular mechanism. However, its regulating signaling pathways remain to be elucidated. We investigated the influence of specific inhibitors of G protein-coupled receptors (GPCR), endocytosis pathways, extracellular signal-regulated kinases 1/2 (ERK1/2) signaling, p38 signaling, mitochondrial respiration and Na+/K+-ATPase activity on the uptake, mitochondria-targeting and -activating properties of F-Hst1. We performed a siRNA knockdown (KD) to assess the effect of Sigma-2 receptor (S2R) /Transmembrane Protein 97 (TMEM97)—a recently identified target protein of Hst1. We also adopted live cell imaging to monitor the whole intracellular trafficking process of F-Hst1. Our results showed that the inhibition of cellular respiration hindered the internalization of F-Hst1. The inhibitors of GPCR, ERK1/2, phagocytosis, and clathrin-mediated endocytosis (CME) as well as siRNA KD of S2R/TMEM97 significantly reduced the uptake, which was accompanied by the nullification of the promoting effect of F-Hst1 on cell metabolic activity. Only the inhibitor of CME and KD of S2R/TMEM97 significantly compromised the mitochondria-targeting of Hst1. We further showed the intracellular trafficking and targeting process of F-Hst1, in which early endosome plays an important role. Overall, phagocytosis, CME, GPCR, ERK signaling, and S2R/TMEM97 are involved in the internalization of Hst1, while only CME and S2R/TMEM97 are critical for its subcellular targeting. The inhibition of either internalization or mitochondria-targeting of Hst1 could significantly compromise its mitochondria-activating property.

Although opioids and nonsteroidal anti-inflammatory drugs (NSAIDs) are the most common drugs used in persistent pain treatment; they have shown many side effects. The development of new analgesics endowed with mu opioid receptor/delta opioid receptor (MOR/DOR) activity represents a promising alternative to MOR-selective compounds. Moreover, new mechanisms, such as sigma-1 receptor (σ1R) antagonism, could be an opioid adjuvant strategy. The in vitro σ1R and σ2R profiles of previous synthesized MOR/DOR agonists (−)-2R/S-LP2 (1), (−)-2R-LP2 (2), and (−)-2S-LP2 (3) were assayed. To investigate the pivotal role of N-normetazocine stereochemistry, we also synthesized the (+)-2R/S-LP2 (7), (+)-2R-LP2 (8), and (+)-2S-LP2 (9) compounds. (−)-2R/S-LP2 (1), (−)-2R-LP2 (2), and (−)-2S-LP2 (3) compounds have Ki values for σ1R ranging between 112.72 and 182.81 nM, showing a multitarget opioid/σ1R profile. Instead, (+)-2R/S-LP2 (7), (+)-2R-LP2 (8), and (+)-2S-LP2 (9) isomers displayed a nanomolar affinity for σ1R, with significative selectivity vs. σ2R and opioid receptors. All isomers were evaluated using an in vivo formalin test. (−)-2S-LP2, at 0.7 mg/kg i.p., showed a significative and naloxone-reversed analgesic effect. The σ1R selective compound (+)-2R/S-LP2 (7), at 5.0 mg/kg i.p., decreased the second phase of the formalin test, showing an antagonist σ1R profile. The multitarget or single target profile of assayed N-normetazocine derivatives could represent a promising pharmacological strategy to enhance opioid potency and/or increase the safety margin.

A series of σ2R compounds containing benzimidazolone and diazacycloalkane cores was synthesized and evaluated in radioligand binding assays. Replacing the piperazine moiety in a lead compound with diazaspiroalkanes and the fused octahydropyrrolo[3,4-b] pyrrole ring system resulted in a loss in affinity for the σ2R. On the other hand, the bridged 2,5-diazabicyclo[2.2.1]heptane, 1,4-diazepine, and a 3-aminoazetidine analog possessed nanomolar affinities for the σ2R. Computational chemistry studies were also conducted with the recently published crystal structure of the σ2R/TMEM97 and revealed that hydrogen bond interactions with ASP29 and π-stacking interactions with TYR150 were largely responsible for the high binding affinity of small molecules to this protein.

Retinal degenerative diseases are the major factors leading to severe visual impairment and even irreversible blindness worldwide. The therapeutic approach for retinal degenerative diseases is one extremely urgent and hot spot in science research. The sigma-1 receptor is a novel, multifunctional ligand-mediated molecular chaperone residing in endoplasmic reticulum (ER) membranes and the ER-associated mitochondrial membrane (ER-MAM); it is widely distributed in numerous organs and tissues of various species, providing protective effects on a variety of degenerative diseases. Over three decades, considerable research has manifested the neuroprotective function of sigma-1 receptor in the retina and has attempted to explore the molecular mechanism of action. In the present review, we will discuss neuroprotective effects of the sigma-1 receptor in retinal degenerative diseases, mainly in aspects of the following: the localization in different types of retinal neurons, the interactions of sigma-1 receptors with other molecules, the correlated signaling pathways, the influence of sigma-1 receptors to cellular functions, and the potential therapeutic effects on retinal degenerative diseases.

σ-1 receptors (σ1R) modulate nociceptive signaling, driving the search for selective antagonists to take advantage of this promising target to treat pain. In this study, a new series of benzylpiperazinyl derivatives has been designed, synthesized, and characterized for their affinities toward σ1R and selectivity over the σ-2 receptor (σ2R). Notably, 3-cyclohexyl-1-{4-[(4-methoxyphenyl)methyl]piperazin-1-yl}propan-1-one (15) showed the highest σ1R receptor affinity (Ki σ1 = 1.6 nM) among the series with a significant improvement of the σ1R selectivity (Ki σ2/Ki σ1 = 886) compared to the lead compound 8 (Ki σ2/Ki σ1 = 432). Compound 15 was further tested in a mouse formalin assay of inflammatory pain and chronic nerve constriction injury (CCI) of neuropathic pain, where it produced dose-dependent (3-60 mg/kg, i.p.) antinociception and anti-allodynic effects. Moreover, compound 15 demonstrated no significant effects in a rotarod assay, suggesting that this σ1R antagonist did not produce sedation or impair locomotor responses. Overall, these results encourage the further development of our benzylpiperazine-based σ1R antagonists as potential therapeutics for chronic pain.

Sigma-1 receptors (Sig-1Rs) are endoplasmic reticulum (ER) chaperones implicated in neuropathic pain. Here we examine if the Sig-1R may relate to neuropathic pain at the level of dorsal root ganglia (DRG). We focus on the neuronal excitability of DRG in a “spare nerve injury” (SNI) model of neuropathic pain in rats and find that Sig-1Rs likely contribute to the genesis of DRG neuronal excitability by decreasing the protein level of voltage-gated Cav2.2 as a translational inhibitor of mRNA. Specifically, during SNI, Sig-1Rs translocate from ER to the nuclear envelope via a trafficking protein Sec61β. At the nucleus, the Sig-1R interacts with cFos and binds to the promoter of 4E-BP1, leading to an upregulation of 4E-BP1 that binds and prevents eIF4E from initiating the mRNA translation for Cav2.2. Interestingly, in Sig-1R knockout HEK cells, Cav2.2 is upregulated. In accordance with those findings, we find that intra-DRG injection of Sig-1R agonist (+)pentazocine increases frequency of action potentials via regulation of voltage-gated Ca2+ channels. Conversely, intra-DRG injection of Sig-1R antagonist BD1047 attenuates neuropathic pain. Hence, we discover that the Sig-1R chaperone causes neuropathic pain indirectly as a translational inhibitor. Supplementary Information The online version contains supplementary material available at 10.1007/s12035-020-02276-8.

There is an urgent need to identify therapies that prevent SARS-CoV-2 infection and improve the outcome of COVID-19 patients. Although repurposed drugs with favorable safety profiles could have significant benefit, widely available prevention or treatment options for COVID-19 have yet to be identified. Efforts to identify approved drugs with invitro activity against SARS-CoV-2 resulted in identification of antiviral sigma-1 receptor ligands, including antihistamines in the histamine-1 receptor binding class. We identified antihistamine candidates for repurposing by mining electronic health records of usage in population of more than 219,000 subjects tested for SARS-CoV-2. Usage of diphenhydramine, hydroxyzine and azelastine was associated with reduced incidence of SARS-CoV-2 positivity in subjects greater than age 61. We found diphenhydramine, hydroxyzine and azelastine to exhibit direct antiviral activity against SARS-CoV-2 invitro. Although mechanisms by which specific antihistamines exert antiviral effects is not clear, hydroxyzine, and possibly azelastine, bind Angiotensin Converting Enzyme-2 (ACE2) and the sigma-1 receptor as off-targets. Clinical studies are needed to measure the effectiveness of diphenhydramine, hydroxyzine and azelastine for disease prevention, for early intervention, or as adjuvant therapy for severe COVID-19.

The sigma 1 receptor (S1R) is a molecular chaperone protein located in the endoplasmic reticulum and plasma membranes and has been shown to play important roles in various pathological disorders including pain and, as recently discovered, COVID-19. Employing structure- and QSAR-based drug design strategies, we rationally designed, synthesized, and biologically evaluated a series of novel triazole-based S1R antagonists. Compound 10 exhibited potent binding affinity for S1R, high selectivity over S2R and 87 other human targets, acceptable in vitro metabolic stability, slow clearance in liver microsomes, and excellent blood-brain barrier permeability in rats. Further in vivo studies in rats showed that 10 exhibited negligible acute toxicity in the rotarod test and statistically significant analgesic effects in the formalin test for acute inflammatory pain and paclitaxel-induced neuropathic pain models during cancer chemotherapy. These encouraging results promote further development of our triazole-based S1R antagonists as novel treatments for pain of different etiologies.

Genetic mutations related to ALS, a progressive neurological disease, have been discovered in the gene encoding σ-1 receptor (σ1R). We previously reported that σ1RE102Q elicits toxicity in cells. The σ1R forms oligomeric states that are regulated by ligands. Nevertheless, little is known about the effect of ALS-related mutations on oligomer formation. Here, we transfected NSC-34 cells, a motor neuronal cell line, and HEK293T cells with σ1R-mCherry (mCh), σ1RE102Q-mCh, or nontagged forms to investigate detergent solubility and subcellular distribution using immunocytochemistry and fluorescence recovery after photobleaching. The oligomeric state was determined using crosslinking procedure. σ1Rs were soluble to detergents, whereas the mutants accumulated in the insoluble fraction. Within the soluble fraction, peak distribution of mutants appeared in higher sucrose density fractions. Mutants formed intracellular aggregates that were co-stained with p62, ubiquitin, and phosphorylated pancreatic eukaryotic translation initiation factor-2-α kinase in NSC-34 cells but not in HEK293T cells. The aggregates had significantly lower recovery in fluorescence recovery after photobleaching. Acute treatment with σ1R agonist SA4503 failed to improve recovery, whereas prolonged treatment for 48 h significantly decreased σ1RE102Q-mCh insolubility and inhibited apoptosis. Whereas σ1R-mCh formed monomers and dimers, σ1RE102Q-mCh also formed trimers and tetramers. SA4503 reduced accumulation of the four types in the insoluble fraction and increased monomers in the soluble fraction. The σ1RE102Q insolubility was diminished by σ1R-mCh co-expression. These results suggest that the agonist and WT σ1R modify the detergent insolubility, toxicity, and oligomeric state of σ1RE102Q, which may lead to promising new treatments for σ1R-related ALS.

Background: No pharmacological treatment has been demonstrated to provide a functional benefit for persons with Huntington’s disease (HD). Pridopidine is a sigma-1-receptor agonist shown to have beneficial effects in preclinical models of HD. Objective: To further explore the effect of pridopidine on Total Functional Capacity (TFC) in the recent double-blind, placebo-controlled PRIDE-HD study. Methods: We performed post-hoc analyses to evaluate the effect of pridopidine on TFC at 26 and 52 weeks. Participants were stratified according to baseline TFC score and analyzed using repeated measures (MMRM) and multiple imputation assuming missing not-at-random (MNAR) and worst-case scenarios. Results: The pridopidine 45 mg bid dosage demonstrated a beneficial effect on TFC for the entire population at week 52 of 0.87 (nominal p = 0.0032). The effect was more pronounced for early HD participants (HD1/HD2, TFC = 7–13), with a change from placebo of 1.16 (nominal p = 0.0003). This effect remained nominally significant using multiple imputation with missing not at random assumption as a sensitivity analysis. Responder analyses showed pridopidine 45 mg bid reduced the probability of TFC decline in early HD patients at Week 52 (nominal p = 0.02). Conclusion: Pridopidine 45 mg bid results in a nominally significant reduction in TFC decline at 52 weeks compared to placebo, particularly in patients with early-stage HD.

Sigma-1 receptor (Sigmar1) is a specific chaperone located in the mitochondria-associated endoplasmic reticulum membrane (MAM) and plays a role in several physiological processes. However, the role of Sigmar1 in bone homeostasis remains unknown. Here, we show that mice lacking Sigmar1 exhibited severe osteoporosis in an ovariectomized model. In contrast, overexpression of Sigmar1 locally alleviated the osteoporosis phenotype. Treatment with Sigmar1 agonists impaired both human and mice osteoclast formation in vitro. Mechanistically, SERCA2 was identified to interact with Sigmar1 based on the immunoprecipitation-mass spectrum (IP-MS) and co-immunoprecipitation (co-IP) assays, and Q615 of SERCA2 was confirmed to be the critical residue for their binding. Furthermore, Sigmar1 promoted SERCA2 degradation through Hrd1/Sel1L-dependent ER-associated degradation (ERAD). Ubiquitination of SERCA2 at K460 and K541 was responsible for its proteasomal degradation. Consequently, inhibition of SERCA2 impeded Sigmar1 deficiency enhanced osteoclastogenesis. Moreover, we found that dimemorfan, an FDA-approved Sigmar1 agonist, effectively rescued bone mass in various established bone-loss models. In conclusion, Sigmar1 is a negative regulator of osteoclastogenesis, and activation of Sigmar1 by dimemorfan may be a potential treatment for osteoporosis in clinical practice.

Many studies involving compounds that enhance histamine release, such as histamine H

Background Angiogenesis plays a critical role on post-infarction heart failure (PIHF), the presence of which facilitates additional blood supply to maintain the survival of residual cardiomyocytes. The sigma-1 receptor (S1R) has been substantiated to stimulate angiogenesis, with the effect on a model of PIHF remaining unknown. Aims This study aims to investigate the effects of S1R on PIHF and the underlying mechanisms involved. Methods Rats were implemented left anterior descending artery ligation followed by rearing for 6 weeks to induce a phenotype of heart failure. Daily intraperitoneal injection of S1R agonist or antagonist for 5 weeks was applied from 2nd week after surgery. The effects exerted by S1R were detected by echocardiography, hemodynamic testing, western blot, Sirius red dyeing, ELISA, immunohistochemistry and fluorescence. We also cultured HUVECs to verify the mechanisms in vitro. Results Stimulation of S1R significantly ameliorated the cardiac function resulted from PIHF, in addition to the observation of reduced fibrosis in the peri-infarct region and the apoptosis of residual cardiomyocytes, which were associated with augmentation of microvascular density in peri-infarct region through activation of the JAK2/STAT3 pathway. We also indicated that suppression of JAK2/STAT3 pathway by specific inhibitor in vitro reversed the pro-angiogenic effects of S1R on HUVECs, which further confirmed that angiogenesis, responsible for PIHF amelioration, by S1R stimulation was in a JAK2/STAT3 pathway-dependent manner. Conclusion S1R stimulation improved PIHF-induced cardiac dysfunction and ventricular remodeling through promoting angiogenesis by activating the JAK2/STAT3 pathway.

Endothelial dysfunction is an important mechanism involved in myocardial ischemia-reperfusion (I/R) injury. We aimed to explore the effects of Oxycodone on myocardial I/R injury in vivo and in vitro to reveal its mechanisms related to Sigma-1 Receptor (SIGMAR1). A rat model of I/R-induced myocardial injury was developed. The ischemic area and myocardial histopathological changes after oxycodone addition were evaluated by TTC staining and HE staining. LDH, CK-MB and cTnI levels were used to assess myocardial function. Then, the endothelial integrity was reflected by the expressions of ZO-1, Claudin-1 and Occludin. Afterward, ELISA, RT-qPCR, western blot and immunofluorescence assays were adopted for the detection of inflammation-related genes. SIGMAR1 expression in myocardial tissues induced by I/R and cardiac microvascular endothelial cells (CMECs) under hypoxic/reoxygenation (H/R) was determined using RT-qPCR and western blotting. Subsequently, after SIGMAR1 silencing or BD1047 addition (a SIGMAR1 antagonist), cell apoptosis and endothelial integrity were analyzed in the presence of Oxycodone in H/R-stimulated CMECs. Results indicated that Oxycodone decreased the ischemic area and improved myocardial function in myocardial I/R injury rat. Oxycodone improved myocardial histopathological injury and elevated endothelial integrity, evidenced by upregulated ZO-1, Claudin-1 and Occludin expressions. Moreover, inflammatory response was alleviated after Oxycodone administration. Molecular docking suggested that SIGMAR1 could directly bind to Oxycodone. Oxycodone elevated SIGMAR1 expression and SIGMAR1 deletion or BD1047 addition attenuated the impacts of Oxycodone on apoptosis and endothelial integrity of CMECs induced by H/R. Collectively, Oxycodone alleviates myocardial I/R injury in vivo and in vitro by binding to SIGMAR1.

Sigma-1 receptor (S1R) is an important endoplasmic reticulum chaperone with various functions in health and disease. The purpose of the current work was to elucidate the involvement of S1R in cancer energy metabolism under its basal, activated, and inactivated states. For this, two cancer cell lines that differentially express S1R were treated with S1R agonist, (+)-SKF10047, and antagonist, BD1047. The effects of the agonist and antagonist on cancer energy metabolism were studied using single-cell fluorescence microscopy analysis of real-time ion and metabolite fluxes. Our experiments revealed that S1R activation by agonist increases mitochondrial bioenergetics of cancer cells while decreasing their reliance on aerobic glycolysis. S1R antagonist did not have a major impact on mitochondrial bioenergetics of tested cell lines but increased aerobic glycolysis of S1R expressing cancer cell line. Our findings suggest that S1R plays an important role in cancer energy metabolism and that S1R ligands can serve as tools to modulate it.

The sigma-1 receptor (σ1R) is a non-opioid transmembrane receptor which has been implicated in many diseases, including neurodegenerative disorders and cancer. After more than forty years of research, substantial progress has been made in understanding this unique receptor, yet the molecular mechanism of its ligand entry pathway remains uncertain. Published structures of human σ1R reveal its homotrimeric organization of a cupin-fold β-barrel body that contains the ligand binding site, a carboxy-terminal V-shaped two-helix bundle, and a single amino-terminal transmembrane helix, while simulation studies have suggested a ligand entry pathway that is generated by conformational rearrangements of the cupin-fold domain. Here, we present multiple crystal structures, including an open-like conformation, of σ1R from Xenopus laevis. Together with functional binding analysis our data suggest that access to the σ1R ligand binding site is likely achieved by protein conformational changes that involve the carboxy-terminal two-helix bundle, rather than structural changes in the cupin-fold domain.

Peripheral nerve injuries lead to the loss of motor, sensory and autonomic functions in the territories supplied by the injured nerve. Currently, nerve injuries are managed by surgical repair procedures, and there are no effective drugs in the clinic for improving the capacity of axonal regeneration. Sigma-1 receptor (Sig-1R) is an endoplasmic reticulum chaperon protein involved in many functions, including neuroprotection and neuroplasticity. A few previous studies using Sig-1R ligands reported results that suggest this receptor as a putative target to enhance regeneration. The aim of this study was to evaluate the possible effects of Sig-1R ligands on axonal regeneration in a sciatic nerve section and repair model in mice. To this end, mice were treated either with the Sig-1R agonist PRE-084 or the antagonist BD1063, and a Sig-1R knock-out (KO) mice group was also studied. The electrophysiological and histological data showed that treatment with Sig-1R ligands, or the lack of this protein, did not markedly modify the process of axonal regeneration and target reinnervation after sciatic nerve injury. Nevertheless, the nociceptive tests provided results indicating a role of Sig-1R in sensory perception after nerve injury, and immunohistochemical labeling indicated a regulatory role in inflammatory cell infiltration in the injured nerve.

Methamphetamine-associated cardiomyopathy is the leading cause of death linked with illicit drug use. Here we show that Sigmar1 is a therapeutic target for methamphetamine-associated cardiomyopathy and defined the molecular mechanisms using autopsy samples of human hearts, and a mouse model of “binge and crash” methamphetamine administration. Sigmar1 expression is significantly decreased in the hearts of human methamphetamine users and those of “binge and crash” methamphetamine-treated mice. The hearts of methamphetamine users also show signs of cardiomyopathy, including cellular injury, fibrosis, and enlargement of the heart. In addition, mice expose to “binge and crash” methamphetamine develop cardiac hypertrophy, fibrotic remodeling, and mitochondrial dysfunction leading to contractile dysfunction. Methamphetamine treatment inhibits Sigmar1, resulting in inactivation of the cAMP response element-binding protein (CREB), decreased expression of mitochondrial fission 1 protein (FIS1), and ultimately alteration of mitochondrial dynamics and function. Therefore, Sigmar1 is a viable therapeutic agent for protection against methamphetamine-associated cardiomyopathy., Chowdhury Abdulla and Richa Aishwarya et al. report that methamphetamine exposure in mice leads to cardiomyopathy via the signaling receptor Sigmar1. They find that methamphetamine inhibits Sigmar1, which alters mitochondrial function, and show that their findings are consistent with observations in hearts from human methamphetamine users.

In this paper, the benzo-cracking approach was applied to the potent sigma1 (σ1) receptor antagonist 1 to afford the less conformationally constrained 1,3-dioxane derivatives 2 and 3. To evaluate the effect of the increase in the distance between the two hydrophobic structural elements that flank the basic function, the cis and trans diastereomers of 4 and 5 were also prepared and studied. Compounds 2 and 3 showed affinity values at the σ1 receptor significantly higher than that of the lead compound 1. In particular, 3 displayed unprecedented selectivity over the σ2 receptor, the phencyclidine site of the NMDA receptor, and opioid receptor subtypes, as well as over the dopamine transporter. Docking results supported the structure–activity relationship studies. Due to its interesting biological profile, derivative 3, selected for an in vivo study in a validated preclinical model of binge eating, was able to counteract the overeating of palatable food only in binging rats, without affecting palatable food intake in the control group and anxiety-like and depression-related behaviors in female rats. This result strengthened the involvement of the σ1 receptor in the compulsive-like eating behavior and supported the σ1 receptor as a promising target for the management of eating disorders.

Microglia maintain brain homeostasis as the main immune cells in the central nervous system. Activation of sigma-1 receptor (Sig1R) plays neuroprotective and anti-inflammatory roles in microglia. Recent studies showed that Sig1R expression level has been reduced in the brain of the patients with neurodegenerative diseases including Alzheimer's disease. However, the mechanisms underlying the down regulation of the Sig1R has not been clear. Treatment of rat primary cultured microglia with the inflammogen lipopolysaccharide (LPS) significantly decreased the expression of Sig1R mRNA in a concentration and time-dependent manner. The effects of LPS were blocked by pretreatment with TAK-242, a toll-like receptor 4 (TLR4) antagonist. Furthermore, inhibitors of transforming growth factor beta-activated kinase 1 (TAK1), p38 mitogen-activated protein kinase (MAPK) and histone deacetylase 6 (HDAC6) restored the LPS-induced downregulation of Sig1R. Thus, the current findings demonstrate that TLR4 activation leads to the downregulation of the Sig1R expression via TLR4-TAK1-p38 MAPK pathway and the inhibition of HDAC6 can increase Sig1R expression in microglia. The current findings suggest that downregulation of Sig1R may contribute to neuroinflammation-induced microglial dysfunction, regulation of microglial Sig1R may be novel therapeutic drug candidates for neurodegenerative and neuroinflammatory diseases.

Sigma non-opioid intracellular receptor 1 (sigma-1 receptor), a non-opioid transmembrane protein, is located on cellular mitochondrial membranes and endoplasmic reticulum. Current research has demonstrated that sigma-1 receptor is related to human degenerative diseases. This study is focused on the effects of sigma-1 receptor on the pathophysiological process of diminished ovarian reserve (DOR) and granulosa cells (GCs) apoptosis. Sigma-1 receptor concentration in follicular fluid (FF) and serum were negatively correlated with basal follicle-stimulating hormone (FSH) and positively correlated with anti-mullerian hormone (AMH), antral follicle count (AFC). Sigma-1 receptor reduction in GCs was accompanied by endoplasmic reticulum stress (ERS)-mediated apoptosis in women with DOR. Plasmid transfection was used to establish SIGMAR1-overexpressed and SIGMAR1-knockdown human granulosa-like tumor (KGN) cell and thapsigargin (TG) was used to induce ERS KGN cells. We found that KGN cells treated with endogenous sigma-1 receptor ligand dehydroepiandrosterone (DHEA) and sigma-1 receptor agonist PRE-084 showed similar biological effects to SIGMAR1-overexpressed KGN cells and opposite effects to SIGMAR1-knockdown KGN cells. DHEA may improve DOR patients' pregnancy outcomes by upregulating sigma-1 receptor and downregulating ERS-mediated apoptotic genes in GCs. Thus, sigma-1 receptor may be a potential ovarian reserve biomarker, and ligand-mediated sigma-1 receptor activation could be a future approach for DOR therapy.

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.

The sigma 1 receptor (S1R) is widely expressed in the CNS and is mainly located on the endoplasmic reticulum. The S1R is involved in the regulation of many neurotransmission systems and, indirectly, in neurodegenerative diseases. The S1R may therefore represent an interesting neuronal biomarker in neurodegenerative diseases such as Parkinson's (PD) or Alzheimer's diseases (AD). Here we present the characterisation of the S1R-specific 18F-labelled tracer 18F-IAM6067 in two animal models and in human brain tissue. Methods: Wistar rats were used for PET-CT imaging (60 min dynamic acquisition) and metabolite analysis (1, 2, 5, 10, 20, 60 min post-injection). To verify in vivo selectivity, haloperidol, BD1047 (S1R ligand), CM398 (S2R ligand) and SB206553 (5HT2B/C antagonist) were administrated for pre-saturation studies. Excitotoxic lesions induced by intra-striatal injection of AMPA were also imaged by 18F-IAM6067 PET-CT to test the sensitivity of the methods in a well-established model of neuronal loss. Tracer brain uptake was also verified by autoradiography in rats and in a mouse model of PD (intrastriatal 6-hydroxydopamine (6-OHDA) unilateral lesion). Finally, human cortical binding was investigated by autoradiography in three groups of subjects (control subjects with Braak ≤2, and AD patients, Braak >2 & ≤4 and Braak >4 stages). Results: We demonstrate that despite rapid peripheral metabolism of 18F-IAM6067, radiolabelled metabolites were hardly detected in brain samples. Brain uptake of 18F-IAM6067 showed differences in S1R anatomical distribution, namely from high to low uptake: pons-raphe, thalamus medio-dorsal, substantia nigra, hypothalamus, cerebellum, cortical areas and striatum. Pre-saturation studies showed 79-90% blockade of the binding in all areas of the brain indicated above except with the 5HT2B/C antagonist SB206553 and S2R ligand CM398 which induced no significant blockade, indicating good specificity of 18F-IAM6067 for S1Rs. No difference between ipsi- and contralateral sides of the brain in the mouse model of PD was detected. AMPA lesion induced a significant 69% decrease in 18F-IAM6067 uptake in the globus pallidus matching the neuronal loss as measured by NeuN, but only a trend to decrease (-16%) in the caudate putamen despite a significant 91% decrease in neuronal count. Moreover, no difference in the human cortical binding was shown between AD groups and controls. Conclusion: This work shows that 18F-IAM6067 is a specific and selective S1R radiotracer. The absence or small changes in S1R detected here in animal models and human tissue warrants further investigations and suggests that S1R might not be the anticipated ideal biomarker for neuronal loss in neurodegenerative diseases such as AD and PD.

The Wolfram syndrome is a rare autosomal recessive disease affecting many organs with life-threatening consequences; currently, no treatment is available. The disease is caused by mutations in the WSF1 gene, coding for the protein wolframin, an endoplasmic reticulum (ER) transmembrane protein involved in contacts between ER and mitochondria termed as mitochondria-associated ER membranes (MAMs). Inherited mutations usually reduce the protein’s stability, altering its homeostasis and ultimately reducing ER to mitochondria calcium ion transfer, leading to mitochondrial dysfunction and cell death. In this study, we found that activation of the sigma-1 receptor (S1R), an ER-resident protein involved in calcium ion transfer, could counteract the functional alterations of MAMs due to wolframin deficiency. The S1R agonist PRE-084 restored calcium ion transfer and mitochondrial respiration in vitro, corrected the associated increased autophagy and mitophagy, and was able to alleviate the behavioral symptoms observed in zebrafish and mouse models of the disease. Our findings provide a potential therapeutic strategy for treating Wolfram syndrome by efficiently boosting MAM function using the ligand-operated S1R chaperone. Moreover, such strategy might also be relevant for other degenerative and mitochondrial diseases involving MAM dysfunction.

A series of novel σ1 receptor ligands with a 4-(2-aminoethyl)piperidine scaffold was prepared and biologically evaluated. The underlying concept of our project was the improvement of the lipophilic ligand efficiency of previously synthesized potent σ1 ligands. The key steps of the synthesis comprise the conjugate addition of phenylboronic acid at dihydropyridin-4(1H)-ones 7, homologation of the ketones 8 and introduction of diverse amino moieties and piperidine N-substituents. 1-Methylpiperidines showed particular high σ1 receptor affinity and selectivity over the σ2 subtype, whilst piperidines with a proton, a tosyl moiety or an ethyl moiety exhibited considerably lower σ1 affinity. Molecular dynamics simulations with per-residue binding free energy deconvolution demonstrated that different interactions of the basic piperidine-N-atom and its substituents (or the cyclohexane ring) with the lipophilic binding pocket consisting of Leu105, Thr181, Leu182, Ala185, Leu186, Thr202 and Tyr206 are responsible for the different σ1 receptor affinities. Recorded logD7.4 and calculated clogP values of 4a and 18a indicate low lipophilicity and thus high lipophilic ligand efficiency. Piperidine 4a inhibited the growth of human non-small cell lung cancer cells A427 to a similar extent as the σ1 antagonist haloperidol. 1-Methylpiperidines 20a, 21a and 22a showed stronger antiproliferative effects on androgen negative human prostate cancer cells DU145 than the σ1 ligands NE100 and S1RA.

Orofacial pain disorders are predominately experienced by women. Progesterone, a major ovarian hormone, is neuroprotective and antinociceptive. We recently reported that progesterone attenuates estrogen-exacerbated orofacial pain behaviors, yet it remains unclear what anatomical substrate underlies progesterone’s activity in the trigeminal system. Progesterone has been reported to exert protective effects through actions at intracellular progesterone receptors (iPR), membrane-progesterone receptors (mPR), or sigma 1 receptors (Sig-1R). Of these, the iPR and Sig-1R have been reported to have a role in pain. Progesterone can also have antinociceptive effects through its metabolite, allopregnanolone. Two enzymes, 5α-reductase and 3α-hydroxysteroid dehydrogenase (3α-HSD), are required for the metabolism of progesterone to allopregnanolone. Both progesterone and allopregnanolone rapidly attenuate pain sensitivity, implicating action of either progesterone at Sig-1R and/or conversion to allopregnanolone which targets GABAA receptors. In the present study, we investigated whether Sig-1 Rs are expressed in nociceptors within the trigeminal ganglia of cycling female rats and whether the two enzymes required for progesterone metabolism to allopregnanolone, 5α-reductase and 3α-hydroxysteroid dehydrogenase, are also present. Adult female rats from each stage of the estrous cycle were rapidly decapitated and the trigeminal ganglia collected. Trigeminal ganglia were processed by either fluorescent immunochemistry or western blotting to for visualization and quantification of Sig-1R, 5α-reductase, and 3α-hydroxysteroid dehydrogenase. Here we report that Sig-1Rs and both enzymes involved in progesterone metabolism are highly expressed in a variety of nociceptive sensory neuron populations in the female rat trigeminal ganglia at similar levels across the four stages of the estrous cycle. These data indicate that trigeminal sensory neurons are an anatomical substrate for the reported antinociceptive activity of progesterone via Sig-1R and/or conversion to allopregnanolone.

In our continuing effort to develop novel sigma receptor (SR) ligands, we present the design, synthesis and binding studies of a small library of aminopropylcarboxamide derivatives, obtained from a deconstruction of the piperidine ring of previously synthesized piperidine-based compounds. The best results were achieved with benzofuran (5c, 5g) and quinoline (5a, 5e) derivatives. These compounds revealed the highest affinity for both receptor subtypes. In particular, the 3,4-dimethoxyphenyl derivatives 5e and 5g showed the highest selectivity profile for S2R, especially the quinoline derivative 5e exhibited a 35-fold higher affinity for S2R subtype. The cytotoxic activity of aforementioned compounds was evaluated against SKBR3 and MCF7 cell lines, widely used for breast cancer studies. Whereas the potency of 5g was similar that of Siramesine and Haloperidol in both cell lines, compounds 5a, 5c and 5e exhibited a potency at least comparable to that of Haloperidol in SKBR3 cells. A molecular modelling evaluation towards the S2R binding site, confirmed the strong interaction of compound 5e thus justifying its highest S2R affinity.

We are pleased to acknowledge the generous support of the National Science Center, Poland, granted based on decision No. 2020/36/C/NZ7/00284. Support by ERNEST COST Action 18133 is also acknowledged. K. Szczepan ' ska is supported by the Foundation of Polish Science within the START scholarship. Financial support by the graduate school "Receptor Dynamics" of the Elite Network of Bavaria (ENB) is gratefully acknowledged (N. Rosier, S. Pockes). M. C. Ruiz Cantero was supported by the Training University Lecturers program (FPU) of the Spanish Ministry of Economy and Competitiveness (MINECO). We also acknowledge funding from the Spanish State Research Agency (10.13039/501100011033) under the auspices of MINECO (grant number PID2019-108691RB-I00 to E. J. Cobos) as well as from the University of Catania, PIA.CE.RI. 2020-2022 Linea di intervento 3 Starting Grant project CARETO (grant 57722172136 to E. Amata)., In an attempt to extend recent studies showing that some clinically evaluated histamine H3 receptor (H3R) antagonists possess nanomolar affinity at sigma-1 receptors (σ1R), we selected 20 representative structures among our previously reported H3R ligands to investigate their affinity at σRs. Most of the tested compounds interact with both sigma receptors to different degrees. However, only six of them showed higher affinity toward σ1R than σ2R with the highest binding preference to σ1R for compounds 5, 11, and 12. Moreover, all these ligands share a common structural feature: the piperidine moiety as the fundamental part of the molecule. It is most likely a critical structural element for dual H3/σ1 receptor activity as can be seen by comparing the data for compounds 4 and 5 (hH3R Ki = 3.17 and 7.70 nM, σ1R Ki = 1531 and 3.64 nM, respectively), where piperidine is replaced by piperazine. We identified the putative protein−ligand interactions responsible for their high affinity using molecular modeling techniques and selected compounds 5 and 11 as lead structures for further evaluation. Interestingly, both ligands turned out to be high-affinity histamine H3 and σ1 receptor antagonists with negligible affinity at the other histamine receptor subtypes and promising antinociceptive activity in vivo. Considering that many literature data clearly indicate high preclinical efficacy of individual selective σ1 or H3R ligands in various pain models, our research might be a breakthrough in the search for novel, dual-acting compounds that can improve existing pain therapies. Determining whether such ligands are more effective than single-selective drugs will be the subject of our future studies., National Science Centre, Poland 2020/36/C/NZ7/00284, ERNEST COST Action 18133, Foundation of Polish Science within the START scholarship graduate school "Receptor Dynamics" of the Elite Network of Bavaria (ENB), Training University Lecturers program (FPU) of the Spanish Ministry of Economy and Competitiveness (MINECO), Spanish State Research Agency under the auspices of MINECO PID2019-108691RB-I00, University of Catania, PIA.CE.RI, project CARETO 57722172136

Motor neuron diseases (MNDs) include sporadic and hereditary neurological disorders characterized by progressive degeneration of motor neurons (MNs). Sigma-1 receptor (Sig-1R) is a protein enriched in MNs, and mutations on its gene lead to various types of MND. Previous studies have suggested that Sig-1R is a target to prevent MN degeneration. In this study, two novel synthesized Sig-1R ligands, coded EST79232 and EST79376, from the same chemical series, with the same scaffold and similar physicochemical properties but opposite functionality on Sig-1R, were evaluated as neuroprotective compounds to prevent MN degeneration. We used an in vitro model of spinal cord organotypic cultures under chronic excitotoxicity and two in vivo models, the spinal nerve injury and the superoxide dismutase 1 (SOD1)G93A mice, to characterize the effects of these Sig-1R ligands on MN survival and modulation of glial reactivity. The antagonist EST79376 preserved MNs in vitro and after spinal nerve injury but was not able to improve MN death in SOD1G93A mice. In contrast, the agonist EST79232 significantly increased MN survival in the three models of MN degeneration evaluated and had a mild beneficial effect on motor function in SOD1G93A mice. In vivo, Sig-1R ligand EST79232 had a more potent effect on preventing MN degeneration than EST79376. These data further support the interest in Sig-1R as a therapeutic target for neurodegeneration.

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-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 This research was funded by ESTEVE Pharmaceuticals (Grant number 066/13 23/01/2017) and by the Vice-Chancellorship of Research of the University of Girona (Grant number MPCUdG2016/087)

Alzheimer's disease (AD) is the most common form of dementia characterized by cognitive dysfunctions. Pharmacological interventions to slow the progression of AD are intensively studied. A potential direction targets neuronal sigma-1 receptors (S1Rs). S1R ligands are recognized as promising therapeutic agents that may alleviate symptom severity of AD, possibly via preventing amyloid-β-(Aβ-) induced neurotoxicity on the endoplasmic reticulum stress-associated pathways. Furthermore, S1Rs may also modulate adult neurogenesis, and the impairment of this process is reported to be associated with AD. We aimed to investigate the effects of two S1R agonists, dimethyltryptamine (DMT) and PRE084, in an Aβ-induced in vivo mouse model characterizing neurogenic and anti-neuroinflammatory symptoms of AD, and the modulatory effects of S1R agonists were analyzed by immunohistochemical methods and western blotting. DMT, binding moderately to S1R but with high affinity to 5-HT receptors, negatively influenced neurogenesis, possibly as a result of activating both receptors differently. In contrast, the highly selective S1R agonist PRE084 stimulated hippocampal cell proliferation and differentiation. Regarding neuroinflammation, DMT and PRE084 significantly reduced Aβ

In an attempt to extend recent studies showing that some clinically evaluated histamine H3 receptor (H3R) antagonists possess nanomolar affinity at sigma-1 receptors (σ1R), we selected 20 representative structures among our previously reported H3R ligands to investigate their affinity at σRs. Most of the tested compounds interact with both sigma receptors to different degrees. However, only six of them showed higher affinity toward σ1R than σ2R with the highest binding preference to σ1R for compounds 5, 11, and 12. Moreover, all these ligands share a common structural feature: the piperidine moiety as the fundamental part of the molecule. It is most likely a critical structural element for dual H3/σ1 receptor activity as can be seen by comparing the data for compounds 4 and 5 (hH3R Ki = 3.17 and 7.70 nM, σ1R Ki = 1531 and 3.64 nM, respectively), where piperidine is replaced by piperazine. We identified the putative protein–ligand interactions responsible for their high affinity using molecular modeling techniques and selected compounds 5 and 11 as lead structures for further evaluation. Interestingly, both ligands turned out to be high-affinity histamine H3 and σ1 receptor antagonists with negligible affinity at the other histamine receptor subtypes and promising antinociceptive activity in vivo. Considering that many literature data clearly indicate high preclinical efficacy of individual selective σ1 or H3R ligands in various pain models, our research might be a breakthrough in the search for novel, dual-acting compounds that can improve existing pain therapies. Determining whether such ligands are more effective than single-selective drugs will be the subject of our future studies.

Sigma-1 receptor (Sig1R) is an endoplasmic reticulum (ER)-related membrane protein, that forms heteromers with other cellular proteins. As the mechanism of action of this chaperone protein remains unclear, the aim of the present study was to detect and analyze the intracellular dynamics of Sig1R in live cells using super-resolution imaging microscopy. For that, the Sig1R-yellow fluorescent protein conjugate (Sig1R-YFP) together with fluorescent markers of cell organelles were transfected into human ovarian adenocarcinoma (SK-OV-3) cells with BacMam technology. Sig1R-YFP was found to be located mainly in the nuclear envelope and in both tubular and vesicular structures of the ER but was not detected in the plasma membrane, even after activation of Sig1R with agonists. The super-resolution radial fluctuations approach (SRRF) performed with a highly inclined and laminated optical sheet (HILO) fluorescence microscope indicated substantial overlap of Sig1R-YFP spots with KDEL-mRFP, slight overlap with pmKate2-mito and no overlap with the markers of endosomes, peroxisomes, lysosomes, or caveolae. Activation of Sig1R with (+)-pentazocine caused a time-dependent decrease in the overlap between Sig1R-YFP and KDEL-mRFP, indicating that the activation of Sig1R decreases its colocalization with the marker of vesicular ER and does not cause comprehensive translocations of Sig1R in cells.

The sigma-1 receptor (Sig-1R) plays an important role in spinal pain transmission by increasing phosphorylation of the N-methyl-D-aspartate (NMDA) receptor GluN1 subunit (pGluN1). As a result Sig-1R has been suggested as a novel therapeutic target for prevention of chronic pain. Here we investigated whether interleukin-1β (IL-1β) modulates the expression of the Sig-1R in spinal astrocytes during the early phase of nerve injury, and whether this modulation affects spinal pGluN1 expression and the development of neuropathic pain following chronic constriction injury (CCI) of the sciatic nerve. Repeated intrathecal (i.t.) administration of IL-1β from days 0-3 post-surgery significantly reduced the increased pGluN1 expression at the Ser896 and Ser897 sites in the ipsilateral spinal cord, as well as, the development of mechanical allodynia and thermal hyperalgesia in the ipsilateral hind paw of CCI mice, which were restored by co-administration of IL-1 receptor antagonist with IL-1β. Sciatic nerve injury increased the expression of Sig-1R in astrocytes of the ipsilateral spinal cord, and this increase was suppressed by i.t. administration of IL-1β. Agonistic stimulation of the Sig-1R with PRE084 restored pGluN1 expression and the development of mechanical allodynia that were originally suppressed by IL-1β in CCI mice. Collectively these results demonstrate that IL-1β administration during the induction phase of neuropathic pain produces an analgesic effect on neuropathic pain development by controlling the expression of Sig-1R in spinal astrocytes.

The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and invitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.