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The sigma-1 (σ 1 ) receptor plays a significant role in many normal physiological functions and pathological disease states, and as such represents an attractive therapeutic target for both agonists and antagonists. Here, we describe a novel series of phenoxyalkylpiperidines based on the lead compound 1-[ω-(4-chlorophenoxy)ethyl]-4-methylpiperidine (1a) in which the degree of methylation at the carbon atoms alpha to the piperidine nitrogen was systematically varied. The affinity at σ 1 and σ 2 receptors and at Δ 8 -Δ 7 sterol isomerase (SI) ranged from subnanomolar to micromolar K i values. While the highest-affinity was displayed at the σ 1 , the increase of the degree of methylation in the piperidine ring progressively decreased the affinity. The subnanomolar affinity 1a and 1-[ω-(4-methoxyphenoxy)ethyl]-4-methylpiperidine (1b) displayed potent anti-amnesic effects associated with σ 1 receptor agonism, in two memory tests. Automated receptor-small-molecule ligand docking provided a molecular structure-based rationale for the agonistic effects of 1a and 1b. Overall, the class of the phenoxyalkylpiperidines holds potential for the development of high affinity σ 1 receptor agonists, and compound 1a, that appears as the best in class (exceeding by far the activity of the reference compound PRE-084) deserves further investigation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. 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., (© 2021. The Author(s).)

In the human brain, aging is characterized by progressive neuronal loss, leading to disruption of synapses and to a degree of failure in neurotransmission. However, there is increasing evidence to support the notion that the aged brain has a remarkable ability to reorganize itself, with the aim of preserving its physiological activity. It is important to develop objective markers able to characterize the biological processes underlying brain aging in the intact human, and to distinguish them from brain degeneration associated with many neurological diseases. Transcranial magnetic stimulation (TMS), coupled with electromyography or electroencephalography (EEG), is particularly suited to this aim, due to the functional nature of the information provided, and thanks to the ease with which it can be integrated with behavioral manipulation. In this review, we aimed to provide up to date information about the role of TMS and TMS-EEG in the investigation of brain aging. In particular, we focused on data about cortical excitability, connectivity and plasticity, obtained by using readouts such as motor evoked potentials and transcranial evoked potentials. Overall, findings in the literature support an important potential contribution of TMS to the understanding of the mechanisms underlying normal brain aging. Further studies are needed to expand the current body of information and to assess the applicability of TMS findings in the clinical setting.

These unprecedented times have forced the scientific community to gather to face the COVID-19 pandemic. Efforts in diverse directions have been made. A multi-university team has focused on the identification of the host (human) proteins interacting with SARS-CoV-2 viral proteins, with the aim of hampering these interactions that may cause severe COVID-19 symptoms. Sigma-1 and sigma-2 receptors surprisingly belong to the "druggable" host proteins found, with the pan-sigma receptor modulator PB28 displaying the most potent anti-SARS-CoV-2 activity in in vitro assays. Being 20-fold more active than hydroxychloroquine, without cardiac side effects, PB28 is a promising antiviral candidate worthy of further investigation. Our research group developed PB28 in 1996 and have thoroughly characterized its biological properties since then. Structure-affinity relationship (SAfiR) studies at the sigma receptor subtypes were also undertaken with PB28 as the lead compound. We herein report our knowledge of PB28 to share information that may help to gain insight into the antiviral action of this compound and sigma receptors, while providing structural hints that may speed up the translation into therapeutics of this class of ligands., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Abate, Niso, Abatematteo, Contino, Colabufo and Berardi.)

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 ORF9b, an interaction we structurally characterized using cryo-electron microscopy. Combining genetically validated host factors with both COVID-19 patient genetic data and medical billing records identified molecular mechanisms and potential drug treatments that merit further molecular and clinical study., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

PB28, a cyclohexylpiperazine derivative, could be a potential strategy for Covid 19 because in a recent study it has been found more active than hydroxychloroquine without interaction with cardiac proteins. PB28 has been designed, developed, and biologically evaluated in the past decade in our research group. A possible mechanism to explain its surprising anti-COVID-19 activity is suggested.., Competing Interests: The authors declare no competing financial interest.

Introduction: Maternal satisfaction regarding care during delivery is an indicator of maternity service's quality., Methods: We conducted an observational study between May and August 2018, using an online questionnaire for women who have delivered in the last 3 years. Data was processed using descriptive and bivariate analysis, considering satisfaction as outcome., Results: Sample includes 1229 women. About 73% had a spontaneous vaginal delivery, of which 29.3% received a Kristeller manoeuvre and 34,4% episiotomy. Three women out of 10 complaint about lack of involving in the decision-making process, 13.9% of women believe that their delivery's experience can negatively influence their decision to have another child, and 19.8% would like not to give birth again in the same hospital. Four women out of 10 are only partially satisfied with the care received and 6.4% is not satisfied at all. Satisfaction is significantly associated to the following variables: age more than 25 years old, positive environment during labour, gentle attitude of healthcare provider, respect of needs, respect of dignity, woman's inclusion in the decision making process, presence of a trustworthy person during the labour/delivery, vaginal delivery, positive delivery's experience, episiotomy, Kristeller manoeuvre, skin-to-skin contact, support during breastfeeding and postpartum period, choice to come back in the hospital for another delivery., Conclusion: We should implement strategies to promote the mother-partner-child triad as a central focus during delivery and allocate human resources in more efficient ways.

(1) Background: The structure of the Sigma 2 receptor/TMEM97 (σ2RTMEM97) has recently been reported. (2, 3) Methods and results: We used genetic and biochemical approaches to identify the molecular mechanism downstream of σ2R/TMEM97. The novel σ2R/TMEM97 fluorescent ligand, NO1, reduced the proliferation and survival of the triple negative breast cancer cell lines (TNBC: MDA-MB-231 and MDA-MB-468 cell lines), due to NO1-induced apoptosis. Greater bioaccumulation and faster uptake of NO1 in MDA-MB-231 cells compared to MCF10A or MCF7 cell lines were also shown. Accordingly, elevated σ2R/TMEM97 expression was confirmed by Western blotting. In contrast to NO1, other σ2R/TMEM97 ligands, such as SM21 and PB28, enhanced MDA-MB-231 cell proliferation and migration. Store-operated calcium entry (SOCE) is crucial for different cancer hallmarks. Here, we show that NO1, but not other σ2R/TMEM97 ligands, reduced SOCE in MDA-MB-231 cells. Similarly, TMEM97 silencing in MDA-MB-231 cells also impaired SOCE. NO1 administration downregulated STIM1-Orai1 interaction, probably by impairing the positive regulatory effect of σ2R/TMEM97 on STIM1, as we were unable to detect interaction with Orai1. (4) Conclusion: σ2R/TMEM97 is a key protein for the survival of triple negative breast cancer cells by promoting SOCE; therefore, NO1 may become a good pharmacological tool to avoid their proliferation., Competing Interests: Authors declare not conflict of interest.

Aim: The σ 1 receptor is a druggable target involved in many physiological processes and diseases. To clarify its physiology and derive therapeutic benefit, nine analogs based on the σ 1 antagonist PB212 were synthesized replacing the 4-methylpiperidine with basic moieties of varying size and degree of conformational freedom. Results & methodology: 3-Phenylpyrrolidine, 4-phenylpiperidine or granatane derivatives displayed the highest affinity ( K i .#x00A0;= 0.12, 0.31 or 1.03 nM). Calcium flux assays in MCF7σ 1 cells indicated that the highest σ 1 receptor affinity are σ 1 antagonists. Molecular models provided a structural basis for understanding the σ 1 affinity and functional activity of the analogs and incorporated Glennon's σ 1 pharmacophore model. Conclusion: Herein, we identify new compounds exploitable as therapeutic drug leads or as tools to study σ 1 receptor physiology.

P-glycoprotein (Pgp) is highly expressed on blood-brain barrier (BBB) and glioblastoma (GB) cells, particularly on cancer stem cells (SC). Pgp recognizes a broad spectrum of substrates, limiting the therapeutic efficacy of several chemotherapeutic drugs in eradicating GB SC. Finding effective and safe inhibitors of Pgp that improve drug delivery across the BBB and target GB SC is open to investigation. We previously identified a series of thiosemicarbazone compounds that inhibit Pgp with an EC 50 in the nanomolar range, and herein, we investigate the efficacy of three of them in bypassing Pgp-mediated drug efflux in primary human BBB and GB cells. At 10 nM, the compounds were not cytotoxic for the brain microvascular endothelial hCMEC/D3 cell line, but they markedly enhanced the permeability of the Pgp-substrate doxorubicin through the BBB. Thiosemicarbazone derivatives increased doxorubicin uptake in GB, with greater effects in the Pgp-rich SC clones than in the differentiated clones derived from the same tumor. All compounds increased intratumor doxorubicin accumulation and consequent toxicity in GB growing under competent BBB, producing significant killing of GB SC. The compounds crossed the BBB monolayer. The most stable derivative, 10a , had a half-life in serum of 4.2 h. The coadministration of doxorubicin plus 10a significantly reduced the growth of orthotopic GB-SC xenografts, without eliciting toxic side effects. Our work suggests that the thiosemicarbazone compounds are able to transform doxorubicin, a prototype BBB-impermeable drug, into a BBB-permeable drug. Bypassing Pgp-mediated drug efflux in both BBB and GB SC, thiosemicarbazones might increase the success of chemotherapy in targeting GB SC, which represent the most aggressive and difficult components to eradicate.

Identification of sigma-2 receptor (sig-2R) has been controversial. Nevertheless, interest in sig-2R is high for its overexpression in tumors and potentials in oncology. Additionally, sig-2R antagonists inhibit Aβ binding at neurons, blocking the cognitive impairments of Alzheimer's disease. The most representative classes of sig-2R ligands are herein treated with focus on compounds that served to study sig-2R biology and to produce sig-2R: fluorescent ligands; multifunctional anticancer agents; and targeting nanoparticles. Although fluorescent ligands serve as 'green' pharmacological tools, sig-2R-multifunctional conjugates and sig-2R-targeted nanoparticles show how sig-2R targeting increases the activity of anticancer drugs in tumors with reduced toxicity. Altogether, this review draws a picture of the multiple approaches of sig-2R ligands in cancer therapy and as Alzheimer's disease modifying disease agents.

If one of the major aspirations of postcolonial theory is to re-establish a balance in the relationship between the (former) colonizer and the colonized by engaging the voices of the "subaltern", and on the other hand to illuminate how power relations of the present are embedded in history (Mills 2007), we argue that important theoretical insights might inform research by anchoring post-colonial theory within a sociopsychological framework. While there is a growing corpus of sociopsychological research articles focusing on how major geopolitical events and historical processes bear on people's lives, we aim to investigate the theoretical potential of postcolonial theory within the disciplines aiming at a sociopsychological approach. By focusing on the social dynamics of power imbalances, post-colonial theory finds its operational meaning: the feelings stemming from actions committed in the past are indeed crucial in determining reparatory attitudes and policies towards members of former colonized groups. Firstly, drawing from the sociopsychological scientific production related to consequences of colonial past, seen in recent years as a growing research interest in the field, we will explore patterns and trends through a thematic analysis of literature. Social Psychology as well as adjacent disciplines can greatly benefit from this theoretical fertilization, especially in the way post-colonial ideologies relate to the symbolic promotion versus exclusion of indigenous culture (Sengupta et al., International Journal of Intercultural Relations, 36(4), 506-517, 2012). Furthermore, by comparing and contrasting the ideological cosmologies relating to this particular topic, this study aims to establish the state of knowledge in the field, to identify how research methods and thematic fields are paired, to find "gaps" and create spaces for research that become integrative of postcolonial theory. While focusing on academic production, we also hope to contribute to develop the idea of cosmopolitism within academia but also beyond academic doors.

The Sigma-1 receptor (Sig-1R) has been described as a pluripotent modulator of distinct physiological functions and its involvement in various central and peripheral pathological disorders has been demonstrated. However, further investigations are required to understand the complex role of the Sig-1R as a molecular chaperon. A specific PET radioligand would provide a powerful tool in Sig-1R related studies. As part of our efforts to develop a Sig-1R PET radioligand that shows antagonistic properties, we investigated the suitability of 1-(4-(6-methoxynaphthalen-1-yl)butyl)-4-methylpiperidine (designated PB212) for imaging Sig-1R. PB212 is a Sig-1R antagonist and exhibits subnanomolar affinity ( K i = 0.030 nM) towards Sig-1R as well as good to excellent selectivity over Sig-2R. The radiolabelling of [ 11 C]PB212 was accomplished by O-methylation of the phenolic precursor using [ 11 C]MeI. In vitro autoradiography with [ 11 C]PB212 on WT and Sig-1R KO mouse brain tissues revealed high non-specific binding, however using rat spleen tissues from CD1 mice and Wistar rats, high specific binding was observed. The spleen is known to have a high expression of Sig-1R. In vivo PET experiments in Wistar rats also showed high accumulation of [ 11 C]PB212 in the spleen. Injection of Sig-1R binding compounds, haloperidol (1 mg/kg) or fluspidine (1 mg/kg) shortly before [ 11 C]PB212 administration induced a drastic reduction of radiotracer accumulation, confirming the specificity of [ 11 C]PB212 towards Sig-1R in the spleen. The results obtained herein indicate that although [ 11 C]PB212 is not suitable for imaging Sig-1R in the brain, it is a promising candidate for the detection and quantification of Sig-1Rs in the periphery., Competing Interests: None.

In this study, a new regenerative strategy to treat several neurodegenerative diseases is suggested by the use of a multitarget approach induced by our small molecule, MC111. Considering the importance of P-gp and BCRP expression on stem cell differentiation and the involvement of TLR4 on neurodegeneration processes, we investigated the effect of MC111, belonging to our library of P-gp active compounds on: (i) TLR4 signaling; (ii) P-gp and BCRP activity and expression; (iii) neurite sprouting. The observed findings exerted by MC111, open a new scenario for a multitarget and regenerative approach in neurodegenerative diseases encouraging the in vivo evaluation of MC111 as new tool in neuroreparative medicine., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The aggressiveness of pancreatic cancer urgently requires more efficient treatment options. Because the sigma-2 (σ 2 ) receptor was recently proposed as a promising target for pancreatic cancer therapy, we explored our previously developed multifunctional thiosemicarbazones, designed to synergistically impair cell energy levels, by targeting σ 2 and P-gp proteins and chelating Iron. A deconstruction approach was herein applied by removing one function at a time from the potent multifunctional thiosemicarbazones 1 and 2, to investigate the contribution to cytotoxicity of each target involved. The results from in vitro (panel of pancreatic tumor cells) and in vivo experiments (C57BL/6 bearing KP02 tumor), suggest that while the multifunctional activity was not required for the antitumor activity of these thiosemicarbazones, σ 2 -targeting appeared to allow alternative tumor cell death mechanisms, leading to potent and less toxic off-targets toxicities compared to other thiosemicarbazones devoid of σ 2 -targeting., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

The expression of sigma-2 receptor (S2R) was assayed in blood and bladder samples from healthy cattle and in blood and bladder of cattle with deltapapillomavirus-associated urothelial tumors. Samples of bladder from cattle with neoplasia had significantly higher S2R than samples of bladder from healthy cattle (95% CI 0.31-0.82, P < 0.05). In addition, significantly higher S2R was detected in the blood of cattle with bladder cancer than blood from healthy cattle (95% CI 0.22-0.41, P < 0.05). The results provide evidence that increased expression of SR2 in blood could be useful as circulating biomarker for bladder cancer in cattle. PGRMC1 protein levels were also found to be increased in blood and bladder from cattle with cancer and increased expression of PGRMC1 transcripts was detected by quantitative real time PCR in samples from cattle neoplasia. Furthermore, electron microscopy revealed phagophores and numerous autophagosomes, ultrastructural hallmark of autophagy., (© 2017 John Wiley & Sons Ltd.)

Background: The intracellular [Ca 2+ ] is modulated by σ receptors. An important component of the cellular machinery governing the intracellular [Ca 2+ ] is Store-Operated Calcium Entry (SOCE). Here we want to investigate whether ligands of σ receptors affect SOCE., Methods: The intracellular [Ca 2+ ] was monitored, with the fluorescent Ca 2+ -sensitive probe Fura-2, in four cell lines with a different expression of σ receptors, namely MCF7 (expressing σ 1 receptors with a low density and overexpressing σ 2 receptors), MCF7σ 1 (overexpressing σ 1 receptors), SK-N-SH, and HT-29., Results: When thapsigargin was used to deplete intracellular Ca 2+ stores, in a Ca 2+ -free incubation medium, the Ca 2+ influx (following Ca 2+ re-addition) was significantly increased by 1μM (+)-pentazocine (σ 1 receptor agonist) in MCF7σ 1 (by 22.5%) and SK-N-SH (by 45.6%), but not in HT-29 and MCF7 cells. We have used, as a second approach, the "Mn 2+ quenching" protocol. In MCF7σ 1 cells, after thapsigargin treatment, the fluorescence quenching induced by Mn 2+ influx (evidence of Ca 2+ influx) was significantly increased (by 25.8%) by 1μM (+)-pentazocine, significantly decreased (by 18.0%) by BD1063 (σ 1 receptor antagonist), and not affected by the presence of both ligands. These effects were not observed in MCF7 cells. Finally, in MCF7 cells, 1μM PB28 (σ 2 receptor agonist), did not affect both the Ca 2+ response after Ca 2+ re-addition and the fluorescence quenching induced by Mn 2+ influx., Conclusions: We propose that the σ 1 receptor agonist (+)-pentazocine increases SOCE in MCF7σ 1 and SK-N-SH cell lines. The σ 2 receptor agonist PB28 does not affect SOCE in MCF7 cells., (Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

A controversial relationship between sigma-2 and progesterone receptor membrane component 1 (PGRMC1) proteins, both representing promising targets for the therapy and diagnosis of tumors, exists since 2011, when the sigma-2 receptor was reported to be identical to PGRMC1. Because a misidentification of these proteins will lead to biased future research hampering the possible diagnostic and therapeutic exploitation of the two targets, there is the need to solve the debate on their identity. With this aim, we have herein investigated uptake and distribution of structurally different fluorescent sigma-2 receptor ligands by flow cytometry and confocal microscopy in MCF7 cells, where together with intrinsic sigma-2 receptors, PGRMC1 was constitutively present or alternatively silenced or overexpressed. HCT116 cells, with constitutive or silenced PGRMC1, were also studied. These experiments showed that the fluorescent sigma-2 ligands bind to their receptor irrespective of PGRMC1 expression. Furthermore, isothermal titration calorimetry was conducted to examine if DTG and PB28, two structurally distinct nanomolar affinity sigma-2 ligands, bind to purified PGRMC1 proteins that have recently been revealed to form both apo-monomeric and heme-mediated dimeric forms. While no binding to apo-PGRMC1 monomer was detected, a micromolar affinity to heme-mediated dimerized PGRMC1 was demonstrated in DTG but not in PB28. The current data provide evidence that sigma-2 receptor and PGRMC1 are not identical, paving the pathway for future unbiased research in which these two attractive targets are treated as different proteins while the identification of the true sigma-2 protein further needs to be pursued., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Despite considerable efforts by scientific research, pancreatic cancer is the fourth leading cause of cancer related mortalities. Sigma-2 receptors, which are overexpressed in several tumors, represent promising targets for triggering selective pancreatic cancer cells death., Methods: We selected five differently structured high-affinity sigma-2 ligands (PB28, PB183, PB221, F281 and PB282) to study how they affect the viability of diverse pancreatic cancer cells (human cell lines BxPC3, AsPC1, Mia PaCa-2, and Panc1 and mouse Panc-02, KCKO and KP-02) and how this is reflected in vivo in a tumor model., Results: Important cytotoxicity was shown by the compounds in the aggressive Panc02 cells, where cytotoxic activity was caspase-3 independent for four of the five compounds. However, both cytotoxicity and caspase-3 activation involved generation of Reactive Oxygen Species (ROS), which could be partially reverted by the lipid antioxidant α-tocopherol, but not by the hydrophilic N-acetylcysteine (NAC) indicating crucial differences in the intracellular sites exposed to oxidative stress induced by sigma-2 receptor ligands. Importantly, all the compounds strongly increased the production of mitochondrial superoxide radicals except for PB282. Despite a poor match between in vitro and the in vivo efficacy, daily treatment of C57BL/6 mice bearing Panc02 tumors resulted in promising effects with PB28 and PB282 which were similar compared to the current standard-of-care chemotherapeutic gemcitabine without showing signs of systemic toxicities., Conclusions: Overall, this study identified differential sensitivities of pancreatic cancer cells to structurally diverse sigma-2 receptor ligands. Of note, we identified the mitochondrial superoxide pathway as a previously unrecognized sigma-2 receptor-activated process, which encourages further studies on sigma-2 ligand-mediated cancer cell death for the targeted treatment of pancreatic tumors.

Starting from our lead compound MC70 displaying high P-glycoprotein (P-gp) inhibition activity but low selectivity, a new class of coumarine derivatives was studied to develop selective and fluorescent P-gp ligands. In this series, the biphenyl moiety of MC70 was replaced with the coumarine fluorophore as a bioisostere of the biphenyl nucleus in order to improve the selectivity toward P-gp and the fluorescent properties for in vitro studies. Moreover, the presence and position of substituents on the coumarine nucleus were probed to develop suitable fluorescent probes to study the expression and activity of P-gp in living cells. The best result was found for compound 4c, which exerts a good P-gp activity profile (EC50  = 13 μM) as substrate and a high selectivity toward the pump since it is inactive toward MRP1., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Despite their controversial physiology, sigma-1 (σ1) receptors are intriguing targets for the development of therapeutic agents for central nervous system diseases. With the aim of providing versatile pharmacological tools to study σ1 receptors, we developed three σ1 fluorescent tracers by functionalizing three well characterized σ1 ligands with a fluorescent tag. A good compromise between σ1 binding affinity and fluorescent properties was reached, and the σ1 specific targeting of the novel tracers was demonstrated by confocal microscopy and flow cytometry. These novel ligands were also successfully used in competition binding studies by flow cytometry, showing their utility in nonradioactive binding assays as an alternative strategy to the more classical radioligand binding assays. To the best of our knowledge these are the first σ1 fluorescent ligands to be developed and successfully employed in living cells, representing promising tools to strengthen σ1 receptors related studies., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

P-glycoprotein is an ATP-binding cassette transporter involved in drug absorption, distribution and excretion. It pumps a wide range of xenobiotic compounds out of the cells and plays a crucial role in Multi Drug Resistance. Moreover, recent studies have demonstrated that changes in P-gp function and/or expression at the blood brain barrier are implicated in the pathogenesis of neurological disorders such as therapy-refractory epilepsy, Alzheimer's and Parkinson's disease. In the last decades the studies have been addressed to the discovery of potent P-gp inhibitors able to revert pharmacoresistance and to the development of PET tracers to detect P-gp activity and expression for an early diagnosis and therapy monitoring of neurodegenerative disease. However, clinical trials have reported only limited success in reversing MDR and radiolabeled ligands were not actually useful to study differences of transporter function in different brain regions due to their low brain uptake. The difficulties into the discovery of new ligands is due to the use of different experimental assays, to the fact that P-gp is highly flexible protein with different binging sites and available crystallographic structures for the protein have inadequate resolution. To overcome these limitations research groups prefer computational approaches such as homology models in their structure-based design or ligand-based methodologies. A recent approach aimed to identify ligands which can interrupt ATP-binding and hydrolysis by P-gp, by interacting at the NBDs of the protein. In this review results from radiolabeled, substrates and inhibitors, for monitoring the activity and expression of P-gp, respectively, are presented.

σ2 Receptor subtype is overexpressed in a variety of human tumors, with σ2 agonists showing antiproliferative effects towards tumor cells through multiple pathways that depend both on the tumor cell type and on the molecule type. Therefore, σ2 receptor is an intriguing target for tumor diagnosis and treatment despite the fact that that it has not yet been cloned. One of the last attempts to characterize σ2 receptors led to identify it as the progesterone receptor membrane component 1 (PGRMC1). Although still controversial, such identity appears to have been accepted. We the aim of contributing to solve this controversy, in this work we stably silenced or overexpressed PGRMC1 protein in human MCF7 adenocarcinoma cells. Western blotting analyses were performed to quantify the presence of PGRMC1 protein on each of the three MCF7 cell lines variants, while scatchard analyses with radioligand were performed in order to determine the expression of the σ2 receptors. In order to correlate the antiproliferative effect of σ2 receptor agonist with PGRMC1 density, some σ2 ligands were administered to each of the three MCF7 cells variants. The results suggested that PGRMC1 and σ2 receptors are two different molecular entities., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Although sigma-2 (σ2 ) receptors are still enigmatic proteins, they are promising targets for tumor treatment and diagnosis. With the aim of clarifying their role in oncology, we developed a σ2 -selective fluorescent tracer (compound 5) as a specific tool to study σ2 receptors. By using flow cytometry with 5, we performed competition binding studies on three different cell lines where we also detected the content of the σ2 receptors, avoiding the inconvenient use of radioligands. Comparison with a previously developed mixed σ1 /σ2 fluorescent tracer (1) also allowed for the detection of σ1 receptors within these cells. Results obtained by flow cytometry with tracers 1 and 5 were confirmed by standard methods (western blot for σ1 , and Scatchard analysis for σ2 receptors). Thus, we have produced powerful new tools for research on the σ whose reliability and adaptability to a number of fluorescence techniques will be useful to elucidate the roles of σ receptors in oncology., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Both subtypes of sigma (σ) receptors, σ₁ and σ₂, are over-expressed in many cancers with σ₂ proposed as a biomarker of tumor proliferation. We are interested in developing a high affinity selective σ₂ radioligand for in vivo monitoring of proliferative status of solid tumors and response to anti-cancer therapies. 1-Cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperazine (PB28) represents one of the lead candidates in the development of σ receptor ligands for therapeutic and diagnostic applications. However, the utility of PB28 is limited due to its relatively high lipophilicity., Methods: A more hydrophilic analogue (-)-(S)-1 was radiolabeled with (11)C via standard O-alkylation. In vitro autoradiography with [(11)C](-)-(S)-1 was done using rat brain slices. PET imaging was performed in mice bearing EMT6, C6 or PC-3 tumors after i.v. injection of [(11)C](-)-(S)-1., Results: [(11)C](-)-(S)-1 was produced in 53%±7% isolated decay-corrected yield with radiochemical and chemical purity over 99% and specific activity greater than 100 GBq/μmol. In vitro autoradiography with [(11)C(-)-(S)-1 resulted in a heterogeneous binding of the tracer in the rat brain with the highest radioactivity signals in the cortex region followed by cerebellum. This binding was successfully blocked by 10 μM of either haloperidol, (+)-(R)-1 or PB28. For C6 xenografts low target-to-nontarget ratio and high non-specific binding did not allow clear tumor visualization. No accumulation was visible in EMT6 tumor or in PC-3 tumor. Rat and mouse brain uptake was low and homogeneous while stronger signal was detected in the spinal cord. High accumulation of radioactivity was observed in liver and intestine suggesting hepatobiliary clearance., Conclusions: Despite excellent in vitro properties, [(11)C](-)-(S)-1 did not provide high enough specific binding in vivo and is, therefore, not a useful PET tracer for imaging σ₂ expression in tumors., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Microglial cells are responsible for clearing and maintaining the central nervous system (CNS) microenvironment. Upon brain damage, they move toward injuries to clear the area by engulfing dying neurons. However, in the context of many neurological disorders chronic microglial responses are responsible for neurodegeneration. Therefore, it is important to understand how these cells can be "switched-off" and regain their ramified state. Current research suggests that microglial inflammatory responses can be inhibited by sigma (σ) receptor activation. Here, we take advantage of the optical transparency of the zebrafish embryo to study the role of σ1 receptor in microglia in an intact living brain. By combining chemical approaches with real time imaging we found that treatment with PB190, a σ1 agonist, blocks microglial migration toward injuries leaving cellular baseline motility and the engulfment of apoptotic neurons unaffected. Most importantly, by taking a reverse genetic approach, we discovered that the role of σ1in vivo is to "switch-off" microglia after they responded to an injury allowing for these cells to leave the site of damage. This indicates that pharmacological manipulation of σ1 receptor modulates microglial responses providing new approaches to reduce the devastating impact that microglia have in neurodegenerative diseases., (Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Generations of modulators of the efflux pump P-glycoprotein (P-gp) have been produced as tools to counteract the Multidrug Resistance (MDR) phenomenon in tumor therapy, but clinical trials were not successful so far. With the aim of contributing to the development of novel P-gp modulators, we started from recently studied high-affinity sigma-2 (σ2) receptor ligands that showed also potent interaction with P-gp. For σ2 receptors high-affinity binding, a basic N-atom is a strict requirement. Therefore, we reduced the basic character of the N-atom present in these ligands, and we obtained potent P-gp modulators with poor or null σ2 receptor affinity. We also evaluated whether modulation of P-gp by these novel compounds involved consumption of ATP (as P-gp substrates do), as a source of energy to support the efflux. Surprisingly, even small structural changes resulted in opposite behavior, with amide 13 depleting ATP, in contrast to its isomer 18. Two compounds, 15 and 25, emerged for their potent activity at P-gp, and deserve further investigations as tools for P-gp modulation., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

6,7-Dimethoxytetrahydroisoquinoline is widely used as basic moiety in σ2 receptor ligands, in order to provide σ2versus σ1 selectivity. This same moiety is also widely exploited in modulators of P-glycoprotein (P-gp) efflux pump, so that mixed σ2/P-gp agents are often obtained. Deconstruction of 6,7-dimethoxytetrahydroisoquinoline moiety present in the potent mixed σ2/P-gp agent 6,7-dimethoxy-2-[4-[1-(4-fluorophenyl)-1H-indol-3-yl]butyl]-1,2,3,4-tetrahydroisoquinoline (1) could lead to the separation of σ2 affinity from P-gp activity. Therefore, phenethylamino-, benzylamino- and indanamine series were obtained. The NH group was also methylated in the N-phenethylamino series, and ethylated in the benzylamino series, to better match 6,7-dimethoxytetrahydroisoquinoline. The σ2 affinity drastically decreased with the increase of conformational freedom, whereas alkylation of the NH-group was beneficial for σ2 receptor interaction. By contrast, deconstruction of 6,7-dimethoxytetrahydroisoquinoline slightly reduced P-gp activity, with dimethoxy-substituted derivatives displaying potent P-gp interaction. Therefore, 'ring-opened' 6,7-dimethoxytetrahydroisoquinoline derivatives represent a promising strategy to obtain P-gp selective agents devoid of σ2 receptor affinity., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)