Haloperidol Metabolite II Valproate Ester (S)-(-)-MRJF22: Preliminary Studies as a Potential Multifunctional Agent Against Uveal Melanoma.

Increased angiogenesis and vascular endothelial growth factor (VEGF) levels contribute to higher metastasis and mortality in uveal melanoma (UM), an aggressive malignancy of the eye in adults. (±)-MRJF22, a prodrug of the sigma (σ) ligand haloperidol metabolite II conjugated with the histone deacetylase (HDAC) inhibitor valproic acid, has previously demonstrated a promising antiangiogenic activity. Herein, the asymmetric synthesis of (R)-(+)-MRJF22 and (S)-(-)-MRJF22 was performed to investigate their contribution to (±)-MRJF22 antiangiogenic effects in human retinal endothelial cells (HREC) and to assess their therapeutic potential in primary human uveal melanoma (UM) 92-1 cell line. While both enantiomers displayed almost identical capabilities to reduce cell viability than the racemic mixture, (S)-(-)-MRJF22 exhibited the highest antimigratory effects in endothelial and tumor cells. Given the fundamental contribution of cell motility to cancer progression, (S)-(-)-MRJF22 may represent a promising candidate for novel antimetastatic therapy in patients with UM.

Sigma Receptor Ligands Carrying a Nitric Oxide Donor Nitrate Moiety: Synthesis, In Silico, and Biological Evaluation.

We report the development of molecular hybrids in which a nitrate group serving as nitric oxide (NO) donor is covalently joined to σ receptor ligands to give candidates for double-targeted cancer therapy. The compounds have been evaluated in radioligand binding assay at both σ receptors and selected compounds tested for NO release. Compounds 9, 15, 18, 19, and 21 were subjected to MTT test. Compound 15 produced a significant reduction of MCF-7 and Caco-2 cellular viability with comparable IC50 as doxorubicin, being also not toxic for fibroblast HFF-1 cells. Compound 15 has shown a σ1 receptor antagonist/σ2 receptor agonist profile. Two derivatives of compound 15 lacking the nitrate group did not induce a reduction of MCF-7 cellular viability, suggesting a potential synergistic effect between the σ receptors and the NO-mediated events. Overall, the combination of NO donor and σ receptors ligands provided compounds with beneficial effects for the treatment of cancer.

Synthesis, in vitro and in vivo characterization of new benzoxazole and benzothiazole-based sigma receptor ligands.

A new set of 5-chlorobenzoxazole- and 5-chlorobenzothiazole-based derivatives containing the azepane ring as a basic moiety was designed, synthesized and evaluated through binding assays to measure their affinity and selectivity towards σ1 and σ2 receptors. Compounds 19, 22 and 24, with a four units spacer between the bicyclic scaffold and the azepane ring, showed nanomolar affinity towards both receptor subtype and the best Ki values (Ki σ1 = 1.27, 2.30, and 0.78 and Ki σ2 = 7.9, 3.8, and 7.61 nM, respectively). Evaluation of cytotoxic and apoptotic effects in MCF-7 human cancer cells was useful to assess σ2 receptor activity, while an in vivo mice model of inflammatory pain allowed to analyze σ1 receptor pharmacological properties. In vitro and in vivo results suggested that compound 19 is a σ1/σ2 agonist, compound 24 a σ1 antagonist/σ2 agonist, whereas compound 22 might act as σ1 antagonist/σ2 partial agonist. Due to their pharmacological profile, a potential therapeutic application in cancer of aforesaid novel σ1/σ2 receptor ligands, especially 22 and 24, is proposed.

Targeting heme Oxygenase-1 with hybrid compounds to overcome Imatinib resistance in chronic myeloid leukemia cell lines.

Heme oxygenase-1 (HO-1) is a cytoprotective enzyme and a survival-enhancing factor in a number of cancers. Chronic myeloid leukemia (CML) is a blood cancer caused by pathological kinase activity of the BCR-ABL protein, currently treated with tyrosine kinase inhibitors (TKIs) such as Imatinib (IM). However, resistance to TKIs persists in a number of patients and HO-1 overexpression has been linked with the induction of chemoresistance in CML. With this in mind, in this study, we designed and synthesized the first series of hybrid compounds obtained by combining the structures of IM, as BCR-ABL inhibitor, with imidazole-based HO-1 inhibitors. We found that many hybrids were able to inhibit the enzymatic activity of both targets and to reduce the viability of CML-IM resistant cells, showing that a single molecular entity may reduce the resistance phenomenon.

Novel Structural Insight into Inhibitors of Heme Oxygenase-1 (HO-1) by New Imidazole-Based Compounds: Biochemical and In Vitro Anticancer Activity Evaluation.

In this paper, the design, synthesis, and molecular modeling of a new azole-based HO-1 inhibitors was reported, using compound 1 as a lead compound, in which an imidazole moiety is linked to a hydrophobic group by means of an ethanolic spacer. The tested compounds showed a good inhibitor activity and possessed IC50 values in the micromolar range. These results were obtained by targeting the hydrophobic western region. Molecular modeling studies confirmed a consolidated binding mode in which the nitrogen of the imidazolyl moiety coordinated the heme ferrous iron, meanwhile the hydrophobic groups were located in the western region of HO-1 binding pocket. Moreover, the new compounds were screened for in silico ADME-Tox properties to predict drug-like behavior with convincing results. Finally, the in vitro antitumor activity profile of compound 1 was investigated in different cancer cell lines and nanomicellar formulation was synthesized with the aim of improving compound's 1 water solubility. Finally, compound 1 was tested in melanoma cells in combination with doxorubicin showing interesting synergic activity.

Molecular modeling studies of pseudouridine isoxazolidinyl nucleoside analogues as potential inhibitors of the pseudouridine 5'-monophosphate glycosidase.

In this paper, we investigated the hypothesis that pseudouridine isoxazolidinyl nucleoside analogues could act as potential inhibitors of the pseudouridine 5'-monophosphate glycosidase. This purpose was pursued using molecular modeling and in silico ADME-Tox profiling. From these studies emerged that the isoxazolidinyl derivative 1 5'-monophosphate can be effectively accommodated within the active site of the enzyme with a ligand efficiency higher than that of the natural substrate. In this context, the poor nucleofugality of the N-protonated isoxazolidine prevents or slows down, the first mechanistic step proposed for the degradation of the pseudouridine 5'-monophosphate glycosidase, leading to the enzyme inhibition. Finally, the results of the physicochemical and ADME-Tox informative analysis pointed out that compound 1 is weakly bounded to plasma protein, only moderately permeate the blood-brain barrier, and is non-carcinogen in rat and mouse. To the best of our knowledge, this is the first paper that introduces the possibility of inhibition of pseudouridine 5'-monophosphate glycosidase by a molecule that competing with the natural substrate hinders the glycosidic C-C bond cleavage.

Novel Sigma-1 receptor antagonists: from opioids to small molecules: what is new?

Sigma-1 (σ1) receptor has been identified as a chaperone protein that interacts with other proteins, such as N-methyl-D-aspartate (NMDA) and opioid receptors, modulating their activity. σ1 receptor antagonists have been developed to obtain useful compounds for the treatment of psychoses, pain, drug abuse and cancer. Some interesting compounds such as E-5842 (5) and MS-377 (24), haloperidol and piperazine derivatives, respectively, were endowed with high affinity for σ1 receptors (Ki σ1 = 4 and 73 nM; Ki σ2 = 220 and 6900, respectively). They were developed for the treatment of psychotic disorders and 5 also underwent Phase II clinical trials suggesting interesting potential therapeutic applications. Here, σ1 receptor antagonists have been grouped based on chemical structure and reviewed according to structure-activity relationship and potential therapeutic role.

Sigma-1 and Sigma-2 receptor ligands induce apoptosis and autophagy but have opposite effect on cell proliferation in uveal melanoma.

Uveal melanoma is the most common primary intraocular tumor in adults, with about 1200-1500 new cases occurring per year in the United States. Metastasis is a frequent occurrence in uveal melanoma, and outcomes are poor once distant spread occurs and no clinically significant chemotherapeutic protocol is so far available. The aim of the present study was to test the effect of various σ1 and σ2 receptor ligands as a possible pharmacological strategy for this rare tumor. Human uveal melanoma cells (92.1) were treated with various concentrations of different σ2 ligands (haloperidol and haloperidol metabolite II) and σ1 ligand ((+)-pentazocine) at various concentrations (1, 10 and 25 µM) and time points (0, 4 h, 8 h, 24 h and 48 h). Cell proliferation and migration were evaluated respectively by continuous cell monitoring by xCELLigence analysis, clonogenic assay and wound healing. Apoptosis and autophagy were also measured by cytofluorimetric and microscopy analysis. Our results showed that σ2 receptor ligands significantly reduced cell proliferation whereas (+)-pentazocine exhibited opposite results. All tested ligands showed significant decrease in cell migration. Interestingly, both σ1 and σ2 receptor ligands showed significant increase of autophagy and apoptosis at all concentrations. Taken all together these results suggest that sigma receptors mediates opposite biological effects but they also share common pharmacological effect on apoptosis and autophagy in uveal melanoma. In conclusion, these data provide the first evidence that sigma receptors may represent a "druggable" target to develop new chemotherapic agent for uveal melanoma.

Novel Sigma Receptor Ligand-Nitric Oxide Photodonors: Molecular Hybrids for Double-Targeted Antiproliferative Effect.

This contribution reports the synthesis and evaluation of novel hybrid compounds that conjugate a sigma (σ) receptor pharmacophore and a nitric oxide (NO) photodonor. All compounds preserve their capability to generate NO under visible light and possess overall σ receptor nanomolar affinity, with one of them (8b) exhibiting remarkable σ2 receptor selectivity. Compounds 8b, 11a, and 11b were tested on tumorigenic MCF-7 and A2058 cells expressing high levels of σ2 and σ1 receptor, respectively. Considerable loss of cell viability was detected under light excitation, while negligible effects in the dark were detected. Moreover, they did not show any significant cytotoxicity in the dark or under irradiation on nontumorigenic NCTC-2544 keratinocytes. NO-induced reduction of cellular viability was demonstrated by in-cell NO detection and total nitrite estimation. For the first time, a combination of σ receptor moieties and a NO photodonor is reported, providing distinctive ligands potentially useful for cancer management.

Synthesis and evaluation of haloperidol metabolite II prodrugs as anticancer agents.

The use of haloperidol metabolite II (HP-metabolite II) prodrugs is an emerging strategy in the treatment of cancer. HP-metabolite II exhibits antiproliferative properties at micromolar concentrations inducing apoptosis in different types of cancer. Thus, the application of the prodrug approach appears as a useful method leading to much more desirable pharmacokinetic and pharmacodynamic properties. Some studies have shown that the esterification of the hydroxyl group of HP-metabolite II with 4-phenylbutiric acid (4-PBA) or valproic acid enhances the anticancer therapeutic potency. The current progresses in the design, synthesis and evaluation of anticancer activity of HP metabolite II prodrugs will be discussed in this review.

Structure-Activity Relationships within a Series of σ1 and σ2 Receptor Ligands: Identification of a σ2 Receptor Agonist (BS148) with Selective Toxicity against Metastatic Melanoma.

A new series of spirocyclic σ receptor (σR) ligands were prepared and studied. Most were found to have a high affinity and selectivity for σ1 R; three compounds were shown to be σ1 R agonists, while another proved to be the only σ1 R antagonist. Only one of the σ1 R agonists (BS148) also exhibited σ2 R selectivity and was able to inhibit the growth of metastatic malignant melanoma cell lines without affecting normal human melanocytes. The antiproliferative activity of this compound suggested an σ2 R agonist profile. Further, preliminary investigations indicated that the mechanism of metastatic malignant melanoma cell death induced by BS148 is due, at least in part, to apoptosis.

Recent advances in drug discovery of phototherapeutic non-porphyrinic anticancer agents.

In the search of novel strategies for the treatment of cancer, photodynamic therapy (PDT) has emerged as an effective, safe for repeated use, and non-invasive method. This technique involves the use of two major non-toxic components, a photosensitizer (PS) and a visible or near-infrared (NIR) light source, combined to induce cellular damage in an oxygen-dependent or -independent manner. Macrocyclic compounds, involving porphyrin and their derivatives, represent the major class of PS agents used in PDT. However, due to the drawbacks associated with these PS, like photosensitivity, dark toxicity, and low wavelength absorbance, new classes of PS appear to be needed. This review summarizes over the recent advances in drug discovery of non-porphyrinic PS suitable as anticancer therapeutics in PDT. The different compounds are grouped by chemical classes and discussed in terms of phototoxicity, together with the critical aspects of design and structure-activity relationship.

Role of the Nrf2/HO-1 axis in bronchopulmonary dysplasia and hyperoxic lung injuries.

Bronchopulmonary dysplasia (BPD) is a chronic illness that usually originates in preterm newborns. Generally, BPD is a consequence of respiratory distress syndrome (RDS) which, in turn, comes from the early arrest of lung development and the lack of pulmonary surfactant. The need of oxygen therapy to overcome premature newborns' compromised respiratory function generates an increasing amount of reactive oxygen species (ROS), the onset of sustained oxidative stress (OS) status, and inflammation in the pulmonary alveoli deputies to respiratory exchanges. BPD is a severe and potentially life-threatening disorder that in the most serious cases, can open the way to neurodevelopmental delay. More importantly, there is no adequate intervention to hamper or treat BPD. This perspective article seeks to review the most recent and relevant literature describing the very early stages of BPD and hyperoxic lung injuries focussing on nuclear factor erythroid derived 2 (Nrf2)/heme oxygenase-1 (HO-1) axis. Indeed, Nrf2/HO1 activation in response to OS induced lung injury in preterm concurs to the induction of certain number of antioxidant, anti-inflammatory, and detoxification pathways that seem to be more powerful than the activation of one single antioxidant gene. These elicited protective effects are able to counteract/mitigate all multifaceted aspects of the disease and may support novel approaches for the management of BPD.

Novel Caffeic Acid Phenethyl Ester (Cape) Analogues as Inducers of Heme Oxygenase-1.

BACKGROUND: Heme Oxygenase-1 (HO-1) is a metabolic enzyme strongly involved in processes including cytoprotection, modulation of inflammatory response, anti-oxidative functions, regulation of cellular proliferation, angiogenesis, cardiovascular homeostasis, and immuno-modulation. HO-1 induction and/or activation is able to counterbalance, at least in part, oxidative stress and inflammation. For this reason, HO-1 can be regarded as an attractive target to ameliorate different stress-related pathologies. Caffeic acid phenethyl ester, a natural polyphenolic compound, behaves as HO-1 inducer and possesses a plethora of beneficial effects under oxidative stress conditions. OBJECTIVES: A small focused series of caffeic acid phenethyl ester (Cape) analogues was designed and synthesized with the aim of obtaining more potent HO-1 inducers. METHOD: The capacity of these new compounds to modify the levels of HO-1 was evaluated in human mesenchymal stem cells (hMSCs) derived from bone marrow. RESULTS AND CONCLUSION: Some of tested compounds were found to be good HO-1 inducers and 3-(3,4- dihydroxyphenyl)-(2E)-2-propenoic acid 2-(3,4-dimethoxyphenyl) ethyl ester (VP961) was the most potent. VP961 tested to measure HO-1 protein expression and HO activity in in vitro system resulted more potent than the parent compound Cape both as inducer and as direct activator of the enzyme. VP961, selected as lead compound for further characterization, showed antioxidant properties in a model of H2O2-mediated ROS production and cytoprotective effects in a model of H2O2 cells viability impairment. To the best of our knowledge, VP961 is the first known compound able to activate directly HO-1 enzyme and to induce at the same time its protein expression.

S2RSLDB: a comprehensive manually curated, internet-accessible database of the sigma-2 receptor selective ligands.

BACKGROUND: Sigma (σ) receptors are accepted as a particular receptor class consisting of two subtypes: sigma-1 (σ1) and sigma-2 (σ2). The two receptor subtypes have specific drug actions, pharmacological profiles and molecular characteristics. The σ2 receptor is overexpressed in several tumor cell lines, and its ligands are currently under investigation for their role in tumor diagnosis and treatment. The σ2 receptor structure has not been disclosed, and researchers rely on σ2 receptor radioligand binding assay to understand the receptor's pharmacological behavior and design new lead compounds. DESCRIPTION: Here we present the sigma-2 Receptor Selective Ligands Database (S2RSLDB) a manually curated database of the σ2 receptor selective ligands containing more than 650 compounds. The database is built with chemical structure information, radioligand binding affinity data, computed physicochemical properties, and experimental radioligand binding procedures. The S2RSLDB is freely available online without account login and having a powerful search engine the user may build complex queries, sort tabulated results, generate color coded 2D and 3D graphs and download the data for additional screening. CONCLUSION: The collection here reported is extremely useful for the development of new ligands endowed of σ2 receptor affinity, selectivity, and appropriate physicochemical properties. The database will be updated yearly and in the near future, an online submission form will be available to help with keeping the database widely spread in the research community and continually updated. The database is available at http://www.researchdsf.unict.it/S2RSLDB.

Effect of MRJF4 on C6 Glioma Cells Proliferation and Migration.

BACKGROUND: MRJF4, a novel haloperidol metabolite II prodrug, was obtained through the esterification of the secondary hydroxyl group of haloperidol metabolite II with 4-phenylbutyric acid. The activities of (±)-MRJF4 and its two enantiomers [(+)-MRJF4 and (-)-MRJF4] as tumor specific inducers of pro-apoptotic genes were evaluated on malignant C6 glioma cells. In particular, changes in Nf-κB signaling pathway, activity of nitric oxide synthases (NOS), metalloproteinases (MMPs), and membrane adhesion proteins were investigated. RESULTS: IκBα reduced phosphorylation and iNOS lowered activity could be correlated with the previously demonstrated decreased proliferation and tumor progression of C6 cells upon 24 h of treatment with all the three compounds. Integrin ß1 decreased expression, at the same experimental time, seems to support lower C6 cells migrative capability and the consequent reduced invasiveness of these cells upon treatment with (±)-MRJF4 and its enantiomers. CONCLUSION: These results suggest that this multi-target prodrug and its two enantiomers might be a valuable clinical tool for the treatment of metastatic glioblastoma.

Antiangiogenic Effect of (±)-Haloperidol Metabolite II Valproate Ester [(±)-MRJF22] in Human Microvascular Retinal Endothelial Cells.

(±)-MRJF22 [(±)-2], a novel prodrug of haloperidol metabolite II (sigma-1 receptor antagonist/sigma-2 receptor agonist ligand) obtained by conjugation to valproic acid (histone deacetylase inhibitor) via an ester bond, exhibits antiangiogenic activity, being able to reduce human retinal endothelial cell (HREC) viability in a comparable manner to bevacizumab. Moreover, (±)-2 was able to significantly reduce viable cells count, endothelial cell migration, and tube formation in vascular endothelial growth factor A (VEGF-A) stimulated HREC cultures.

(+)-Pentazocine reduces oxidative stress and apoptosis in microglia following hypoxia/reoxygenation injury.

BACKGROUND: Sigma-1 receptors (σ1R) are highly expressed in neurons as well as microglia and have been shown to modulate the inflammatory response in the central nervous system and thus may serve as possible target for neuroprotective strategies. The aim of the present study was to test the effect of (+)-pentazocine, a putative σ 1R agonist, in an in vitro model of microglia activation. METHODS: Microglia (BV2 cells) was exposed (3h) to 1% oxygen and reoxygenation was allowed for 24h. Cells were treated with different concentrations (1, 10, 25 and 50µM) of (+)-pentazocine in the presence or absence of NE-100 (1µM), a well established σ1R antagonist. Cell viability and apoptosis were measured by cytofluorimetric analysis, whereas oxidative stress was evaluated by reduced glutathione (GSH) content and mitochondrial potential analysis. RESULTS: Our results showed that (+)-pentazocine was able to increase cell viability and restore mitochondrial potential at all concentrations whereas only 1 and 10µM were able to reduce significantly apoptotic cell death, to restore reduced glutathione intracellular content and prevent ERK1/2 phosphorylation. All these effects were abolished by concomitant treatment with NE-100. CONCLUSIONS: (+)-pentazocine exhibits significant dose dependent protective effects in our in vitro model of microglial activation thus suggesting that σ1R may represent a possible target for neuroprotection.

Identification of a potent and selective σ1 receptor agonist potentiating NGF-induced neurite outgrowth in PC12 cells.

Herein we report the synthesis, drug-likeness evaluation, and in vitro studies of new sigma (σ) ligands based on arylalkenylaminic scaffold. For the most active olefin the corresponding arylalkylamine was studied. Novel arylalkenylamines generally possess high σ(1) receptor affinity (K(i) values <25 nM) and good σ(1)/σ(2) selectivity (K(i)σ(2) >100). Particularly, the piperidine derivative (E)-17 and its arylalkylamine analog (R,S)-33 were observed to be excellent σ(1) receptor ligands (K(i)=0.70 and 0.86 nM, respectively) and to display significantly high selectivity over σ(2), µ-, and κ-opioid receptors and phencyclidine (PCP) binding site of the N-methyl-d-aspartate (NMDA) receptors. Moreover in PC12 cells (R,S)-33 promoted the nerve growth factor (NGF)-induced neurite outgrowth and elongation. Co-administration of the selective σ(1) receptor antagonist BD-1063 totally counteracted this effect, confirming that σ(1) receptors are involved in the (R,S)-33 modulation of the NGF effect in PC12 cells and suggesting a σ(1) agonist profile. As a part of our work, a threedimensional σ(1) pharmacophore model was also developed employing GALAHAD methodology. Only active compounds were used for deriving this model. The model included two hydrophobes and a positive nitrogen as relevant features and it was able to discriminate between molecules with and without affinity toward σ(1) receptor subtype.