Chaperone-Dependent Mechanisms as a Pharmacological Target for Neuroprotection.

Modern pharmacotherapy of neurodegenerative diseases is predominantly symptomatic and does not allow vicious circles causing disease development to break. Protein misfolding is considered the most important pathogenetic factor of neurodegenerative diseases. Physiological mechanisms related to the function of chaperones, which contribute to the restoration of native conformation of functionally important proteins, evolved evolutionarily. These mechanisms can be considered promising for pharmacological regulation. Therefore, the aim of this review was to analyze the mechanisms of endoplasmic reticulum stress (ER stress) and unfolded protein response (UPR) in the pathogenesis of neurodegenerative diseases. Data on BiP and Sigma1R chaperones in clinical and experimental studies of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease are presented. The possibility of neuroprotective effect dependent on Sigma1R ligand activation in these diseases is also demonstrated. The interaction between Sigma1R and BiP-associated signaling in the neuroprotection is discussed. The performed analysis suggests the feasibility of pharmacological regulation of chaperone function, possibility of ligand activation of Sigma1R in order to achieve a neuroprotective effect, and the need for further studies of the conjugation of cellular mechanisms controlled by Sigma1R and BiP chaperones.

Pharmacological Analysis of GABAA Receptor and Sigma1R Chaperone Interaction: Research Report I-Investigation of the Anxiolytic, Anticonvulsant and Hypnotic Effects of Allosteric GABAA Receptors' Ligands.

Two groups of facts have been established in previous drug development studies of the non-benzodiazepine anxiolytic fabomotizole. First, fabomotizole prevents stress-induced decrease in binding ability of the GABAA receptor's benzodiazepine site. Second, fabomotizole is a Sigma1R chaperone agonist, and exposure to Sigma1R antagonists blocks its anxiolytic effect. To prove our main hypothesis of Sigma1R involvement in GABAA receptor-dependent pharmacological effects, we performed a series of experiments on BALB/c and ICR mice using Sigma1R ligands to study anxiolytic effects of benzodiazepine tranquilizers diazepam (1 mg/kg i.p.) and phenazepam (0.1 mg/kg i.p.) in the elevated plus maze test, the anticonvulsant effects of diazepam (1 mg/kg i.p.) in the pentylenetetrazole-induced seizure model, and the hypnotic effects of pentobarbital (50 mg/kg i.p.). Sigma1R antagonists BD-1047 (1, 10, and 20 mg/kg i.p.), NE-100 (1 and 3 mg/kg i.p.), and Sigma1R agonist PRE-084 (1, 5, and 20 mg/kg i.p.) were used in the experiments. Sigma1R antagonists have been found to attenuate while Sigma1R agonists can enhance GABAARs-dependent pharmacological effects.

Low molecular weight NGF mimetic GK-2 normalizes the parameters of glucose and lipid metabolism and exhibits a hepatoprotective effect on a prediabetes model in obese Wistar rats.

Signs of metabolic syndrome and prediabetes preceding type 2 diabetes are modelled in an experiment using a high-fat diet (HFD). The aim of this work was to study the effect of a low molecular weight systemically active nerve growth factor mimetic, compound GK-2 (hexamethylenediamide bis[N-monosuccinyl-L-glutamyl-L-lysine]), on indicators of abdominal obesity, basal blood glucose level, glucose tolerance, cholesterol and triglyceride blood levels, as well as the morphological structure of the liver in male Wistar rats fed a HFD. Rats were divided into three groups: one of them received standard food (control) and two others were fed a HFD containing 45% fat, 35% carbohydrates and 20% protein, with a total caloric value of 516 kcal/100 g, over 12 weeks. Starting from the ninth week, for the next 4 weeks, one of the HFD groups was treated orally with saline whilst the other group was treated orally with GK-2 at a dose of 5 mg/kg. GK-2 was found to reduce the basal glycaemia level and improve glucose tolerance, as well as to reduce the blood level of cholesterol by 30% and that of triglycerides by 28% in comparison with the saline-treated HFD animals. GK-2 reduced the degree of abdominal obesity to the level of the healthy animals and eliminated morphological abnormalities in the liver caused by the HFD. The results of the study determine the feasibility of further GK-2 research as a potential agent for prediabetes treatment.

Involvement of Chaperone Sigma1R in the Anxiolytic Effect of Fabomotizole.

Sigma-1 receptor (chaperone Sigma1R) is an intracellular protein with chaperone functions, which is expressed in various organs, including the brain. Sigma1R participates in the regulation of physiological mechanisms of anxiety (Su, T. P. et al., 2016) and reactions to emotional stress (Hayashi, T., 2015). In 2006, fabomotizole (ethoxy-2-[2-(morpholino)-ethylthio]benzimidazole dihydrochloride) was registered in Russia as an anxiolytic (Seredenin S. and Voronin M., 2009). The molecular targets of fabomotizole are Sigma1R, NRH: quinone reductase 2 (NQO2), and monoamine oxidase A (MAO-A) (Seredenin S. and Voronin M., 2009). The current study aimed to clarify the dependence of fabomotizole anxiolytic action on its interaction with Sigma1R and perform a docking analysis of fabomotizole interaction with Sigma1R. An elevated plus maze (EPM) test revealed that the anxiolytic-like effect of fabomotizole (2.5 mg/kg i.p.) administered to male BALB/c mice 30 min prior EPM exposition was blocked by Sigma1R antagonists BD-1047 (1.0 mg/kg i.p.) and NE-100 (1.0 mg/kg i.p.) pretreatment. Results of initial in silico study showed that fabomotizole locates in the active center of Sigma1R, reproducing the interactions with the site's amino acids common for established Sigma1R ligands, with the ΔGbind value closer to that of agonist (+)-pentazocine in the 6DK1 binding site.

Neuroprotective Properties of Quinone Reductase 2 Inhibitor M-11, a 2-Mercaptobenzimidazole Derivative.

The ability of NQO2 to increase the production of free radicals under enhanced generation of quinone derivatives of catecholamines is considered to be a component of neurodegenerative disease pathogenesis. The present study aimed to investigate the neuroprotective mechanisms of original NQO2 inhibitor M-11 (2-[2-(3-oxomorpholin-4-il)-ethylthio]-5-ethoxybenzimidazole hydrochloride) in a cellular damage model using NQO2 endogenous substrate adrenochrome (125 µM) and co-substrate BNAH (100 µM). The effects of M-11 (10-100 µM) on the reactive oxygen species (ROS) generation, apoptosis and lesion of nuclear DNA were evaluated using flow cytometry and single-cell gel electrophoresis assay (comet assay). Results were compared with S29434, the reference inhibitor of NQO2. It was found that treatment of HT-22 cells with M-11 results in a decline of ROS production triggered by incubation of cells with NQO2 substrate and co-substrate. Pre-incubation of HT-22 cells with compounds M-11 or S29434 results in a decrease of DNA damage and late apoptotic cell percentage reduction. The obtained results provide a rationale for further development of the M-11 compound as a potential neuroprotective agent.

Analysis of Antidepressant-like Effects and Action Mechanisms of GSB-106, a Small Molecule, Affecting the TrkB Signaling.

Induction of BDNF-TrkB signaling is associated with the action mechanisms of conventional and fast-acting antidepressants. GSB-106, developed as a small dimeric dipeptide mimetic of BDNF, was previously shown to produce antidepressant-like effects in the mouse Porsolt test, tail suspension test, Nomura water wheel test, in the chronic social defeat stress model and in the inflammation-induced model of depression. In the present study, we evaluated the effect of chronic per os administration of GSB-106 to Balb/c mice under unpredictable chronic mild stress (UCMS). It was observed for the first time that long term GSB-106 treatment (1 mg/kg, 26 days) during ongoing UCMS procedure ameliorated the depressive-like behaviors in mice as indicated by the Porsolt test. In addition, chronic per os administration of GSB-106 resulted in an increase in BDNF levels, which were found to be decreased in the prefrontal cortex and hippocampus of mice after UCMS. Furthermore, prolonged GSB-106 treatment was accompanied by an increase in the content of pTrkB706/707 in the prefrontal cortex and by a pronounced increase in the level of pTrkB816 in both studied brain structures of mice subjected to UCMS procedure. In summary, the present data show that chronic GSB-106 treatment produces an antidepressant-like effect in the unpredictable chronic mild stress model, which is likely to be associated with the regulation of the BDNF-TrkB signaling.

Chaperone Sigma1R and Antidepressant Effect.

This review analyzes the current scientific literature on the role of the Sigma1R chaperone in the pathogenesis of depressive disorders and pharmacodynamics of antidepressants. As a result of ligand activation, Sigma1R is capable of intracellular translocation from the endoplasmic reticulum (ER) into the region of nuclear and cellular membranes, where it interacts with resident proteins. This unique property of Sigma1R provides regulation of various receptors, ion channels, enzymes, and transcriptional factors. The current review demonstrates the contribution of the Sigma1R chaperone to the regulation of molecular mechanisms involved in the antidepressant effect.

Deferred Administration of Afobazole Induces Sigma1R-Dependent Restoration of Striatal Dopamine Content in a Mouse Model of Parkinson's Disease.

Previously, we demonstrated that the immediate administration of multitarget anxiolytic afobazole slows down the progression of neuronal damage in a 6-hydroxidodamine (6-OHDA) model of Parkinson's disease due to the activation of chaperone Sigma1R. The aim of the present study is to evaluate the therapeutic potential of deferred afobazole administration in this model. Male ICR mice received a unilateral 6-OHDA lesion of the striatum. Fourteen days after the surgery, mice were treated with afobazole, selective Sigma1R agonist PRE-084, selective Sigma1R antagonist BD-1047, and a combination of BD-1047 with afobazole or PRE-084 for another 14 days. The deferred administration of afobazole restored the intrastriatal dopamine content in the 6-OHDA-lesioned striatum and facilitated motor behavior in rotarod tests. The action of afobazole accorded with the effect of Sigma1R selective agonist PRE-084 and was blocked by Sigma1R selective antagonist BD-1047. The present study illustrates the Sigma1R-dependent effects of afobazole in a 6-OHDA model of Parkinson's disease and reveals the therapeutic potential of Sigma1R agonists in treatment of the condition.

The New Dipeptide TSPO Ligands: Design, Synthesis and Structure-Anxiolytic Activity Relationship.

The translocator protein (TSPO, 18 kDa) plays an important role in the synthesis of neurosteroids by promoting the transport of cholesterol from the outer to the inner mitochondrial membrane, which is the rate-limiting step in neurosteroidogenesis. Stimulation of TSPO by appropriate ligands increases the level of neurosteroids. The present study describes the design, synthesis and investigation of anxiolytic-like effects of a series of N-acyl-tryptophanyl-containing dipeptides. These novel dipeptide TSPO ligands were designed with the original drug-based peptide design strategy using alpidem as non-peptide prototype. The anxiolytic activities were investigated in Balb/C mice using the illuminated open-field and elevated plus-maze tests in outbred laboratory mice ICR (CD-1). Dipeptide GD-102 (N-phenylpropionyl-l-tryptophanyl-l-leucine amide) in the dose range of 0.01-0.5 mg/kg intraperitoneally (i.p.) has a pronounced anxiolytic activity. The anxiolytic effect of GD-102 was abolished by PK11195, a specific TSPO antagonist. The structure-activity relationship study made it possible to identify a pharmacophore fragment for the dipeptide TSPO ligand. It was shown that l,d-diastereomer of GD-102 has no activity, and the d,l-isomer has less pronounced activity. The anxiolytic activity also disappears by replacing the C-amide group with the methyl ester, a free carboxyl group or methylamide. Consecutive replacement of each amino acid residue with glycine showed the importance of each of the amino acid residues in the structure of the ligand. The most active and technologically available compound GD-102, was selected for evaluation as a potential anxiolytic drug.

Activation of σ1 and σ2 receptors by afobazole increases glial cell survival and prevents glial cell activation and nitrosative stress after ischemic stroke.

Activation of sigma receptors at delayed time points has been shown to decrease injury following ischemic stroke. The mixed σ1/σ2 receptor agonist, 5-ethoxy-2-[2-(morpholino)-ethylthio]benzimidazole (afobazole), provides superior long-term outcomes compared to other σ ligands in the rat middle cerebral artery occlusion (MCAO) stroke model. Experiments using the MCAO model were carried out to determine the molecular mechanism involved in the beneficial effects of afobazole. Administration of afobazole (3 mg/kg) at delayed time points post-stroke significantly increased the number of microglia and astrocytes detected in the ipsilateral hemisphere at 96 h post-surgery. Morphological analysis of the microglia indicated that a greater number of these cells were found in the ramified resting state in MCAO animals treated with afobazole relative to MCAO vehicle controls. Similarly, fewer reactive astrocytes were detected in the injured hemisphere of afobazole-treated animals. Both the enhanced survival and reduced activation of glial cells were abolished by co-application of either a σ1 (BD-1063) or a σ2 (SM-21) receptor antagonist with afobazole. To gain further insight into the mechanisms by which afobazole lessens stroke injury, we probed the brain sections for markers of neuroinflammation (tumor necrosis factor α) and nitrosative stress (S-nitrosocysteine). Data show that afobazole significantly reduces S-nitrosocysteine levels, but does not alter tumor necrosis factor α expression 96 h after an ischemic stroke. Taken together our data indicate that afobazole acting via both σ1 and σ2 receptors decreases stroke injury by enhancing glial cell survival, blocking ischemia-induced glial cell activation, and decreasing nitrosative stress.

Design, Synthesis and Anticonvulsant Activity of Cinnamoyl Derivatives of 3,4,6,7,8,9-hexahydrodibenzo[b,d]furan-1-(2H)-one Oxime.

BACKGROUND: Epilepsy continues to be a significant global health problem and the search for new drugs for its treatment remains an urgent task. 5-HT2 and GABAA-receptors are among promising biotargets for the search for new anticonvulsants. METHODS: New potential 5-HT2 and GABAA ligands in the series of substituted cinnamoyl derivatives of 3,4,6,7,8,9-hexahydrodibenzo[b,d]furan-1-(2H)-one oxime were designed using pharmacophore model and molecular docking analysis. The synthesis of new compounds was carried out from 3,4,6,7,8,9-hexahydrodibenzo[b,d]furan-1(2H)-one oxime and substituted cinnamoyl chlorides. The anticonvulsant activity of new substances has been established using the maximal electroshock seizure test. RESULTS: Several synthesized substituted cinnamoyl derivatives of 3,4,6,7,8,9-hexahydrodibenzo [b,d]furan-1-(2H)-one oxime significantly reduced the severity of convulsive manifestations and completely prevented the death of animals after MES. The structure-activity relationship was investigated. The most effective compound was found to be GIZH-348 (1g) (3,4,6,7,8,9-hexahydrodibenzo[ b,d]furan-1(2Н)-one О-(4-chlorophenyl)acryloyl)oxime) at the doses of 10-20 mg/kg. CONCLUSION: Molecular and pharmacophore modelling methods allowed us to create a new group of substituted cinnamoyl derivatives of 3,4,6,7,8,9-hexahydrodibenzo[b,d]furan-1-(2H)-one oxime with anticonvulsant activity.

Stimulation of sigma receptors with afobazole blocks activation of microglia and reduces toxicity caused by amyloid-ß25-35.

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the leading cause of senile dementia in the United States. Accumulation of amyloid-ß (Aß) and the effects of this peptide on microglial cells contribute greatly to the etiology of AD. Experiments were carried out to determine whether the pan-selective σ-receptor agonist afobazole can modulate microglial response to the cytotoxic Aß fragment, Aß25-35. Treatment with afobazole decreased microglial activation in response to Aß, as indicated by reduced membrane ruffling and cell migration. The effects of afobazole on Aß25-35-evoked migration were concentration dependent and consistent with σ-receptor activation. When afobazole was coapplied with either BD-1047 [N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine dihydrobromide] or rimcazole, which are σ-1- and σ-2-selective antagonists, respectively, the inhibition of Aß25-35-induced migration by afobazole was reduced. Prolonged exposure of microglia to Aß25-35 resulted in glial cell death that was associated with increased expression of the proapoptotic protein Bax and the death protease caspase-3. Coapplication of afobazole with Aß25-35 decreased the number of cells expressing both Bax and caspase-3 and resulted in a concomitant enhancement in cell survival. Although afobazole inhibited activation of microglia cells by Aß25-35, it preserved normal functional responses in these cells after exposure to the amyloid peptide. Intracellular calcium increases induced by ATP were depressed in microglia after 24-hour exposure to Aß25-35. However, coincubation in afobazole returned these responses to near control levels. Therefore, stimulation of σ-1 and σ-2 receptors by afobazole prevents Aß25-35 activation of microglia and inhibits Aß25-35-associated cytotoxicity, suggesting that afobazole may be useful for AD therapeutics.

Afobazole activation of σ-1 receptors modulates neuronal responses to amyloid-ß25-35.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a continual decline of cognitive function. No therapy has been identified that can effectively halt or reverse its progression. One hallmark of AD is accumulation of the amyloid-ß peptide (Aß), which alone induces neuronal injury via various mechanisms. Data presented here demonstrate that prolonged exposure (1-24 hours) of rat cortical neurons to Aß25-35 results in an increase in basal intracellular Ca(2+) concentration ([Ca(2+)]i), and that coincubation with the compound afobazole inhibits these [Ca(2+)]i increases. The effect of afobazole on [Ca(2+)]i is due to activation of σ-1 receptors but could not be mimicked by a second pan-selective σ receptor agonist, 1,3-di-o-tolylguanidine (DTG). Afobazole was also found to lessen nitric oxide (NO) production in response to Aß25-35 application but did not affect elevations in reactive oxygen species elicited by the Aß fragment. The reductions in [Ca(2+)]i and NO perturbation produced by afobazole were associated with a decrease in neuronal cell death, whereas DTG failed to enhance cell survival. Examining the molecular mechanisms involved in the increased neuronal survival demonstrates that afobazole incubation results in lower expression of the proapoptotic protein Bax and the death protease caspase-3, while at the same time increasing expression of the antiapoptotic protein, Bcl-2. Given the importance of Aß neurotoxicity in AD etiology, the findings reported here suggest that afobazole may be an effective AD therapeutic agent. Furthermore, σ-1 receptors may represent a useful target for AD treatment, although not all σ ligands appear to be equally beneficial.

The neuropeptide cycloprolylglycine produces antidepressant-like effect and enhances BDNF gene expression in the mice cortex.

BACKGROUND: Cycloprolylglycine (CPG) is an endogenous dipeptide with a wide range of psychotropic activity and putative therapeutic potential for depression. A small but growing body of data suggests that antidepressant-like effect of CPG is associated with neuroplastic changes in the brain or 5-HT system modulation. However, the mechanisms of the dipeptide action remain elusive. AIMS: Here, we characterize the effects of chronic CPG administration on behavior and genes expression of antidepressants sensitive catalepsy (ASC) mice strain, characterized by depressive-like behavior. METHODS: ASC mice were injected with saline, fluoxetine (10 mg/kg/day), or CPG (1 and 2 mg/kg/day) during 2 weeks. Behavior was studied using the open field test, novel object test, elevated plus maze test, forced swim test, and tail suspension test (TST). The expressions of genes coding BDNF, CREB, 5-HT1A and 5-HT2A receptors, TPH2, and SERT in the brain were measured with quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Chronic intraperitoneal administration of 1 and 2 mg/kg of CPG revealed the significant antidepressant-like effect by decreasing immobility time in the TST. At the same time, CPG did not negatively affect locomotor activity, cognition, or anxiety. In the real-time quantitative polymerase chain reaction (PCR) assay, chronic CPG treatment (2 mg/kg for 14 days) increased Bdnf mRNA level in the frontal cortex. CONCLUSIONS: Our findings extend the evidence for the effectiveness of CPG to reduce depressive-like behaviors. The antidepressant-like effect of CPG is mediated, as least in part, by BDNF-dependent mechanism. The exact mechanism remains to be elucidated, and further studies are warranted.

GVS-111 prevents oxidative damage and apoptosis in normal and Down's syndrome human cortical neurons.

The neuroprotective activity of a novel N-acylprolyl-containing dipeptide analog of the nootropic 2-oxo-1-pyrrolidine acetamide (Piracetam) designated as GVS-111 (DVD-111/Noopept) was tested in two in vitro models of neuronal degeneration mediated by oxidative stress: normal human cortical neurons treated with H(2)O(2), and Down's syndrome (DS) cortical neurons. Incubation of normal cortical neurons with 50 microM H(2)O(2) for 1h resulted in morphological and structural changes consistent with neuronal apoptosis and in the degeneration of more than 60% of the neurons present in the culture. GVS-111 significantly increased neuronal survival after H(2)O(2)-treatment displaying a dose-dependent neuroprotective activity from 10nM to 100 microM, and an IC(50) value of 1.21+/-0.07 microM. GVS-111 inhibited the accumulation of intracellular free radicals and lipid peroxidation damage in neurons treated with H(2)O(2) or FeSO(4), suggesting an antioxidant mechanism of action. GVS-111 exhibited significantly higher neuroprotection compared to the standard cognition enhancer Piracetam, or to the antioxidants Vitamin E, propyl gallate and N-tert-butyl-2-sulpho-phenylnitrone (s-PBN). In DS cortical cultures, chronic treatment with GVS-111 significantly reduced the appearance of degenerative changes and enhanced neuronal survival. The results suggest that the neuroprotective effect of GVS-111 against oxidative damage and its potential nootropic activity may present a valuable therapeutic combination for the treatment of mental retardation and chronic neurodegenerative disorders.