Negative Modulatory Effect Research Articles

Ribonucleotides differentially modulate oral glutamate detection thresholds.

The savory or umami taste of the amino acid glutamate is synergistically enhanced by the addition of the purines inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP) disodium salt. We hypothesized that the addition of purinergic ribonucleotides, along with the pyrimidine ribonucleotides, would decrease the absolute detection threshold of (increase sensitivity to) l-glutamic acid potassium salt (MPG). To test this, we measured both the absolute detection threshold of MPG alone and with a background level (3mM) of 5 different 5'-ribonucleotides. The addition of the 3 purines IMP, GMP, and adenosine 5'-monophosphate (AMP) lowered the MPG threshold in all participants (P < 0.001), indicating they are positive modulators or enhancers of glutamate taste. The average detection threshold of MPG was 2.08mM, and with the addition of IMP, the threshold was decreased by approximately 1.5 orders of magnitude to 0.046mM. In contrast to the purines, the pyrimidines uridine 5'-monophosphate (UMP) and cytidine 5'-monophosphate (CMP) yielded different results. CMP reliably raised glutamate thresholds in 10 of 17 subjects, suggesting it is a negative modulator or diminisher of glutamate taste for them. The rank order of effects on increasing sensitivity to glutamate was IMP > GMP> AMP >> UMP// CMP. These data confirm that ribonucleotides are modulators of glutamate taste, with purines enhancing sensitivity and pyrimidines displaying variable and even negative modulatory effects. Our ability to detect the co-occurrence of glutamate and purines is meaningful as both are relatively high in evolutionarily important sources of nutrition, such as insects and fermented foods.

Characterization of allosteric modulators that disrupt androgen receptor co-activator protein-protein interactions to alter transactivation-Drug leads for metastatic castration resistant prostate cancer.

Three series of compounds were prioritized from a high content screening campaign that identified molecules that blocked dihydrotestosterone (DHT) induced formation of Androgen Receptor (AR) protein-protein interactions (PPIs) with the Transcriptional Intermediary Factor 2 (TIF2) coactivator and also disrupted preformed AR-TIF2 PPI complexes; the hydrobenzo-oxazepins (S1), thiadiazol-5-piperidine-carboxamides (S2), and phenyl-methyl-indoles (S3). Compounds from these series inhibited AR PPIs with TIF2 and SRC-1, another p160 coactivator, in mammalian 2-hybrid assays and blocked transcriptional activation in reporter assays driven by full length AR or AR-V7 splice variants. Compounds inhibited the growth of five prostate cancer cell lines, with many exhibiting differential cytotoxicity towards AR positive cell lines. Representative compounds from the 3 series substantially reduced both endogenous and DHT-enhanced expression and secretion of the prostate specific antigen (PSA) cancer biomarker in the C4-2 castration resistant prostate cancer (CRPC) cell line. The comparatively weak activities of series compounds in the H3-DHT and/or TIF2 box 3 LXXLL-peptide binding assays to the recombinant ligand binding domain of AR suggest that direct antagonism at the orthosteric ligand binding site or AF-2 surface respectively are unlikely mechanisms of action. Cellular enhanced thermal stability assays (CETSA) indicated that compounds engaged AR and reduced the maximum efficacy and right shifted the EC50 of DHT-enhanced AR thermal stabilization consistent with the effects of negative allosteric modulators. Molecular docking of potent representative hits from each series to AR structures suggest that S1-1 and S2-6 engage a novel binding pocket (BP-1) adjacent to the orthosteric ligand binding site, while S3-11 occupies the AR binding function 3 (BF-3) allosteric pocket. Hit binding poses indicate spaces and residues adjacent to the BP-1 and BF-3 pockets that will be exploited in future medicinal chemistry optimization studies. Small molecule allosteric modulators that prevent/disrupt AR PPIs with coactivators like TIF2 to alter transcriptional activation in the presence of orthosteric agonists might evade the resistance mechanisms to existing prostate cancer drugs and provide novel starting points for medicinal chemistry lead optimization and future development into therapies for metastatic CRPC.

COVID-19 AND ARTERIAL STIFFNESS IN VACCINATED YOUNG ADULTS WITHOUT PRIOR CO-MORBIDITIES. A CROSS-SECTIONAL ANALYSIS OF THE FIT@4COVID COHORT STUDY

Objective: The long-term consequences of COVID-19 amongst survivors of acute infection are unknown, particularly on the cardiovascular system. Given the ability of the virus to affect the arterial wall, the question emerges to whether COVID-19 has a negative modulating effect of the vascular aging trajectory, conveying accelerated arterial stiffening and endothelial dysfunction, and thus, contributing to an increased CV risk over the life course. Objective: The main aim of this study was to ascertain the consequences of COVID-19 on arterial stiffness, by measuring PWV and thereby evaluating the presence of EVA. Secondary objectives of the study include investigating the effect of the SARS-CoV-2 infection on haemodynamic parameters measured by PWA. Design and method: A cross-sectional analysis of the initial data from the Fit@4COVID cohort was made. Carotid-femoral pulse wave velocity (PWV) and carotid pulse wave analysis (PWA) were evaluated in participants with (Covid19 group) and without (Control group) prior infection with SARS-CoV-2. Groups were matched for age and gender. Blood samples were taken for confirmation of prior exposition to the virus and to study the immune and inflammatory profile. Results: A total of 60 participants, equally divided in two groups, were enrolled, with a mean age of 20 ± 1.0 years. No significant differences were observed between groups concerning anthropometric, sociodemographic and clinical characteristics. Significant differences between-groups were observed for PWV (control: 5.84 ± 0.33 m/s, Covid19: 6.19 ± 0,47 m/s; p &lt; 0.01), even after adjusting for mean arterial pressure (p = 0.019). Significant differences were also depicted for central systolic blood pressure, with 106.40 ± 9.27 mmHg in the control group and 115.00 ± 11.23 mmHg in the Covid19 group (p = .012). Similar results were found when observing brachial systolic blood pressure, central pulse pressure and brachial pulse pressure, all of which showed higher values in the Covid19 group. Conclusions: The cross-sectional analysis of the initial Fit@4COVID data buttresses a relevant impact of the SARS-CoV-2 virus over the arterial wall, even in healthy young adults with mild forms of COVID-19. The long-term effects of COVID-19 over the cardiovascular system remains to be demonstrated.

Analysis of M4 Transmembrane Segments in NMDA Receptor Function: A Negative Allosteric Modulatory Site at the GluN1 M4 is Determining the Efficiency of Neurosteroid Modulation.

Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels that play a crucial role in excitatory synaptic transmission in the central nervous system. Each subunit contributes with three helical transmembrane segments (M1, M3, and M4) and a pore loop (M2) to form the channel pore. Recent studies suggest that the architecture of all eukaryotic iGluRs derives from a common prokaryotic ancestral receptor that lacks M4 and consists only of transmembrane segments M1 and M3. Although significant contribution has emerged in the last years, the role of this additionally evolved transmembrane segment in iGluR assembly and function remains unclear. Here, we have investigated how deletions and mutations of M4 in members of the NMDA receptor (NMDAR) subfamily, the conventional heteromeric GluN1/GluN2 and glycine-gated GluN1/GluN3 NMDARs, affect expression and function in Xenopus oocytes. We show that deletion of M4 in the GluN1, GluN2A, or GluN3A subunit, despite retained receptor assembly and cell surface expression, results in nonfunctional membrane receptors. Coexpression of the corresponding M4 as an isolated peptide in M4-deleted receptors rescued receptor function of GluN1/GluN2A NMDARs without altering the apparent affinity of glutamate or glycine. Electrophysiological analyses of agonist-induced receptor function and its modulation by the neurosteroid pregnenolone sulfate (PS) at mutations of the GluN1-M4/GluN2/3-transmembrane interfaces indicate a crucial role of position M813 in M4 of GluN1 for functional coupling to the core receptor and the negative modulatory effects of PS. Substitution of residues and insertion of interhelical disulfide bridges confirmed interhelical interactions of positions in M4 of GluN1 with residues of transmembrane segments of neighboring subunits. Our results show that although M4s in NMDARs are not important for receptor assembly and surface expression, the residues at the subunit interface are substantially involved in M4 recognition of the core receptor and regulation of PS efficacy. Because mutations in the M4 of GluN1 specifically resulted in loss of PS-induced inhibition of GluN1/GluN2A and GluN1/GluN3A NMDAR currents, our results point to distinct roles of M4s in NMDAR modulation and highlight the importance of the evolutionarily newly evolved M4 for selective in vivo modulation of glutamate- and glycine-activated NMDARs by steroids.

A multi-dosing regimen to enhance the spatial memory of normal rats with α5-containing GABAA receptor negative allosteric modulator L-655,708.

The reported inconsistent effects of negative allosteric modulators of α5-containing GABAA receptors on learning and memory may be attributed to receptor selectivity, effective plasma concentration maintenance, and administration time. This study aimed to compare the effects of L-655,708 administered by single-dosing regimen versus multi-dosing regimen on spatial memory, signaling molecules, and brain functional connectivity. After comparing the maintenance time of the effective plasma concentration of L-655,708 between multi-dosing and single-dosing regimens, we further compared the effects of the administration of the two regimens at different phases (before-learning, during-learning, and before-probe) of the Morris water maze (MWM) test on the performance of learning and memory and the levels of signaling molecules related to learning and memory in hippocampal tissues. Functional connectivity analyses between hippocampal and cortical regions were performed to further clarify the effects of the multi-dosing regimen. The multi-dosing regimen could maintain the effective plasma concentration of L-655,708 much longer than the single-dosing regimen. Only the multi-dosing regimen for L-655,708 administration during the learning period led to significant improvement in spatial memory in the MWM test and increases in levels of glutamate receptors and phosphorylated signaling molecules (p-PKAα, p-CaMKII, and p-CREB-1). Compared with the vehicle control, the multi-dosing regimen increased the functional connectivity of the left hippocampal CA1 with cingulate and motor cortices. A multi-dosing regimen for L-655,708 administered during the learning period is an effective strategy to improve spatial memory, increase signaling molecule levels, and enhance the functional connectivity of the hippocampus.

Reduced Expression of Cerebral Metabotropic Glutamate Receptor Subtype 5 in Men with Fragile X Syndrome.

Glutamatergic receptor expression is mostly unknown in adults with fragile X syndrome (FXS). Favorable behavioral effects of negative allosteric modulators (NAMs) of the metabotropic glutamate receptor subtype 5 (mGluR5) in fmr1 knockout (KO) mouse models have not been confirmed in humans with FXS. Measurement of cerebral mGluR5 expression in humans with FXS exposed to NAMs might help in that effort. We used positron emission tomography (PET) to measure the mGluR5 density as a proxy of mGluR5 expression in cortical and subcortical brain regions to confirm target engagement of NAMs for mGluR5s. The density and the distribution of mGluR5 were measured in two independent samples of men with FXS (N = 9) and typical development (TD) (N = 8). We showed the feasibility of this complex study including MRI and PET, meaning that this challenging protocol can be accomplished in men with FXS with an adequate preparation. Analysis of variance of estimated mGluR5 expression showed that mGluR5 expression was significantly reduced in cortical and subcortical regions of men with FXS in contrast to age-matched men with TD.

P.074 The effects of positive and negative modulators of alpha5 GABAA receptors on social behavior in 5xFAD model of Alzheimer's disease

In the context of industry 4.0 where the use of new technologies is required to meet productivity and profitability demands, this paper addresses 3D scanning, thermal imaging and computer vision to identify pipe clogging caused by waste accumulation in the fertilizer industry. This paper presents a study using thermal images and the use artificial neural network to model the waste distribution in industrial pipes.

Abstract 3455: Preclinical evidences of antitumoral properties of a Yerba mate extract on breast and colon cancer models

Abstract Yerba Mate (Ilex Paraguariensis) belongs to the Aquifoliaceae family and is a native plant of South America. This plant are enriched in bioactive compounds with biological activities. This study investigated the antitumor activity of Yerba Mate extract (YM) using in vitro and in vivo colon and breast cancer models. YM was generated by aqueous extraction and standardized to total phenolics content, antioxidant activity and Chlorogenic acid content. The HPLC analysis of the YM allowed us to quantify the main polyphenols: Chlorogenic acid (66.3 ± 0.05 mg/g dry sample), Rutin (6.783 ± 0.05 mg/g dry sample), Gallic acid (6.665 ± 0.321 mg/g dry sample), Caffeic acid (0.533 ± 0.04 mg/g dry sample) and Quercetin (0.229 ± 0.02 mg/g dry sample). To investigate the antitumoral activity of YM, we first evaluated its effect on cell proliferation of colon (CT26 and COLO205) and breast (MDA-MB 231, MCF7, F3II and 4T1) cancer cell lines. YM reduced tumor cell viability and induced apoptosis. In addition, YM showed negative modulatory effect on cell adhesion and migration of tumor cells and reduced their invasiveness capacity. The use of herbal products requires previous toxicity studies to identify the dose ranges that are safe for subsequent studies. Acute toxicity was evaluated in rats and chronic toxicity test was conducted in rabbits. Animals exposed showed no statistical changes in weight gain. Clinical examination and macroscopic analysis did not show any changes that indicate toxicity. We found a YM LD50 value above 5000 mg/kg. The effect of YM on tumor progression was also studied in vivo using different syngeneic tumor models. Animals received YM by oral administration in a dose of 1.6 g/kg/day before and after tumor cell inoculation. Orthotopic and heterotopic breast cancer models in BALB/c mice was performed. In the heterotopic F3II model the treatment with YM significantly reduced tumor volumes when compared with control group. In addition, the consumption of YM significantly increased the survival of animals in both models. We also studied the inhibition of spontaneous metastatic activity by YM. Diet with YM resulted in a reduced number of lung metastases compared to control in both models. On the other hand, in the heterothopic colorectal cancer model we observed that oral administration of YM reduced angiogenesis, delayed tumor onset and showed a reduction of tumor volume. The effects of the combination of YM extract with 5-fluorouracil (5-FU) was also evaluated in mice. The results suggest that this combination increased susceptibility of the colon cancer cells to the cytotoxicity of 5-Fu. In conclusion, our findings suggest that YM may be a promising agent for the treatment of breast and colon cancer in chemoprevention schemes or in combinatory schedules with standard-of-care therapeutics. Citation Format: Rocío S. García Lázaro, Humberto Lamdan, Norailys Lorenzo Perez, Lorena G. Caligiuri, Andrea L. Berengeno, Hugo H. Ortega, Daniel F. Alonso, Hernan G. Farina. Preclinical evidences of antitumoral properties of a Yerba mate extract on breast and colon cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3455.

The effects of cannabinoid 1 receptor compounds on memory: a meta-analysis and systematic review across species

RationaleWhile cannabis-based medicinal products have been shown to be effective for numerous neurological and psychiatric disorders, the evidence base regarding their adverse cognitive effects is poorly understood. The cannabinoid 1 receptor modulates memory performance via intracellular and extracellular mechanisms that alter synaptic transmission and plasticity. While previous literature has consistently shown that chronic cannabis users exhibit marked cognitive impairments, mixed findings have been reported in the context of placebo-controlled experimental trials. It is therefore unclear whether these compounds inherently alter cognitive processes or whether individuals who are genetically predisposed to use cannabis may have underlying cognitive deficits.ObjectiveWe conducted a meta-analysis to investigate the effects of full and partial cannabinoid 1 receptor (CB1R) agonists, antagonists, and negative allosteric modulators on non-spatial and spatial memory.MethodsIn accordance with the PRISMA guidelines, the EMBASE, MEDLINE, and PsycINFO databases were systematically searched for studies examining the effects of CB1R agonists, antagonists, and negative allosteric modulators on memory performance.ResultsWe systematically reviewed 195 studies investigating the effects of cannabinoid compounds on memory. In humans (N = 35 studies, comprising N = 782 subjects), delta-9-tetrahydrocannabinol (THC) (1.5–5 mg/kg) relative to placebo impaired performance on non-spatial memory tests, whereas only high THC doses (67 mg/kg) impaired spatial memory. Similarly, THC (0.2–4 mg/kg) significantly impaired visuospatial memory in monkeys and non-human primates (N = 8 studies, comprising N = 71 subjects). However, acute THC (0.002–10 mg/kg) had no effect on non-spatial (N = 6 studies, comprising 117 subjects; g = 1.72, 95% confidence interval (CI) − 0.18 to 3.63, p = 0.08) or spatial memory (9 studies, comprising 206 subjects; g = 0.75, 95% confidence interval (CI) − 1.09 to 2.58, p = 0.43). However, acute, full CB1R agonists significantly impaired non-spatial memory (N = 23 studies, 519 subjects; g = − 1.39, 95% CI − 2.72 to − 0.06, p = 0.03). By contrast, the chronic administration of CB1R agonists had no effect on non-spatial memory (N = 5 studies, comprising 146 subjects; g = − 0.05, 95% confidence interval (CI) − 1.32 to 1.22, p = 0.94). Moreover, the acute administration of CB1R antagonists had no effect on non-spatial memory in rodents (N = 9 studies, N = 149 subjects; g = 0.40, 95% CI − 0.11 to 0.92, p = 0.12).ConclusionsThe acute administration of THC, partial CB1R agonist, significantly impaired non-spatial memory in humans, monkeys, and non-human primates but not rodents. However, full CB1R agonists significantly impaired non-spatial memory in a dose-dependent manner but CB1R antagonists had no effect on non-spatial memory in rodents. Moreover, chronic THC administration did not significantly impair spatial or non-spatial memory in rodents, and there is inconclusive evidence on this in humans. Our findings highlight species differences in the effects of cannabinoid compounds on memory.

Four novel interaction partners demonstrate diverse modulatory effects on voltage-gated CaV2.2 Ca2+ channels.

Voltage-gated Ca2+ channels are embedded in a network of protein interactions that are fundamental for channel function and modulation. Different strategies such as high-resolution quantitative MS analyses and yeast-two hybrid screens have been used to uncover these Ca2+ channel nanodomains. We applied the yeast split-ubiquitin system with its specific advantages to search for interaction partners of the CaV2.2 Ca2+ channel and identified four proteins: reticulon 1 (RTN1), member 1 of solute carrier family 38 (SLC38), prostaglandin D2 synthase (PTGDS) and transmembrane protein 223 (TMEM223). Interactions were verified using the yeast split-ubiquitin system and narrowed down to CaV2.2 domain IV. Colocalization studies using fluorescent constructs demonstrated defined regions of subcellular localization. Detailed electrophysiological studies revealed that coexpression of RTN1 modulated CaV2.2 channels only to a minor extent. SLC38 accelerated the cumulative current inactivation during a high-frequency train of brief depolarizing pulses. As neurons expressing CaV2.2 channels were exposed to high-frequency bursts under physiological conditions, observed regulation may have a negative modulatory effect on transmitter release. Coexpression of PTGDS significantly lowered the average current density and slowed the kinetics of cumulative current inactivation. Since the latter effect was not significant, it may only partly compensate the first one under physiological conditions. Expression of TMEM223 lowered the average current density, accelerated the kinetics of cumulative current inactivation and slowed the kinetics of recovery from inactivation. Therefore, TMEM223 and, to a lesser extent, PTGDS, may negatively modulate Ca2+ entry required for transmitter release and/or for dendritic plasticity under physiological conditions.

Regulation of ABCB1 activity by microRNA-200c and microRNA-203a in breast cancer cells: the quest for microRNAs' involvement in cancer drug resistance.

Aim: ABCB1 is a major player in cancer drug resistance. The purpose of this study was to functionally assess the regulation of ABCB1 activity in a doxorubicin-resistant breast cancer cell line by miR-200c and miR-203.Methods: Human breast carcinoma cell lines MCF-7 (Doxorubicin-sensitive and not expressing ABCB1) and KCR (Doxorubicin-resistant and expressing ABCB1) were used to evaluate the expression levels of miR-200c and miR-203 by Real-time quantitative PCR (RT-qPCR). The effects of transient ectopic expression of miRNA-200c and miR-203 on the expression of ABCB1 in KCR and MCF-7 cells was verified by RT-qPCR and Western Blot. The extrusion activity of the ABCB1 pump was analyzed by fluorescence microscopy and flow cytometry through fluorescence substrate retention assays (DiOC2) in the presence and absence of the ABCB1 inhibitor verapamil.Results: RT-qPCR results indicated a 100,000-fold increase in ABCB1 mRNA expression levels in KCR cells compared to MCF-7 cells, and is inversely correlated with the expression of miR-203 and miR-200c. The insertion of miR-200c and miR-203 led to a higher retention of DiOC2 within KCR cells, and slightly reduced the protein levels of ABCB1 in KCR cells, although the high initial expression of ABCB1 masked the reduction in protein levels. The increased intracellular accumulation of the fluorescent due DiOC2 in the presence of the ABCB1 inhibitor verapamil correlated with the inhibition caused by miR-203 and miR-200c in transfected cells.Conclusion: The present study demonstrates that miR-200c and miR-203 exert a negative modulating effect on the activity of ABCB1 associated with doxorubicin resistance.

Comparison of rapid and long-lasting antidepressant effects of negative modulators of α5-containing GABAA receptors and (R)‑ketamine in a chronic social defeat stress model

The negative allosteric modulators (NAMs: L-655,708 and MRK-016) of α5 subunit-containing GABAA receptors are reported to show rapid-acting antidepressant effects in rodents. However, there are no reports comparing these NAMs and (R)‑ketamine, (R)-enantiomer of the rapid-acting antidepressant ketamine, in a chronic social defeat stress (CSDS) model. Here we measured expression of α5 GABAA receptor in the brain regions from CSDS susceptible mice and postmortem brain samples from depressed patients. Expression of α5 GABAA receptor in the prefrontal cortex and hippocampus from CSDS susceptible mice was significantly higher than that of control mice. Furthermore, expression of α5 GABAA receptor in the parietal cortex from depressed patients was also higher than that of control subjects. In the tail suspension and forced swimming tests, (R)‑ketamine and MRK-016 significantly attenuated the increased immobility time in the susceptible mice, compared with the vehicle-treated group. In the sucrose preference test, (R)‑ketamine and MRK-016 significantly enhanced the reduced preference in CSDS susceptible mice two days after a single injection. Unlike (R)‑ketamine, MRK-016 did not attenuate the reduced sucrose preference in susceptible mice 7 days after a single injection. In contrast, L-655,708 did not show antidepressant effects in the same model. In conclusion, this study shows that increased levels of α5 GABAA receptors in the PFC and hippocampus may play a role in depression-like phenotype after CSDS. It is unlikely that MRK-016 has long-lasting antidepressant effects although it elicits rapid-acting antidepressant effects.

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor's biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate.

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor's biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The first AMPA receptor negative modulators based on the tetrahydroquinazoline scaffold

A convenient approach to novel symmetric bispyrimidine diamines comprises SNAr reactions of 4-fluoropyrimidine N-oxide with alkane-α,ω-diamines with subsequent reduction of N-oxide function. The obtained substances were tested in patch-clamp experiments for the influence on the kainateinduced currents in Purkinje neurons and showed negative modulating effect.

Modulation of GABA and glycine receptors in rat pyramidal hippocampal neurones by 3α5β-pregnanolone derivatives

The ability of pregnanolone glutamate (PA-Glu), pregnanolone hemisuccinate (PA-hSuc) and pregnanolone hemipimelate (PA-hPim), neuroactive steroids with a negative modulatory effect on excitatory N-methyl-d-aspartate receptors, to influence the functional activity of inhibitory γ-aminobutyric acid and glycine receptors was estimated. The GABA- and glycine-induced chloride currents (IGABA and IGly) were measured in isolated pyramidal neurons of the rat hippocampus using the patch-clamp technique. Compound PA-Glu was found to potentiate IGABA and to inhibit IGly, while PA-hSuc and PA-hPim inhibited both IGABA and IGly. Moreover, PA-Glu, PA-hSuc, and PA-hPim had a greater effect on desensitization than on the peak amplitude of IGly. At a high concentration of glycine (500 μM), the effect of neurosteroids on the peak amplitude of IGly disappeared, and the acceleration of desensitization remained. The conversion of PA-Glu into androstane glutamate (AND-Glu), an analogue that lacks the C-17 acetyl moiety, completely eliminated the effects on these receptors. Our results indicate that the C-17 acetyl moiety is crucial for the action on IGABA and IGly. Our results indicate that the pregnanolone derivatives, in contrast to the androstane analogues, modulate IGABA and IGly at low micromolar concentrations and this family of neurosteroids can be useful for future structure-activity relationship studies of the steroid modulation of other receptor types.

Alcohol Addiction- Metabotropic Glutamate Receptor Subtype 5 and its Ligands: How They All Come Together?

In the past decade, many studies have highlighted the role of metabotropic glutamate receptor subtype 5 (mGlu5) modulators in attenuating alcohol-related biological effects such as alcohol consumption, alcohol-seeking and relapse-like behaviors. Taken together, these findings suggest that pharmacological agents acting at mGlu5 could be promising tools in curbing inebriation. mGlu5s are present abundantly in brain regions known to be involved in emotion regulation, motivation and drug administration. On a cellular level, they are primarily located at the postsynaptic part of the neuron where the receptor is functionally linked to various downstream proteins that are involved in cell signaling and gene transcription that mediate the alcohol-induced neuroplasticity. As well, the discovery of a functional link between mGlu5 and a specific isozyme, Protein Kinase C epsilon (PKCε) in mediating the attenuating effects of selective negative allosteric modulators of mGlu5 such as methyl- 6(phenylethynyl)pyridine (MPEP) and 3-((2-methyl-4-thiazolyl)ethynyl)pyridine (MTEP) has sparked interesting speculations. In this article, we shall review the following: the effects of acute and chronic alcohol intake on mGlu5 signaling; the effects of mGlu5 ligands on alcohol-related neurobehavioral changes that are currently being studied both at pre-clinical and clinical stages; and the mechanisms underlying the pharmacological effects of these drugs.

Topiramate mitigates 3-nitropropionic acid-induced striatal neurotoxicity via modulation of AMPA receptors

Prevalence of glutamate receptor subunit 2 (GluR2)-lacking alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors is a hallmark of excitotoxicity-related neurodegenerative diseases. Topiramate (TPM) is a structurally novel anticonvulsant with a well-known modulatory effects on AMPA/kainate subtypes of glutamate receptors. The present study aimed at investigating the neuroprotective potential of TPM on 3-nitropropionic acid (3-NP)-induced striatal neurodegeneration and Huntington's disease-like symptoms. Rats were injected with 3-NP (10 mg/kg/i.p.) for 14 days. TPM (50 mg/kg/p.o.) was given once a day, 1 h before 3-NP. TPM amended 3-NP induced changes in neurobehavioral performance, striatal neurotransmitters levels and histopathological injury. 3-NP control rats showed a significant ablation in the mRNA expression of Ca2+-impermeable Glu2R subunit along with an elevation in its regulatory protein (protein interacting with C kinase-1) PICK1, an effect that was largely reversed by TPM. TPM in addition, enhanced the phosphorylation of the protein kinase B/glycogen synthase kinase-3β/cAMP response element binding protein (Akt/GSK-3β/CREB) cue. Moreover, improvement in oxidative status, suppression of caspase-3 activity and restoration of striatal BDNF were noticed following treatment with TPM. The current study revealed that TPM boosted the neuroprotective (Akt/GSK-3β/CREB) pathway by its negative modulatory effect on AMPA glutamate receptors as well as its direct antioxidant property.

Selective Blockade of the Metabotropic Glutamate Receptor mGluR5 Protects Mouse Livers in In Vitro and Ex Vivo Models of Ischemia Reperfusion Injury.

2-Methyl-6-(phenylethynyl)pyridine (MPEP), a negative allosteric modulator of the metabotropic glutamate receptor (mGluR) 5, protects hepatocytes from ischemic injury. In astrocytes and microglia, MPEP depletes ATP. These findings seem to be self-contradictory, since ATP depletion is a fundamental stressor in ischemia. This study attempted to reconstruct the mechanism of MPEP-mediated ATP depletion and the consequences of ATP depletion on protection against ischemic injury. We compared the effects of MPEP and other mGluR5 negative modulators on ATP concentration when measured in rat hepatocytes and acellular solutions. We also evaluated the effects of mGluR5 blockade on viability in rat hepatocytes exposed to hypoxia. Furthermore, we studied the effects of MPEP treatment on mouse livers subjected to cold ischemia and warm ischemia reperfusion. We found that MPEP and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) deplete ATP in hepatocytes and acellular solutions, unlike fenobam. This finding suggests that mGluR5s may not be involved, contrary to previous reports. MPEP, as well as MTEP and fenobam, improved hypoxic hepatocyte viability, suggesting that protection against ischemic injury is independent of ATP depletion. Significantly, MPEP protected mouse livers in two different ex vivo models of ischemia reperfusion injury, suggesting its possible protective deployment in the treatment of hepatic inflammatory conditions.

Impact of Topiramate on Rat Phrenic Nerve-Hemidiaphragm Preparations.

Topiramate has a negative modulatory effect on voltage-gated ion channels involved in neuromuscular junction transmission. To investigate the potential impact of topiramate on muscle contraction, phrenic nerve-hemidiaphragm preparations were used as a neuromuscular junction model. Phrenic nerve-hemidiaphragm preparations were isolated from rats and were mounted in oxygenated Krebs solution. Preparations were stimulated in the presence of topiramate and phenytoin with a rectangular pulse at 0.1 Hertz, 0.3 miliseconds, and 3 miliseconds of duration, forming indirect, direct single, and tetanic muscle contractions, respectively. The expressed tension was isometrically recorded via a force displacement transducer on a polygraph. Topiramate and phenytoin directly and indirectly reduced contractions in a time-dependent manner. By contrast, topiramate, but not phenytoin, showed an excitatory effect on contraction in tetanic potentiation. To our knowledge, our study is the first to show the effects of topiramate on muscle contraction and neuromuscular junction transmission. Topiramate needs to be used with caution in patients with muscle weakness and respiratory problems.