Agonist Properties Research Articles

Beneficial effects of atypical antipsychotics on object recognition deficits after adolescent toluene exposure in mice: involvement of 5-HT1A receptors

ABSTRACT Background: Inhalant (e.g. toluene) misuse by adolescents has been linked to psychosis and persistent cognitive deficits. Identifying effective strategies to improve cognitive deficits following chronic toluene misuse is critical. 5-HT1A receptor has been proposed as a target for the treatment of cognitive deficits. Objectives: We compared the effects of antipsychotics on recognition deficits after adolescent toluene exposure in mice and elucidated the role of 5-HT1A receptors in the cognition-improving effects of antipsychotics. Methods: Male NMRI mice (n = 279) received one injection per day of either toluene (750 mg/kg) or corn oil at postnatal days 35–39 and 42–46. Thereafter, the acute and subchronic effects of haloperidol, aripiprazole, or clozapine on toluene-induced recognition deficits were evaluated by novel object recognition test. Results: Acute administration of aripiprazole (p < .05) and clozapine (p < .01), but not haloperidol, significantly attenuated the toluene-induced recognition deficits. Pretreatment with 5-HT1A receptor antagonist WAY -100,635 (p < .05) blocked their beneficial effects. Moreover, 5-HT1A receptor agonist buspirone (p < .01) ameliorated the toluene-induced recognition deficits, which was reversed by WAY -100,635 (p < .001). Finally, after repeated treatment with clozapine, aripiprazole, and buspirone daily for 14 days, the impaired object recognition in toluene-exposed mice was significantly improved (p < .05) and the beneficial effects lasted for at least 2 weeks (p < .05). Conclusions: The results indicate that clozapine and aripiprazole, which display 5-HT1A agonist properties, restored cognitive deficits in mice induced by adolescent toluene exposure. These findings suggest that these antipsychotics should be further explored as a potential treatment option for cognitive deficits in patients with psychosis associated with toluene exposure.

Manipulation of tissue factor-mediated basal PAR-2 signalling on macrophages determines sensitivity for IFNγ responsiveness and significantly modifies the phenotype of murine DTH.

BackgroundTissue factor (TF) generates proteases that can signal through PAR-1 and PAR-2. We have previously demonstrated PAR-1 signalling primes innate myeloid cells to be exquisitely sensitive to interferon-gamma (IFNγ). In this work we explored how TF mediated PAR-2 signalling modulated responsiveness to IFNγ and investigated the interplay between PAR-1/-2 signalling on macrophages.MethodologyWe characterised how TF through PAR-2 influenced IFNγ sensitivity in vitro using PCR and flow cytometry. and how it influenced oxazolone-induced delayed type hypersensitivity (DTH) responses in vivo. We investigated how basal signalling through PAR-2 influenced PAR-1 signalling using a combination of TF-inhibitors and PAR-1 &-2 agonists and antagonists. Finally, we investigated whether this system could be targeted therapeutically using 3-mercaptopropionyl-F-Cha-Cha-RKPNDK (3-MP), which has actions on both PAR-1 and -2.ResultsTF delivered a basal signal through PAR-2 that upregulated SOCS3 expression and blunted M1 polarisation after IFNγ stimulation, opposing the priming achieved by signalling through PAR-1. PAR-1 and -2 agonists or antagonists could be used in combination to modify this basal signal in vitro and in vivo. 3-MP, by virtue of its PAR-2 agonist properties was superior to agents with only PAR-1 antagonist properties at reducing M1 polarisation induced by IFNγ and suppressing DTH. Tethering a myristoyl electrostatic switch almost completely abolished the DTH response.ConclusionsTF-mediated signalling through PARs-1 and -2 act in a homeostatic way to determine how myeloid cells respond to IFNγ. 3-MP, an agent that simultaneously inhibits PAR-1 whilst delivering a PAR-2 signal, can almost completely abolish immune responses dependent on M1 polarisation, particularly if potency is enhanced by targeting to cell membranes; this has potential therapeutic potential in multiple diseases.

Sphingosine 1-phosphate lyase facilitates cancer progression through converting sphingolipids to glycerophospholipids.

BackgroundIn addition to potent agonist properties for sphingosine 1‐phosphate (S1P) receptors, intracellularly, S1P is an intermediate in metabolic conversion pathway from sphingolipids to glycerolysophospholipids (glyceroLPLs). We hypothesized that this S1P metabolism and its products might possess some novel roles in the pathogenesis of cancer, where S1P lyase (SPL) is a key enzyme.MethodsThe mRNA levels of sphingolipid‐related and other cancer‐related factors were measured in human hepatocellular carcinoma (HCC), colorectal cancer, and esophageal cancer patients’ tumours and in their adjacent non‐tumour tissues. Phospholipids (PL) and glyceroLPLs were measured by using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). In‐vitro experiments were performed in Colon 26 cell line with modulation of the SPL and GPR55 expressions. Xenograft model was used for determination of the cancer progression and for pharmacological influence.ResultsBesides high SPL levels in human HCC and colon cancer, SPL levels were specifically and positively linked with levels of glyceroLPLs, including lysophosphatidylinositol (LPI). Overexpression of SPL in Colon 26 cells resulted in elevated levels of LPI and lysophosphatidylglycerol (LPG), which are agonists of GPR55. SPL overexpression‐enhanced cell proliferation was inhibited by GPR55 silencing. Conversely, inhibition of SPL led to the opposite outcome and reversed by adding LPI, LPG, and metabolites generated during S1P degradation, which is regulated by SPL. The xenograft model results suggested the contribution of SPL and glyceroLPLs to tumour progression depending on levels of SPL and GPR55. Moreover, the pharmacological inhibition of SPL prevented the progression of cancer. The underlying mechanisms for the SPL‐mediated cancer progression are the activation of p38 and mitochondrial function through the LPI, LPG‐GPR55 axis and the suppression of autophagy in a GPR55‐independent manner.ConclusionA new metabolic pathway has been proposed here in HCC and colon cancer, SPL converts S1P to glyceroLPLs, mainly to LPI and LPG, and facilitates cancer development.

Reconsidering the role of glycaemic control in cardiovascular disease risk in type 2 diabetes: A 21st century assessment.

It is well known that the multiple factors contributing to the pathogenesis of type 2 diabetes (T2D) confer an increased risk of developing cardiovascular disease (CVD). Although the relationship between hyperglycaemia and increased microvascular risk is well established, the relative contribution of hyperglycaemia to macrovascular events has been strongly debated, particularly owing to the failure of attempts to reduce CVD risk through normalizing glycaemia with traditional therapies in high-risk populations. The debate has been further fuelled by the relatively recent discovery of the cardioprotective properties of glucagon-like peptide-1 receptor agonists and sodium-glucose cotransporter-2 inhibitors. Further, as guidelines now recommend individualizing glycaemic targets, highlighting the importance of achieving glycated haemoglobin (HbA1c) goals safely, the previously observed negative influences of intensive therapy on CVD risk might not present if trials were repeated using current-day treatments and individualized HbA1c goals. Emerging longitudinal data illuminate the overall effect of excess glucose, the impacts of magnitude and duration of hyperglycaemia on disease progression and risk of CVD complications, and the importance of glycaemic control at or early after diagnosis of T2D for prevention of complications. Herein, we review the role of glucose as a modifiable cardiovascular (CV) risk factor, the role of microvascular disease in predicting macrovascular risk, and the deleterious impact of therapeutic inertia on CVD risk. We reconcile new and old data to offer a current perspective, highlighting the importance of effective, early treatment in reducing latent CV risk, and the timely use of appropriate therapy individualized to each patient's needs.

Quantitative and histological evaluations for the potentiality of pilocarpine on tramadol induced xerostomia and structural alterations in Rats parotid gland

Tramadol is a centrally acting analgesic with weak opioid agonist properties, which also has monoaminergic activity, exerted via inhibition of neuronal uptake of serotonin and norepinephrine. Tramadol is generally well tolerated and the most common adverse events are nausea, dizziness, drowsiness, sweating, vomiting and dry mouth. The effect of intraperitoneal injection of tramadol and pilocarpine were studied in rats on the flow of saliva, histological and histochemical change in the parotid salivary gland. The rats were divided into three groups, first group, rats received saline and used as control. The second group treated with 10 mg tramadol hydrochloride for three days. The third group treated with tramadol hydrochloride for three days followed with 1 mg pilocarpine. The rats were sacrificed at the fourth day after the end of experimental procedure. The administration of tramadol caused decreased in salivary flow rate by approximately 60 %. The parotid histopathological changes include vacuolation and atrophy of acini and excessive widening of the interstitial tissue area which are replaced by extensive fibrosis fibrosis mostly associated with the degenerated ducts and acini and their replaced by inflammatory cells.

Synthetic cannabimimetics with cyclobutyl methyl and norbornyl methyl side chains – Pharmacological data and legislative implications

Structural elucidation and analytical characterization of six newly emerged synthetic cannabimimetics (SCs) carrying a cyclobutyl methyl (CBM) or a norbornyl methyl (NBM) side chain were performed within the ADEBAR project. Pharmacological evaluation allowed for estimating in vivo cannabimimetic activities as required in many countries for the amendment of laws controlling these substances. SCs, one of the largest subgroups of new psychoactive substances (NPS), possess a unique dynamic in Germany, where new variants have been observed to circumvent national legislation. In 2019–2020, the CBM moiety was identified as a new side chain in three SCs, leading to substances not covered by the German new psychoactive substances act (NpSG) at that time. In the following year, indole, indazole, and γ-carbolinone SCs featuring a NBM side chain were detected shortly after an amendment of the NpSG covering the CBM side chain. The introduction of these moieties that were not described in the synthetic cannabinoid research literature prior to their appearance on the drug market underpins the creativity of drug designers, obviously motivated by a recent amendment of the German NpSG. Analytical characterization of six SCs carrying a CBM or a NBM side chain was performed using GC-MS, ATR-FT-(N)IR, GC-sIR, NMR, LC-(HR)MS, and Raman spectroscopy. Binding affinities and functional activities were studied at the cannabinoid receptor 1 (CB1) using filtration-based radioligand assays. All six SCs showed full agonist properties at the CB1 receptor compared to Δ9-THC and CP-55940. The three SCs carrying a CBM group showed binding affinities from 29.4 to 0.65 nM (CBMICA >> CBMINACA > CBMeGaClone). The potencies of the compounds increased in the same order (EC50: 483–40.1 nM), which also reflects the order of their occurrence on the drug market. The norbornyl derivatives exhibit higher affinities (Ki: 1.87–0.25 nM), with the indazole derivative exhibiting the highest affinity. Functional activity data confirmed the indazole being the most potent and efficacious of these three NBM SCs (EC50: 169-–1.78 nM). The binding affinities and potencies were significantly increased by the introduction of the relatively voluminous norbornyl side chain. Only the γ-carbolinone analog exhibited a slight decrease of the binding affinity from 0.65 to 2.4 nM when exchanging the CBM group for the NBM group. Still, potency increased by factor 4 (indole: factor 2, indazole: factor 31). Cumyl-CBMICA showed the lowest binding affinity and functional activity of the six compounds. Interestingly, the average concentration of Cumyl-CBMICA increased from 5 mg/g to 71 mg/g in commercially available ‘herbal blends’ between July 2019 and December 2019 ( n = 14 blends analyzed) and the herbal blend product with the highest concentration was advertised as “strong” in the online shop. The concentrations were likely increased in the herbal blends due to the low potency of Cumyl-CBMICA in vivo, which has also been reported by user feedback given in online forums. New CBM and NBM SCs have been detected by the ADEBAR project in Germany and the six investigated SCs showed to act as full agonists at the CB1 receptor. The pharmacological data underpin their abuse potential and facilitated the legislative amendments to the German NpSG.

Interacting binding insights and conformational consequences of the differential activity of cannabidiol with two endocannabinoid-activated G-protein-coupled receptors

Cannabidiol (CBD), the major non-psychoactive phytocannabinoid present in the plant Cannabis sativa, has displayed beneficial pharmacological effects in the treatment of several neurological disorders including, epilepsy, Parkinson’s disease, and Alzheimer’s disease. In particular, CBD is able to modulate different receptors in the endocannabinoid system, some of which belong to the family of G-protein-coupled receptors (GPCRs). Notably, while CBD is able to antagonize some GPCRs in the endocannabinoid system, it also seems to activate others. The details of this dual contrasting functional feature of CBD, that is, displaying antagonistic and (possible) agonistic ligand properties in related receptors, remain unknown. Here, using computational methods, we investigate the interacting determinants of CBD in two closely related endocannabinoid-activated GPCRs, the G-protein-coupled receptor 55 (GPR55) and the cannabinoid type 1 receptor (CB1). While in the former, CBD has been demonstrated to function as an antagonist, the way by which CBD modulates the CB1 receptor remains unclear. Namely, CBD has been suggested to directly trigger receptor’s activation, stabilize CB1 inactive conformations or function as an allosteric modulator. From microsecond-length unbiased molecular dynamics simulations, we found that the presence of the CBD ligand in the GPR55 receptor elicit conformational changes associated with antagonist-bound GPCRs. In contrast, when the GPR55 receptor is simulated in complex with the selective agonist ML186, agonist-like conformations are sampled. These results are in agreement with the proposed modulatory function of each ligand, showing that the computational techniques utilized to characterize the GPR55 complexes correctly differentiate the agonist-bound and antagonist-bound systems. Prompted by these results, we investigated the role of the CBD compound on the CB1 receptor using similar computational approaches. The all-atom MD simulations reveal that CBD induces conformational changes linked with agonist-bound GPCRs. To contextualize the results we looked into the CB1 receptor in complex with a well-established antagonist. In contrast to the CBD/CB1 complex, when the CB1 receptor is simulated in complex with the ligand antagonist AM251, inactive conformations are explored, showing that the computational techniques utilized to characterize the CB1 complexes correctly differentiate the agonist-bound and antagonist-bound systems. In addition, our results suggest a previously unknown sodium-binding site located in the extracellular domain of the CB1 receptor. From our detailed characterization, we found particular interacting loci in the binding sites of the GPR55 and the CB1 receptors that seem to be responsible for the differential functional features of CBD. Our work will pave the way for understanding the CBD pharmacology at a molecular level and aid in harnessing its potential therapeutic use.

The effects of post-learning alcohol ingestion on human motor memory consolidation.

The neurochemical mechanisms underlying motor memory consolidation remain largely unknown. Based on converging work showing that ethyl alcohol retrogradely enhances declarative memory consolidation, this work tested the hypothesis that post‐learning alcohol ingestion would enhance motor memory consolidation. In a within‐subject and fully counterbalanced design, participants (n = 24; 12M; 12F) adapted to a gradually introduced visual deviation and ingested, immediately after adaptation, a placebo (PBO), a medium (MED) or high (HIGH) dose of alcohol. The alcohol doses were bodyweight‐ and gender‐controlled to yield peak breath alcohol concentrations of 0.00% in the PBO, ~0.05% in the MED and ~0.095% in the HIGH condition. Retention was evaluated 24 h later through reach aftereffects when participants were sober. The results revealed that retention levels were neither significantly nor meaningfully different in both the MED and HIGH conditions as compared to PBO (all absolute Cohen's dz values < ~0.2; small to negligible effects), indicating that post‐learning alcohol ingestion did not alter motor memory consolidation. Given alcohol's known pharmacological GABAergic agonist and NMDA antagonist properties, one possibility is that these neurochemical mechanisms do not decisively contribute to motor memory consolidation. As converging work demonstrated alcohol's retrograde enhancement of declarative memory, the present results suggest that distinct neurochemical mechanisms underlie declarative and motor memory consolidation. Elucidating the neurochemical mechanisms underlying the consolidation of different memory systems may yield insights into the effects of over‐the‐counter drugs on everyday learning and memory but also inform the development of pharmacological interventions seeking to alter human memory consolidation.

Guanabenz ameliorates disease in vanishing white matter mice in contrast to sephin1.

ObjectiveVanishing white matter (VWM) is a leukodystrophy, characterized by stress‐sensitive neurological deterioration and premature death. It is currently without curative treatment. It is caused by bi‐allelic pathogenic variants in the genes encoding eukaryotic initiation factor 2B (eIF2B). eIF2B is essential for the regulation of the integrated stress response (ISR), a physiological response to cellular stress. Preclinical studies on VWM mouse models revealed that deregulated ISR is key in the pathophysiology of VWM and an effective treatment target. Guanabenz, an α2‐adrenergic agonist, attenuates the ISR and has beneficial effects on VWM neuropathology. The current study aimed at elucidating guanabenz's disease‐modifying potential and mechanism of action in VWM mice. Sephin1, an ISR‐modulating guanabenz analog without α2‐adrenergic agonistic properties, was included to separate effects on the ISR from α2‐adrenergic effects.MethodsWild‐type and VWM mice were subjected to placebo, guanabenz or sephin1 treatments. Effects on clinical signs, neuropathology, and ISR deregulation were determined. Guanabenz's and sephin1's ISR‐modifying effects were tested in cultured cells that expressed or lacked the α2‐adrenergic receptor.ResultsGuanabenz improved clinical signs, neuropathological hallmarks, and ISR regulation in VWM mice, but sephin1 did not. Guanabenz's effects on the ISR in VWM mice were not replicated in cell cultures and the contribution of α2‐adrenergic effects on the deregulated ISR could therefore not be assessed.InterpretationGuanabenz proved itself as a viable treatment option for VWM. The exact mechanism through which guanabenz exerts its ameliorating impact on VWM requires further studies. Sephin1 is not simply a guanabenz replacement without α2‐adrenergic effects.

Modulatory Effects of Estradiol and Its Mixtures with Ligands of GPER and PPAR on MAPK and PI3K/Akt Signaling Pathways and Tumorigenic Factors in Mouse Testis Explants and Mouse Tumor Leydig Cells.

The present study was designed to evaluate how estradiol alone or in combination with G protein-coupled estrogen receptor (GPER) agonists and GPER and peroxisome proliferator-activated receptor (PPAR) antagonists alter the expression of tumor growth factor β (TGF-β), cyclooxygenase-2 (COX-2), hypoxia inducible factor 1-alpha (HIF-1α), and vascular endothelial growth factor (VEGF) in mouse testis explants and MA-10 mouse tumor Leydig cells. In order to define the hormone-associated signaling pathway, the expression of MAPK and PI3K/Akt was also examined. Tissue explants and cells were treated with estradiol as well as GPER agonist (ICI 182,780), GPER antagonist (G-15), PPARα antagonist (GW6471), and PPARγ antagonist (T00709072) in various combinations. First, we showed that in testis explants GPER and PPARα expressions were activated by the GPER agonist and estradiol (either alone or in mixtures), whereas PPARγ expression was activated only by GPER agonist. Second, increased TGF-β expression and decreased COX-2 expression were found in all experimental groups of testicular explants and MA-10 cells, except for up-regulated COX-2 expression in estradiol-treated cells, compared to respective controls. Third, estradiol treatment led to elevated expression of HIF-1α and VEGF, while their lower levels versus control were noted in the remaining groups of explants. Finally, we demonstrated the up-regulation of MAPK and PI3Kp85/Akt expressions in estradiol-treated groups of both ex vivo and in vitro models, whereas estradiol in mixtures with compounds of agonistic or antagonistic properties either up-regulated or down-regulated signaling kinase expression levels. Our results suggest that a balanced estrogen level and its action together with proper GPER and PPAR signaling play a key role in the maintenance of testis homeostasis. Moreover, changes in TGF-β and COX-2 expressions (that disrupted estrogen pathway) as well as disturbed GPER-PPAR signaling observed after estradiol treatment may be involved in testicular tumorigenesis.

Treating Patients with Aripiprazol: A Safe Gamble?

IntroductionAripiprazole (ARI) is an atypical antipsychotic drug with D2 partial agonist properties, usually prescribed to treat mood disorders (major depression or bipolar disorder) and schizophrenic disorder (schizophrenia or schizoaffective disorder). Dopamine receptor agonists, as is ARI, have been implicated in some cases of impulse-control problems, such as gambling disorder (GD), increased spending, hypersexuality and compulsive eating.ObjectivesCurrently, it is hypothesized that aripiprazole may cause impulse-control problems because it can produce a hyperdopaminergic state in the mesolimbic pathway (reward system) through its predominant action on dopamine D3 receptors. We intend to do a non-systematic review of the scientific information regarding this subject.MethodsThe authors revised the published literature about this topic, selecting relevant articles, systematic reviews and case reports, with the topic words: “aripiprazol”, “gambling disorder” and “dopamine receptor” in scientific data base.ResultsOverall, a few cases of ARI-induced pathological gambling as well as ARI-induced hypersexuality have been reported. In one study it was verified that comorbid psychiatric and substance use disorders were common among those who have experienced GD or worsened GD after beginning ARI treatment. In another study, it was verified that the group of patients who reported this alleged side-effect were mostly young (mean age, 33.6 years), mostly men (88.2%) and most lived alone.ConclusionsAttributing to dopamine agonists the only factor that can explain the onset of GD is simplistic and dangerous. Many other potential risk factors, including individual vulnerability factors (temperament, genetics) as well as environmental factors, must be considered.DisclosureNo significant relationships.

CXCR4 as a novel target in immunology: moving away from typical antagonists.

CXCR4 has been a target of interest in drug discovery for numerous years. However, so far, most if not all studies focused on finding antagonists of CXCR4 function. Recent studies demonstrate that targeting a minor allosteric pocket of CXCR4 induces an immunomodulating effect in immune cells expressing CXCR4, connected to the TLR pathway. Compounds binding in this minor pocket seem to be functionally selective with inverse agonistic properties in selected GPCR signaling pathways (Gi activation), but additional signaling pathways are likely to be involved in the immunomodulating effects. In depth research into these CXCR4-targeted immunomodulators could lead to novel treatment options for (auto)-immune diseases.

Synthesis of 2-chloropurine ribosides with chiral amino acid amides at C6 and their evaluation as A1 adenosine receptor agonists

A series of purine ribonucleosides bearing chiral amino acid amides at the C6 position of 2-chloropurine was synthesized. Molecular docking of the synthesized analogs of 2-chloroadenosine by their affinity for A1 adenosine receptors (A1ARs) was conducted. The investigation of A1AR stimulating activity of synthesized nucleosides was carried out in a model of an isolated mouse atrium. We have shown that derivatives with tyrosine, valine, and serine residues exhibit the properties of A1AR partial agonists. Animal experiments in the open field test have shown that these compounds have different profiles of psychoactive action. These nucleosides have an ophthalmic hypotensive effect and reduce intraocular pressure in a manner slightly inferior to that of timolol and brimonidine. The synthesized nucleosides can be the basis for further design and synthesis of new A1AR agonists.

Addressing Peroxisome Proliferator-Activated Receptor-gamma in 3-Nitropropionic Acid-Induced Striatal Neurotoxicity in Rats

Telmisartan (TEL) is an angiotensin II type 1 receptor blocker and a partial activator of peroxisome proliferator-activated receptor-gamma (PPARγ), which regulates inflammatory and apoptotic pathways. Increasing evidence has demonstrated the PPARγ agonistic property of TEL in several brain disorders. This study aims to explore the neuroprotective impact of TEL in 3-nitropropionic acid (3-NP)-induced neurotoxicity in rats. The PPARγ effect of TEL was affirmed by using the PPARγ agonist pioglitazone (PIO), and the antagonist GW9662. 3-NP led to a significant reduction in body weight alongside motor and cognitive functioning. The striata of the 3-NP-treated rats showed energy-deficit, microglia-mediated inflammatory reactions, apoptotic damage as well as histopathological lesions. PIO and TEL improved motor and cognitive perturbations induced by 3-NP, as confirmed by striatal histopathological examination, energy restoration, and neuronal preservation. Both drugs improved mitochondrial biogenesis evidenced by elevated mRNA expression of PPARγ, PGC-1α, and TFAM, alongside increased striatal ATP and SDH. The mitochondrial effect of TEL was beyond PPARγ activation. As well, their anti-inflammatory effect was attributed to suppression of microglial activation, and protein expression of pS536 p65 NF-κB with marked attenuation of striatal inflammatory mediator's release. Anti-inflammatory cytokine IL-10 expression was concurrently increased. TEL effectively participated in neuronal survival as it promoted phosphorylation of Akt/GSK-3β, further increased Bcl-2 expression, and inhibited cleavage of caspase-3. Interestingly, co-treatment with GW9662 partially revoked the beneficial effects of TEL. These findings recommend that TEL improves motor and cognitive performance, while reducing neuronal inflammation and apoptosis in 3-NP-induced neurotoxicity via a PPARγ-dependent mechanism.Graphical

Neuroprotective properties of GLP-1 receptor agonists and SGLT-2 inhibitors in experimental stroke

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Development of an Automated, High‐Throughput Assay to Detect Angiotensin AT2‐Receptor Agonistic Activity by Nitric Oxide Measurement In Vitro

BackgroundThe angiotensin AT2‐receptor (AT2R) is a key component within the protective arm of the renin‐angiotensin system inducing vasodilation by nitric oxide (NO) production. Currently, no high‐throughput assay is available to identify AT2R agonists in vitro, which could explain the low number of AT2R selective ligands in drug development programs.ObjectiveTo design and validate a high‐throughput method for detection of AT2R activation in vitro based on NO release.MethodsHuman aortic endothelial cells (HAEC) were seeded into 96‐well plates at a density of 5000 cells per well. 48 hours after seeding, cells were loaded for 30 minutes with the NO probe, DAF‐FM diacetate (5µM). Cells were stimulated for 15 minutes with the AT2R‐agonist C21 (1µM) or with classical NO‐releasing agents as positive controls: bradykinin (BK, 0.1µM), acetylcholine (ACh, 1µM) or Ang‐(1‐7) (MasR agonist, 0.1µM). Cells were fixed and fluorescence signals captured by fluorescence microscopy with automated, built‐in image analysis (ImageXpress Pico, Molecular Devices, USA). AT2R‐transfected (AT2R‐CHO) or non‐transfected (NT‐CHO) Chinese hamster ovary cells were cultured and loaded with DAF‐FM diacetate according to the same protocol as HAEC. Concentration‐response curves were obtained for C21, BK, Ang II and Ang‐(1‐5) (0.1nM to 10µM each) using the automated protocol for ImageXpress Pico described above on AT2R‐CHO. To evaluate whether effects of the compounds in AT2R‐CHO were AT2R specific, the response to these compounds at the highest concentration (10µM) was also tested in NT‐CHO.ResultsUsing primary HAEC we could show that the automated, high‐throughput workflow was able to detect NO release by AT2R agonists as efficiently as by classical NO‐releasing agents [C21: 16.16 ± 6.84%; Ang‐(1‐7): 25.45 ± 3.37%, BK: 19.30 ± 2.92%; ACh: 24.01 ± 4.24%; one‐way ANOVA followed by Dunnett’s multiple comparison test, p≤0.05 vs control, ≥3 independent experiments for all compounds). In AT2R‐CHO, we detected that both C21 and Ang II (AT2R agonists) increased NO release in a concentration‐dependent manner, while BK had no effect (AT2R‐CHO lacks BK receptors) at any tested concentration. Surprisingly Ang‐(1‐5), a degradation product of Ang II thought to be inactive, induced NO release in a concentration‐dependent manner in AT2R‐CHO. In NT‐CHO, none of the compounds stimulated NO release, indicating that the effects by C21, Ang II and Ang‐(1‐5) in AT2R‐CHO were AT2R specific.ConclusionMeasurement of NO release from AT2R‐CHO or HAEC by DAF‐FM diacetate in an automated way is suitable as high‐throughput assay for the identification of AT2R‐agonistic compounds in vitro. Application of the assay revealed that Ang‐(1‐5), which has been regarded as an inactive metabolite of Ang II, has AT2R agonistic properties.

Ligand‐Dependent Reversal Kinetics of Mu Opioid Receptor Agonists by the Novel Antagonist, Methocinnamox

Naloxone (NLX) is a competitive antagonist at the mu opioid receptor and is currently the only pharmacological intervention available to treat an opioid overdose. Unfortunately, NLX is limited by its surmountability and its rapid metabolism/clearance can lead to a reemergence of narcotic effects (renarcotization). Methocinnamox (MCAM) is a long‐acting antagonist at mu receptors both in vivo and in vitro. In addition to its orthosteric antagonism, we have found that MCAM differentially modulates orthosteric properties (affinity and/or efficacy) of mu agonists via an allosteric mechanism (Zamora, Smith, et al. (2021). Pharmacology Research &amp; Perspectives. https://doi.org/10.1002/prp2.887). To further explore the interactions between MCAM and the human mu opioid receptor, we compared the rate by which MCAM and NLX reverse cAMP inhibition by the mu receptor agonists DAMGO and fentanyl. Treatment with either DAMGO or fentanyl (~ 30 x EC50) inhibited forskolin (FSK)‐stimulated intracellular cAMP levels. When administered 20 min after agonist, NLX (400 nM; ~100 x Ki) reversed DAMGO‐ and fentanyl‐mediated inhibition of cAMP production at a similar rate (25.4% ± 2.5 min‐1 vs 26.8% ± 5.5 min‐1, respectively). Since reversal by NLX was driven mostly by dissociation of the agonists, the similar rate of reversal by NLX indicate that DAMGO and fentanyl dissociate from the receptor at similar rates. Reversal by MCAM (100 nM; ~ 100 x Ki) however was ligand‐dependent. The rate of reversal of DAMGO‐mediated inhibition by MCAM was 11.7% ± 1.3 min‐1, whereas reversal of fentanyl was slower (4.1% ± 0.9 min‐1; P&lt;0.001). The ligand‐dependence of MCAM’s reversal kinetics on DAMGO vs fentanyl’s inhibition of cAMP production is consistent with the reported allosteric action of MCAM, and may be explained by modulation of ligand dissociation by MCAM. Together, these data suggest in addition to its orthosteric antagonism, MCAM may allosterically modulate the binding of mu agonists.

The acute dose and baseline amplitude-dependent effects of CDP-choline on deviance detection (MMN) in chronic schizophrenia: A pilot study.

The detection of deviant auditory features is empirically supported as impaired in schizophrenia and has been shown to associate with functional outcome. Modulated by glutamate neurotransmission, this sensory process has also been shown to relate to the α7 nicotinic acetylcholine receptor (nAChR) system, a prioritized molecular target for the development of novel cognition targeted pharmacological treatments. This pilot study assessed the acute effects of CDP-Choline, a choline supplement with α7 nAChR agonist properties, on the mismatch negativity (MMN), an event-related potential index of the detection of an acoustic change, in a sample of individuals diagnosed with chronic schizophrenia. Utilizing a randomized, placebo-controlled, double-blind design, the dose-dependent (500 mg, 1,000 mg, 2,000 mg), baseline amplitude-dependent (low vs. high), and deviant feature-dependent effects of CDP-Choline on the MMN were examined. CDP-choline's effects interacted with dosage, deviance feature, and baseline amplitude with low baseline amplitude patients demonstrating enhanced MMNs, and high baseline amplitude patients demonstrating suppressed MMNs in response to CDP-Choline. These findings offer tentative support for the involvement of the α7 nAChR system in auditory MMN abnormalities in schizophrenia and supports further research assessing the effects of long-term treatment with CDP-Choline in the personalized treatment of auditory deviance processing impairments. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

An insight about the mechanism of action (MoA) of R-bicalutamide on the androgen receptor homodimer using molecular dynamic

R-bicalutamide is a first-line therapy used to treat prostate cancer (PCa) inhibiting the androgen receptor (AR) which plays an important role in the development and the progression of PCa. However, after a protracted drug administration, many patients develop a form of androgen insensitivity since R-bicalutamide starts to exhibit some agonistic properties lead by the W741L AR mutation in the ligand-binding pocket even if the mechanism of the antagonist-agonist switch is still not clear. To study the drug-resistant mechanism, we explored the structural effects of the antagonist R-bicalutamide on the homodimer stability considering both the AR wild-type and W741L employing molecular dynamic (MD) simulations. The results obtained indicate that the binding of R-bicalutamide in the two AR monomers induces a great instability in the homodimer, which may determine the monomer's dissociation preventing AR migration into the nucleus and avoiding the transcriptional activity. If the W741L mutation occurs, the homodimer tends to have a behaviour close to the agonistic system where the two monomers are tightly bound, which may explain the effect of the W741L in drug insensitivity from a structural point of view.

The Role of NRF2 in Bone Metabolism – Friend or Foe?

Bone metabolism is closely related to oxidative stress. As one of the core regulatory factors of oxidative stress, NRF2 itself and its regulation of oxidative stress are both involved in bone metabolism. NRF2 plays an important and controversial role in the regulation of bone homeostasis in osteoblasts, osteoclasts and other bone cells. The role of NRF2 in bone is complex and affected by several factors, such as its expression levels, age, sex, the presence of various physiological and pathological conditions, as well as its interaction with certains transcription factors that maintain the normal physiological function of the bone tissue. The properties of NRF2 agonists have protective effects on the survival of osteogenic cells, including osteoblasts, osteocytes and stem cells. Activation of NRF2 directly inhibits osteoclast differentiation by resisting oxidative stress. The effects of NRF2 inhibition and hyperactivation on animal skeleton are still controversial, the majority of the studies suggest that the presence of NRF2 is indispensable for the acquisition and maintenance of bone mass, as well as the protection of bone mass under various stress conditions. More studies show that hyperactivation of NRF2 may cause damage to bone formation, while moderate activation of NRF2 promotes increased bone mass. In addition, the effects of NRF2 on the bone phenotype are characterized by sexual dimorphism. The efficacy of NRF2-activated drugs for bone protection and maintenance has been verified in a large number of in vivo and in vitro studies. Additional research on the role of NRF2 in bone metabolism will provide novel targets for the etiology and treatment of osteoporosis.