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Purpose: γ-H2AX and 53BP1 are fundamental for cellular DNA damage response (DDR) after radiation exposure and are linked to cell repair, arrest, or apoptosis. We aimed to evaluate whether DDR-markers in peripheral blood lymphocytes (PBLs) may have predictive potential for outcome in metastatic castration-resistant prostate cancer (mCRPC) patients receiving [177Lu]Lu-prostate-specific membrane antigen (PSMA) radioligand therapy (RLT). Methods: We prospectively enrolled 20 men with advanced mCRPC scheduled for PSMA-targeted RLT. Prior to the first cycle of [177Lu]Lu-PSMA RLT, all patients underwent [18F]F-PSMA-1007 positron emission tomography (PET)/computed tomography (CT) for assessment of tumor PSMA expression (assessing maximum standardized uptake value (SUVmax) of all tumor lesions). Blood samples were collected prior to, + 1 h after, and + 24 h after administration of [177Lu]Lu-PSMA, and DDR-markers γ-H2AX and 53BP1 were determined in PBLs through immunocytofluorescence. We then tested the predictive performance of DDR-markers relative to clinical and PET-based parameters for progressive disease (PSA-PD) after 2 cycles. In addition, the predictive value for progression-free survival (PSA-PFS, provided as median and 95% confidence interval [CI]) was explored. Results: Low baseline 53BP1 and γ-H2AX foci (P = 0.17) tended to predict early PSA-PD, whereas low SUVmax was significantly associated with higher risk for PSA-PD (P = 0.04). In Kaplan–Meier analysis, there was a trend towards prolonged PSA-PFS in patients with higher baseline 53BP1 of 6 months (mo; 95%CI, 4–9 mo) compared to 3 mo in patients with low 53BP1 (95% CI, 2–3 mo; P = 0.12). Comparable results were recorded for higher γ-H2AX expression (6 mo [95% CI, 3–9 mo] relative to 3 mo [95% CI, 2–4 mo] in patients with low γ-H2AX; P = 0.12). SUVmax, however, did not demonstrate predictive value (P = 0.29). Consistently, in univariate Cox-regression analysis, baseline 53BP1 foci demonstrated borderline significance for predicting PSA-PFS under [177Lu]Lu-PSMA RLT (P = 0.05). Conclusion: In this prospective study investigating mCRPC patients undergoing [177Lu]Lu-PSMA RLT, low baseline DDR-markers in PBLs tended to predict poor outcome. Although the study group was small and results need further confirmation, these preliminary findings lay the foundation for exploring additive radiosensitizing or treatment intensification in future studies with high-risk individuals scheduled for RLT. [ABSTRACT FROM AUTHOR]

Acquired lipodystrophy is often characterized as an idiopathic subtype of lipodystrophy. Despite suspicion of an immune-mediated pathology, biomarkers such as autoantibodies are generally lacking. Here, we used an unbiased proteome-wide screening approach to identify autoantibodies to the adipocyte-specific lipid droplet protein perilipin 1 (PLIN1) in a murine model of autoimmune polyendocrine syndrome type 1 (APS1). We then tested for PLIN1 autoantibodies in human subjects with acquired lipodystrophy with two independent severe breaks in immune tolerance (including APS1) along with control subjects using a specific radioligand binding assay and indirect immunofluorescence on fat tissue. We identified autoantibodies to PLIN1 in these two cases, including the first reported case of APS1 with acquired lipodystrophy and a second patient who acquired lipodystrophy as an immune-related adverse event following cancer immunotherapy. Lastly, we also found PLIN1 autoantibodies to be specifically enriched in a subset of patients with acquired generalized lipodystrophy (17 of 46 [37%]), particularly those with panniculitis and other features of autoimmunity. These data lend additional support to new literature that suggests that PLIN1 autoantibodies represent a marker of acquired autoimmune lipodystrophies and further link them to a break in immune tolerance. [ABSTRACT FROM AUTHOR]

Agonists of Benzodiazepine (BZD) receptor are exhaustively used in the control of muscle spasms, seizure, anxiety, and insomnia. BZDs have some unwanted effects; therefore, the development of new BZD receptor agonists with better efficacy and fewer unwanted effects is one of the subjects of interest. In this study, based on the pharmacophore/receptor model of the BZD binding site of GABAA receptors, a series of new 2-substituted-5-(4-chloro2-phenoxy)phenyl-1,3,4-oxadiazole derivatives (6a-f) were designed. Energy minima conformers of the designed compounds and diazepam were well matched in conformational analysis and showed proper interaction with the BZD-binding site of the GABAA receptor model (α1β2ϒ2) in docking studies. The designed compounds were synthesized in acceptable yield and evaluated for their in vitro affinity to the benzodiazepine receptor of rat brains by radioligand receptor binding assay. The results demonstrated that the affinities of most of the novel compounds were even higher than diazepam. The novel compound 6a with the best affinity in radioligand receptor binding assay (Ki=0.44 nM and IC50= 0.73±0.17 nM) had considerable hypnotic activity and weak anticonvulsant and anxiolytic effects with no negative effect on memory in animal models. Flumazenil as a selective benzodiazepine receptor antagonist was able to prevent hypnotic and anticonvulsant effects of 6a indicating the role of BZD receptors in these effects. [ABSTRACT FROM AUTHOR]

Cardiovascular diseases remain one of the leading causes of death worldwide. Unfortunately, the available pharmacotherapeutic options have limited effectiveness. Therefore, developing new drug candidates remains very important. We selected six novel arylpiperazine alkyl derivatives of salicylamide to investigate their cardiovascular effects. Having in mind the beneficial role of α1-adrenergic receptors in restoring sinus rhythm and regulating blood pressure, first, using radioligand binding assays, we evaluated the affinity of the tested compounds for α-adrenergic receptors. Our experiments revealed their high to moderate affinity for α1- but not α2-adrenoceptors. Next, we aimed to determine the antiarrhythmic potential of novel derivatives in rat models of arrhythmia induced by adrenaline, calcium chloride, or aconitine. All compounds showed potent prophylactic antiarrhythmic activity in the adrenaline-induced arrhythmia model and no effects in calcium chloride- or aconitine-induced arrhythmias. Moreover, the tested compounds demonstrated therapeutic antiarrhythmic activity, restoring a normal sinus rhythm immediately after the administration of the arrhythmogen adrenaline. Notably, none of the tested derivatives affected the normal electrocardiogram (ECG) parameters in rodents, which excludes their proarrhythmic potential. Finally, all tested compounds decreased blood pressure in normotensive rats and reversed the pressor response to methoxamine, suggesting that their hypotensive mechanism of action is connected with the blockade of α1-adrenoceptors. Our results confirm the antiarrhythmic and hypotensive activities of novel arylpiperazine derivatives and encourage their further investigation as model structures for potential drugs. [ABSTRACT FROM AUTHOR]

Nicotinic acetylcholine receptors (nAChRs) play an important role in the functioning of the central and peripheral nervous systems, and other organs of living creatures. There are several subtypes of nAChRs, and almost all of them are considered as pharmacological targets in different pathological states. The crude venom of the sea anemone Metridium senile showed the ability to interact with nAChRs. Four novel peptides (Ms11a-1–Ms11a-4) with nAChR binding activity were isolated. These peptides stabilized by three disulfide bridges have no noticeable homology with any known peptides. Ms11a-1–Ms11a-4 showed different binding activity towards the muscle-type nAChR from the Torpedo californica ray. The study of functional activity and selectivity for the most potent peptide (Ms11a-3) revealed the highest antagonism towards the heterologous rat α9α10 nAChR compared to the muscle and α7 receptors. Structural NMR analysis of two toxins (Ms11a-2 and Ms11a-3) showed that they belong to a new variant of the inhibitor cystine knot (ICK) fold but have a prolonged loop between the fifth and sixth cysteine residues. Peptides Ms11a-1–Ms11a-4 could represent new pharmacological tools since they have structures different from other known nAChRs inhibitors. [ABSTRACT FROM AUTHOR]

The screened compound DYT-1 from our in-house library was taken as a lead (inhibiting tubulin polymerisation: IC50=25.6 µM, anti-angiogenesis in Zebrafish: IC50=38.4 µM, anti-proliferation against K562 and Jurkat: IC50=6.2 and 7.9 µM, respectively). Further investigation of medicinal chemistry conditions yielded compound 29e (inhibiting tubulin polymerisation: IC50=4.8 µM and anti-angiogenesis in Zebrafish: IC50=3.6 µM) based on tubulin and zebrafish assays, which displayed noteworthily nanomolar potency against a variety of leukaemia cell lines (IC50= 0.09-1.22 µM), especially K562 cells where apoptosis was induced. Molecular docking, molecular dynamics (MD) simulation, radioligand binding assay and cellular microtubule networks disruption results showed that 29e stably binds to the tubulin colchicine site. 29e significantly inhibited HUVEC tube formation, migration and invasion in vitro. Anti-angiogenesis in vivo was confirmed by zebrafish xenograft. 29e also prominently blocked K562 cell proliferation and metastasis in blood vessels and surrounding tissues of the zebrafish xenograft model. Together with promising physicochemical property and metabolic stability, 29e could be considered an effective anti-angiogenesis and -leukaemia drug candidate that binds to the tubulin colchicine site. [ABSTRACT FROM AUTHOR]

P2X4 receptors are ATP-gated cation channels that were proposed as novel drug targets due to their role in inflammation and neuropathic pain. Only few potent and selective P2X4 receptor antagonists have been described to date. Labeled tool compounds suitable for P2X4 receptor binding studies are lacking. Here, we present a novel allosteric P2X4 receptor antagonist possessing high potency in the low nanomolar range. We describe its tritium-labeling resulting in the P2X4-selective radiotracer [ 3 H]PSB-OR-2020 with high specific activity (45 Ci/mmol; 1.67 TBq/mmol). A radioligand binding assay was developed using human embryonic kidney (HEK293) cell membranes recombinantly expressing the human P2X4 receptor. Competition binding studies with structurally diverse P2X4 receptor antagonists revealed different allosteric binding sites indicating that the new class of P2X4 receptor antagonists, to which PSB-OR-2020 belongs, interacts with an unprecedented allosteric site. [ 3 H]PSB-OR-2020 may become a useful tool for research on P2X4 receptors and for promoting drug development., Competing Interests: Declarations Ethical approval Not applicable. Competing interests The authors filed a patent on the new class of P2X4 receptor antagonists including the radioligand described in this study., (© 2024. The Author(s).)

Background: Phosphodiesterase 10A (PDE10A) is expressed almost exclusively in the striatum and its inhibition is suggested to offer potential treatment in disorders associated with basal ganglia. We evaluated the selectivity, cytotoxicity, genotoxicity, pharmacokinetics and potential adverse effects of a novel PDE10A inhibitor, CPL500036, in vivo. Methods: The potency of CPL500036 was demonstrated by microfluidic technology, and selectivity was investigated in a radioligand binding assay against 44 targets. Cardiotoxicity in vitro was evaluated in human ether-a-go-go related gene (hERG)-potassium channel-overexpressing cells by the patch-clamp method and by assessing key parameters in 3D cardiac spheroids. Cytotoxicity was determined in H1299, HepG2 and SH-SY5Y cell lines. The Ames test was used for genotoxicity analyses. During in vivo studies, CPL500036 was administered by oral gavage. CPL500036 exposure were determined by liquid chromatography--tandem mass spectrometry and plasma protein binding was assessed. The bar test was employed to assess catalepsy. Prolactin and glucose levels in rat blood were measured by ELISAs and glucometers, respectively. Cardiovascular safety in vivo was investigated in dogs using a telemetry method. Results: CPL500036 inhibited PDE10A at an IC50 of 1 nM, and interacted only with the muscarinic M2 receptor as a negative allosteric modulator with an IC50 of 9.2 µM. Despite inhibiting hERG tail current at an IC25 of 3.2 μM, cardiovascular adverse effects were not observed in human cardiac 3D spheroids or in vivo. Cytotoxicity in vitro was observed only at > 60 μM and genotoxicity was not recorded during the Ames test. CPL500036 presented good bioavailability and penetration into the brain. CPL500036 elicited catalepsy at 0.6 mg/kg, but hyperprolactinemia or hyperglycemic effects were not observed in doses up to 3 mg/kg. Conclusion: CPL500036 is a potent, selective and orally bioavailable PDE10A inhibitor with a good safety profile distinct from marketed antipsychotics. CPL500036 may be a compelling drug candidate. [ABSTRACT FROM AUTHOR]

Purpose: The radiolabeled gastrin-releasing peptide receptor (GRPR)-targeting antagonist NeoB is a promising radioligand for imaging and therapy of GRPR-expressing malignancies. In the current study, we aimed to discover the target organs of toxicity and the radiotoxic effects to these organs, when repeated dosages of [177Lu]Lu-NeoB are administered to healthy female and male mice. Methods: Animals received either 3 injections, with a 7-day interval, of vehicle (control group 1), 1200 pmol [175Lu]Lu-NeoB (control group 2) or 40 MBq/400 pmol, 80 MBq/800 pmol, and 120 MBq/1200 pmol [177Lu]Lu-NeoB (treatment groups 1, 2, and 3, respectively). At week 5, 19, and 43 after the first injection acute, early, and late organ toxicity, respectively, was determined. For this, histopathological and blood analyses were performed. To correlate the observed toxicity to absorbed dose, we also performed extensive biodistribution and dosimetry studies. Results: The biodistribution study showed the highest absorbed doses in GRPR-expressing pancreas, the liver, and the kidneys (the main organs of excretion). Both control groups and almost all animals of treatment group 1 did not show any treatment-related toxicological effects. Despite the high absorbed doses, no clear microscopic signs of toxicity were found in the pancreas and the liver. Histological analysis indicated kidney damage in the form of hydronephrosis and nephropathy in treatment groups 2 and 3 that were sacrificed at the early and late time point. In the same groups, increased blood urea nitrogen levels were found. Conclusion: In general, repeated administration of [177Lu]Lu-NeoB was tolerated. The most significant radiotoxic effects were found in the kidneys, similar to other clinically applied radioligands. The results of this study underline the potential of [177Lu]Lu-NeoB as a promising option for clinical therapy. [ABSTRACT FROM AUTHOR]

Introduction: In men with metastatic castration-resistant prostate cancer (mCRPC) scheduled for prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT), biochemical response is assessed based on repeated measurements of prostate-specific antigen (PSA) levels. We aimed to determine overall survival (OS) in patients experiencing sustained PSA increase, decrease, or fluctuations during therapy. Materials and methods: In this bicentric study, we included 176 mCRPC patients treated with PSMA-directed RLT. PSA levels were determined using blood samples prior to the first RLT and on the admission days for the following cycles. We calculated relative changes in PSA levels compared to baseline. Kaplan–Meier curves as well as log-rank test were used to compare OS of different subgroups, including patients with sustained PSA increase, decrease, or fluctuations (defined as change after initial decrease or increase after the first cycle). Results: Sixty-one out of one hundred seventy-six (34.7%) patients showed a sustained increase and 86/176 (48.8%) a sustained decrease in PSA levels. PSA fluctuations were observed in the remaining 29/176 (16.5%). In this subgroup, 22/29 experienced initial PSA decrease followed by an increase (7/29, initial increase followed by a decrease). Median OS of patients with sustained decrease in PSA levels was significantly longer when compared to patients with sustained increase of PSA levels (19 vs. 8 months; HR 0.35, 95% CI 0.22–0.56; P < 0.001). Patients with PSA fluctuations showed a significantly longer median OS compared to patients with sustained increase of PSA levels (18 vs. 8 months; HR 0.49, 95% CI 0.30–0.80; P < 0.01), but no significant difference relative to men with sustained PSA decrease (18 vs. 19 months; HR 1.4, 95% CI 0.78–2.49; P = 0.20). In addition, in men experiencing PSA fluctuations, median OS did not differ significantly between patients with initial decrease or initial increase of tumor marker levels (16 vs. 18 months; HR 1.2, 95% CI 0.38–4.05; P = 0.68). Conclusion: Initial increase or decrease of PSA levels is sustained in the majority of patients undergoing RLT. Sustained PSA decrease was linked to prolonged survival and men with PSA fluctuations under treatment experienced comparable survival benefits. As such, transient tumor marker oscillations under RLT should rather not lead to treatment discontinuation, especially in the absence of radiological progression. [ABSTRACT FROM AUTHOR]

Ecdysone agonists are a class of insecticides that activate the ecdysone receptor (EcR) heterodimerized with the ultraspiracle (USP). Here, we report a new luciferase reporter assay for ecdysone agonists. The assay employs mammalian HEK293T cells transiently transfected with the EcR and USP genes of Chilo suppressalis, along with the taiman (Tai) gene of Drosophila melanogaster that encodes a steroid receptor coactivator. This assay system gave results consistent with those of radioligand binding assays and showed sensitivity superior to that of the existing in vitro methods. In addition, use of the heterologous host cells precludes perturbation from intrinsic players of the ecdysone signaling, which is a potential drawback of insect cell-based methods. This reporter system is suitable for detailed structure-activity analysis of ecdysone agonists and will serve as a valuable tool for the rational design of novel insect growth regulators. [ABSTRACT FROM AUTHOR]

An editorial is presented which argues Prostate-specific membrane antigen is a type II transmembrane protein highly expressed in almost all stages of prostate cancer (>90%). It also discusses that PSMA-targeted radionuclide imaging is now signifcantly impacting clinical decisionmaking in about 54 percent of patients.

In this study, we propose a method for the separate determination of both types of adrenergic receptors based on radioligand binding analysis using 125I-iodocyanopindolol, comprising three measurements: (1) without competing ligands, (2) in the presence of selective ligand ICI 118,551 (0.25 μM), and (3) in the presence of two selective ligands, ICI 118,551 and CGP 20712 (0.25 μM each). The technique was tested on a model system of two transgenic cell lines ADL-7A and A2R9 with the expression of recombinant β1- and β2-adrenergic receptors. If the ratio of the number of β1-adrenergic receptors to β2-adrenergic receptors is 1 : 10, the measurement error is about 15%. Analysis of nine cell lines representing different types of blood cells showed the presence of β2-adrenergic receptors in Daudi, Raji, Dami, K-562, HL-60, U-937, and THP-1 cells and their absence in Jurkat and MOLT-4 cells. β1-Adrenergic receptors are reliably recorded only in THP-1 cells of monocytic origin. In other cell lines, with the exception of Dami, the number of β1-adrenergic receptors was found below the detection limit, estimated as 250 molecules per cell. Measurements performed on the peripheral blood mononuclear cells of seven healthy donors showed the presence of β2-adrenergic receptors in the range from 1000 to 2500 molecules per cell, while the content of β1-adrenergic receptors in all cases appeared to be on the border or beyond the detection limit. [ABSTRACT FROM AUTHOR]

Synthetic cannabinoid receptor agonists (SCRAs) continue to make up a significant portion new psychoactive substances (NPS) detected and seized worldwide. Due to their often potent activation of central cannabinoid receptors in vivo, use of SCRAs can result in severe intoxication, in addition to other adverse health effects. Recent detections of AB-4CN-BUTICA, MMB-4CN-BUTINACA, MDMB-4F-BUTICA and MDMB-4F-BUTINACA mark a continuation in the appearance of SCRAs bearing novel tail substituents. The proactive characterization campaign described here has facilitated the detection of several new SCRAs in toxicological case work. Here we detail the synthesis, characterization, and pharmacological evaluation of recently detected SCRAs, as well as a systematic library of 32 compounds bearing head, tail, and core group combinations likely to appear in future. In vitro radioligand binding assays revealed most compounds showed moderate to high affnity at both CB1 (pKi = < 5 to 8.89 ± 0.09M) and CB2 (pKi = 5.49 ± 0.03 to 9.92 ± 0.09M) receptors. In vitro functional evaluation using a fluorescence-based membrane potential assay showed that most compounds were sub-micromolar to sub-nanomolar agonists at CB1 (pEC50 = < 5 to 9.48 ± 0.14M) and CB2 (pEC50 = 5.92 ± 0.16 to 8.64 ± 0.15M) receptors. An in silico receptor-ligand docking approach was utilized to rationalize binding trends for CB2 with respect to the tail substituent, and indicated that rigidity in this region (i.e., 4-cyanobutyl) was detrimental to affnity. [ABSTRACT FROM AUTHOR]

Background: Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen. Results: Here we generated highly soluble and stable recombinant protein constructs GST-CB1414–472 and GST-CB1414-442 containing much of the human CB1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443–473 of the mouse CB1 receptor that corresponds to residues 442–472 in the human homolog. Estimated values of CB1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay. Conclusions: Collectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints. [ABSTRACT FROM AUTHOR]

Matuzumab and nimotuzumab are anti-EGFR monoclonal antibodies that bind to different epitopes of domain III of EGFR. We developed 89Zr-matuzumab as a PET probe for diagnosis/monitoring of response to treatment of a noncompeting anti-EGFR nimotuzumab antibody drug conjugate (ADC) using mouse colorectal cancer (CRC) xenografts. We developed 89Zr-matuzumab and performed quality control in EGFR-positive DLD-1 cells. The KD of matuzumab, DFO-matuzumab and 89Zr-matuzumab in DLD-1 cells was 5.9, 6.2 and 3 nM, respectively. A competitive radioligand binding assay showed that 89Zr-matuzumab and nimotuzumab bound to noncompeting epitopes of EGFR. MicroPET/CT imaging and biodistribution of 89Zr-matuzumab in mice bearing EGFR-positive xenografts (HT29, DLD-1 and MDA-MB-231) showed high uptake that was blocked with pre-dosing with matuzumab but not with the noncompeting binder nimotuzumab. We evaluated nimotuzumab-PEG6-DM1 ADC in CRC cells. IC50 of nimotuzumab-PEG6-DM1 in SNU-C2B, DLD-1 and SW620 cells was dependent on EGFR density and was up to five-fold lower than that of naked nimotuzumab. Mice bearing the SNU-C2B xenograft were treated using three 15 mg/kg doses of nimotuzumab-PEG6-DM1, and 89Zr-matuzumab microPET/CT was used to monitor the response to treatment. Treatment resulted in complete remission of the SNU-C2B tumor in 2/3 mice. Matuzumab and nimotuzumab are noncompeting and can be used simultaneously. [ABSTRACT FROM AUTHOR]

The design and development of analgesics with mixed-opioid receptor interactions has been reported to decrease side effects, minimizing respiratory depression and reinforcing properties to generate safer analgesic therapeutics. We synthesized bis-cyclic guanidine heterocyclic peptidomimetics from reduced tripeptides. In vitro screening with radioligand competition binding assays demonstrated variable affinity for the mu-opioid receptor (MOR), delta-opioid receptor (DOR), and kappa-opioid receptor (KOR) across the series, with compound 1968-22 displaying good affinity for all three receptors. Central intracerebroventricular (i.c.v.) administration of 1968-22 produced dose-dependent, opioid receptor-mediated antinociception in the mouse 55 °C warm-water tail-withdrawal assay, and 1968-22 also produced significant antinociception up to 80 min after oral administration (10 mg/kg, p.o.). Compound 1968-22 was detected in the brain 5 min after intravenous administration and was shown to be stable in the blood for at least 30 min. Central administration of 1968-22 did not produce significant respiratory depression, locomotor effects or conditioned place preference or aversion. The data suggest these bis-cyclic guanidine heterocyclic peptidomimetics with multifunctional opioid receptor activity may hold potential as new analgesics with fewer liabilities of use. [ABSTRACT FROM AUTHOR]

In the brain, microglia continuously scan the surrounding extracellular space in order to respond to damage or infection by becoming activated and participating in neuroinflammation. When activated, microglia increase the expression of translocator protein (TSPO) 18 kDa, thereby making the TSPO expression a marker for neuroinflammation. We used the radiotracer [11C]DAA1106 (a ligand for TSPO) and positron emission tomography (PET) to determine the effect of smoking on availability of this marker for neuroinflammation. Forty-five participants (30 smokers and 15 non-smokers) completed the study and had usable data. Participants underwent a dynamic PET scanning session with bolus injection of [11C]DAA1106 (with smokers in the satiated state) and blood draws during PET scanning to determine TSPO affinity genotype and plasma nicotine levels. Whole-brain standardized uptake values (SUVs) were determined, and analysis of variance was performed, with group (smoker vs non-smoker) and genotype as factors, thereby controlling for genotype. Smokers and non-smokers differed in whole-brain SUVs (P=0.006) owing to smokers having 16.8% lower values than non-smokers. The groups did not differ in injected radiotracer dose or body weight, which were used to calculate SUV. An inverse association was found between whole-brain SUV and reported cigarettes per day (P

Bisphenol A (BPA) and its brominated derivative tetrabromobisphenol A (TBBPA) are high production volume chemicals used in the manufacture of various consumer products. Although regarded as endocrine disruptors, these chemicals are suspected to exert nongenomic actions on muscle function that are not well understood. Using skeletal muscle microsomes, we examined the effects of BPA and TBBPA on ryanodine receptor type 1 (RyR1), dihydropyridine receptor (DHPR), and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA). We assessed the impact of these chemicals on Ca2+ dynamics and signaling in embryonic skeletal myotubes through fluorescent Ca2+ imaging and measurement of resting membrane potential (Vm). TBBPA activated RyR1 and inhibited DHPR and SERCA, inducing a net efflux of Ca2+ from loaded microsomes, whereas BPA exhibited little or no activity at these targets. Regardless, both compounds disrupted the function of intact myotubes. TBBPA diminished and eventually abrogated Ca2+ transients, altered intracellular Ca2+ equilibrium, and caused Vm depolarization. For some cells, BPA caused rapid Ca2+ transient loss without marked changes in cytosolic and sarcoplasmic reticulum Ca2+ levels, likely owing to altered cellular excitability as a result of BPA-induced Vm hyperpolarization. BPA and TBBPA both interfere with skeletal muscle function through divergent mechanisms that impair excitation-contraction coupling and may be exemplary of their adverse outcomes in other muscle types.

[Display omitted] • Halogenated indoles showed high efficacy and potency in activating AhR. • Lead compounds act as AhR ligands, promoting AhR-ARNT heterodimer formation and enhancing CYP1A1 promoter binding. • 4-FI and 7-FI demonstrated anti-inflammatory effects in Caco-2 cell models, suggesting their therapeutic potential. The aryl hydrocarbon receptor (AhR) is a cytosolic ligand-activated transcription factor integral to various physiological and pathological processes. Among its diverse ligands, indole-based compounds have garnered attention due to their significant biological activity and potential therapeutic applications. This study explores the activation of AhR by structurally diverse halogenated indoles. We evaluated the transcriptional activity of AhR and cell viability in the human LS174T-AhR-luc reporter cell line. Among the tested compounds, 4-FI, 7-FI, 6-BrI, 7-BrI, 6-Cl-2-ox, 5-Br-2-ox, and 6-Br-2-ox activated AhR in a concentration-dependent manner, displaying high efficacy and potency. Molecular docking analysis revealed moderate binding affinities of these compounds to the PAS-B domain of AhR, corroborated by competitive radioligand binding assays. Functional assays showed that halogenated indoles induce the formation of AhR-ARNT heterodimer and enhance the binding of the AhR to the CYP1A1 promoter. Additionally, 4-FI and 7-FI exhibited anti-inflammatory properties in Caco-2 cell models, highlighting their potential for therapeutic applications. This study underscores the significance of the type and position of halogen moiety in indole scaffold, suggesting their potential as candidates for developing therapeutics drugs to treat conditions such as inflammatory bowel disease via AhR activation. [ABSTRACT FROM AUTHOR]

Here we assess whether neuropathic pain hypersensitivity is attenuated by spinal administration of the imidazoline I 1 -receptor agonist LNP599 and whether the attenuation involves co-activation of α 2 -adrenoceptors. Spared nerve injury (SNI) model of neuropathy was used to induce mechanical hypersensitivity in male and female rats with a chronic catheter for intrathecal drug administrations. Mechanical sensitivity and heat nociception were assessed behaviorally in the injured limb. Additionally, GTPγS radioligand binding assay, β-arrestin recruitment and intracellular cAMP levels were used for receptor profiling in vitro. LNP599 (imidazoline I 1 receptor agonist) and clonidine (α 2 -adrenoceptor agonist) produced equal dose-related mechanical antihypersensitivity effects in both sexes. LNP599 attenuated heat nociception preferentially in males, while clonidine reduced heat nociception equally in males and females. Carbophenyline (another imidazoline I 1 receptor agonist) had no significant effect on mechanical hypersensitivity or heat nociception in males or females. Mechanical antihypersensitivity and heat antinociception induced by LNP599 in SNI males was prevented by pretreatments with yohimbine or atipamezole (two α 2 -adrenoceptor antagonists) but not by efaroxan (a mixed imidazoline I 1 receptor/α 2 -adrenoceptor antagonist). In vitro assays indicated that LNP599 does not activate α 2A - or other subtypes of α 2 -adrenoceptors. However, LNP599 was a weak partial agonist for 5-HT 2B receptors and bound to sigma-1 and sigma-2 receptors that all are involved in modulation of spinal nociception. The results indicate that the suppression of neuropathic pain hypersensitivity by LNP599 is not due to action on spinal imidazoline I 1 receptors, but rather due to indirect activation of spinal α 2 -adrenoceptors. • Spinally administered imidazoline I 1 receptor ligand LNP599 reduced neuropathic pain. • LNP599-induced pain reduction was reversed by α 2 -adrenoceptor (AR) antagonists. • LNP599-effect was not reversed by I 1 block or recapitulated by another I 1 agonist. • LNP599 did not directly activate α 2 -AR but bound to sigma-1/2 and 5-HT 2B receptors. • Spinal LNP599 reduces neuropathic pain due to indirect activation of α 2 -AR. [ABSTRACT FROM AUTHOR]

Background: In contrast to the thyroid hormones (THs) 3,3′,5-triiodothyronine (T3) and 3,3′,5,5′-tetraiodothyronine (thyroxine or T4), the binding characteristics of the thyroid hormone distributor proteins (THDP), thyroxine-binding globulin (TBG), albumin, and transthyretin in relation to TH metabolites are mostly lacking. In this study, we determined the distribution and binding affinity of TH metabolites to THDP, which is important for adequate interpretation of TH metabolite concentrations. Methods: Distribution of 125I-3,3′-diiodothyronine (3,3′-T2), -T3, -3,3′,5′-triiodothyronine (rT3), -3,3′,5-triiodothyroacetic acid (TA3), and -3,3′,5,5′-tetraiodothyroacetic acid (TA4) to TBG, transthyretin, and albumin was determined by agar gel electrophoresis. The rank order of affinity (IC50) of TBG and transthyretin to thyronine (T0), 3-monoiodothyronine (3-T1), 3,5-diiodothyronine (3,5-T2), 3,3′-T2, T3, rT3, T4, TA3, and TA4 was determined with a radioligand, competitive binding assay. In healthy subjects, associations of serum TBG, transthyretin, and albumin with TH and its metabolites were analyzed using multiple linear regression models, adjusted for sex and age. Results: While T3 and T4 are predominantly bound to TBG, we demonstrated that the predominant THDP of 3,3′-T2 and rT3 is albumin, of TA3 is transthyretin and albumin, and of TA4 is transthyretin. With the radioligand binding assay, we showed that the rank order of affinity was T4>TA4 = rT3>T3>TA3 = 3,3′-T2 > 3-T1 = 3,5-T2>T0 for TBG (IC50-range: 0.36 nM to >100 μM) and TA4>T4 = TA3>rT3>T3 > 3,3′-T2 > 3-T1 > 3,5-T2>T0 for transthyretin (IC50-range: 0.94 nM to >100 μM). TBG, transthyretin, and albumin were not associated with T0, 3-T1, 3,3′-T2, rT3, and TA4. Conclusions: Differences in serum TBG, transthyretin, and albumin concentrations within the reference interval do not influence serum concentrations of T0, 3-T1, 3,3′-T2, rT3, and TA4. Distribution of TH metabolites between THDP differs from T4 and T3, which predominantly bind to TBG. The results from our study have potential clinical importance for adequate interpretation of TH metabolism in (patho)physiology. [ABSTRACT FROM AUTHOR]

Excessive Ca2+ currents via N-methyl-D-aspartate receptors (NMDARs) have been implicated in many disorders. Uncompetitive NMDAR channel blockers are an emerging class of drugs in clinical use for major depressive disorder (MDD) and other neuropsychiatric diseases. The pharmacological characterization of uncompetitive NMDAR blockers in clinical use may improve our understanding of NMDAR function in physiology and pathology. REL-1017 (esmethadone-HCl), a novel uncompetitive NMDAR channel blocker in Phase 3 trials for the treatment of MDD, was characterized together with dextromethorphan, memantine, (±)-ketamine, and MK-801 in cell lines over-expressing NMDAR subtypes using fluorometric imaging plate reader (FLIPR), automated patch-clamp, and manual patch-clamp electrophysiology. In the absence of Mg2+, NMDAR subtypes NR1-2D were most sensitive to low, sub-μM glutamate concentrations in FLIPR experiments. FLIPR Ca2+ determination demonstrated low μM affinity of REL-1017 at NMDARs with minimal subtype preference. In automated and manual patch-clamp electrophysiological experiments, REL-1017 exhibited preference for the NR1-2D NMDAR subtype in the presence of 1 mM Mg2+ and 1 μM L-glutamate. Tau off and trapping characteristics were similar for (±)-ketamine and REL-1017. Results of radioligand binding assays in rat cortical neurons correlated with the estimated affinities obtained in FLIPR assays and in automated and manual patch-clamp assays. In silico studies of NMDARs in closed and open conformation indicate that REL-1017 has a higher preference for docking and undocking the open-channel conformation compared to ketamine. In conclusion, the pharmacological characteristics of REL-1017 at NMDARs, including relatively low affinity at the NMDAR, NR1-2D subtype preference in the presence of 1 mM Mg2+, tau off and degree of trapping similar to (±)-ketamine, and preferential docking and undocking of the open NMDAR, could all be important variables for understanding the rapid-onset antidepressant effects of REL-1017 without psychotomimetic side effects. [ABSTRACT FROM AUTHOR]

A series of σ2R compounds containing benzimidazolone and diazacycloalkane cores was synthesized and evaluated in radioligand binding assays. Replacing the piperazine moiety in a lead compound with diazaspiroalkanes and the fused octahydropyrrolo[3,4-b] pyrrole ring system resulted in a loss in affinity for the σ2R. On the other hand, the bridged 2,5-diazabicyclo[2.2.1]heptane, 1,4-diazepine, and a 3-aminoazetidine analog possessed nanomolar affinities for the σ2R. Computational chemistry studies were also conducted with the recently published crystal structure of the σ2R/TMEM97 and revealed that hydrogen bond interactions with ASP29 and π-stacking interactions with TYR150 were largely responsible for the high binding affinity of small molecules to this protein. [ABSTRACT FROM AUTHOR]

Conformational biosensors to monitor the activation state of G protein-coupled receptors are a useful addition to the molecular pharmacology assay toolbox to characterize ligand efficacy at the level of receptor proteins instead of downstream signaling. We recently reported the initial characterization of a NanoBRET-based conformational histamine H3 receptor (H3R) biosensor that allowed the detection of both (partial) agonism and inverse agonism on living cells in a microplate reader assay format upon stimulation with H3R ligands. In the current study, we have further characterized this H3R biosensor on intact cells by monitoring the effect of consecutive ligand injections in time and evaluating its compatibility with photopharmacological ligands that contain a light-sensitive azobenzene moiety for photo-switching. In addition, we have validated the H3R biosensor in membrane preparations and found that observed potency values better correlated with binding affinity values that were measured in radioligand competition binding assays on membranes. Hence, the H3R conformational biosensor in membranes might be a ready-to-use, high-throughput alternative for radioligand binding assays that in addition can also detect ligand efficacies with comparable values as the intact cell assay. [ABSTRACT FROM AUTHOR]

Cepharanthine is an alkaloid that isolated from Stephania cepharantha Hayata, however，its analgesic properties are unclear and the molecular targets that mediating Cepharanthine-induced analgesia are not explored yet. In the current study, mice pain models including hot plate, acetic acid-induced writhing and formalin tests were conducted to evaluate the antinociceptive actions of Cepharanthine. [3H]-ligand competitive binding assay was applied to determine the binding affinity and selectivity of Cepharanthine at κ, μ and δ opioid receptors. Cepharanthine-induced constipation was investigated using the small intestinal transit test. The results showed that intraperitoneal injection of Cepharanthine produced potent antinociception with an ED 50 value of 24.5 mg/kg in the acetic acid-induced writhing test. In the formalin test, Cepharanthine produced moderate antinociception with the maximum analgesic activity of 42.6 ± 11.3% in phase I and 60.1 ± 7.7% in phase Ⅱ, respectively. Cepharanthine had no effects in the hot plate test. In vitro radioligand binding assay, Cepharanthine exhibited a high affinity for μ opioid receptors with a Ki value of 80 nM, without binding to κ and δ opioid receptors. Correspondingly, Cepharanthine-mediated antinociceptive effects were antagonized by pretreatment with opioid receptor antagonist naloxone. Cepharanthine also decreased the small intestine propulsion rates in the small intestinal transit test. Together, this study firstly demonstrates that Cepharanthine produces potent antinociception in acetic acid-induced visceral pain and moderate antinociception in formalin-induced inflammatory pain, and its mechanism of action may be through activation of μ opioid receptors. • Cepharanthine produces potent antinociception in visceral pain and moderate antinociception in inflammatory pain. • Cepharanthine produces antinociception via acting at μ opioid receptors. • Cepharanthine causes lower constipation in comparison with classical μ agonist morphine. [ABSTRACT FROM AUTHOR]

Purpose: Radioligand therapy (RLT) targeting prostate-specific membrane antigen (PSMA) is emerging as an effective treatment option for metastatic castration-resistant prostate cancer (mCRPC). An imaging-based method to quantify early treatment responses can help to understand and optimize RLT. Methods: We developed a self-triggered probe 2 targeting the colocalization of PSMA and caspase-3 for fluorescence imaging of RLT-induced apoptosis. Results: The probe binds to PSMA potently with a Ki of 4.12 nM, and its fluorescence can be effectively switched on by caspase-3 with a Km of 67.62 μM. Cellular and in vivo studies demonstrated its specificity for imaging radiation-induced caspase-3 upregulation in prostate cancer. To identify the detection limit of our method, we showed that probe 2 could achieve 1.79 times fluorescence enhancement in response to 177Lu-RLT in a medium PSMA-expressing 22Rv1 xenograft model. Conclusion: Probe 2 can potently bind to PSMA, and the fluorescence signal can be sensitively switched on by caspase-3 both in vitro and in vivo. This method may provide an effective tool to investigate and optimize PSMA-RLT. [ABSTRACT FROM AUTHOR]

Purpose: P2X7 receptors have been considered as a promising biomarker for vulnerable atherosclerotic plaques, which are highly expressed by that instability-associated factors such as macrophages. Thus, we aim to investigate the feasibility of using specific P2X7-targeted 18F-labeled tracer 18F-FTTM ((2-chloro-3-[18F]fluorophenyl)[1,4,6,7-tetrahydro-1-(2-pyrimidinyl)-5H-1,2,3-triazolo[4,5-c]pyridin-5-yl]methanone) for PET study of vulnerable atherosclerotic plaques identification. Method: The radioligand 18F-FTTM was achieved based on the copper-mediated radiofluorination of arylstannane. In vitro and in vivo experiments were performed to verify the biochemical properties. Dynamic 18F-FTTM Micro-PET/CT imaging was performed for 1 h on ApoE−/− mice (10, 20, 30 weeks on high-fat diet) and wild-type C57BL/6 J mice on normal diet. Ex vivo PET imaging was conducted to verify the specificity of the radioligand. Serum inflammatory cytokines, lipids, and lipoproteins profiles were detected by ELISA. The lipid distribution and morphology of plaques were evaluated by Oil Red O, HE, Masson, and immunofluorescence stainings. Results: 18F-FTTM was afforded with decay-corrected radiochemical yields of 5–10%, specific activity of 269–320 MBq/nmol (n = 8, EOS), and radiochemical purity of above 99%. 18F-FTTM showed excellent stability in vitro, rapid blood clearance in mice, good affinity to RAW264.7 cells. We observed an increase in both in vivo and ex vivo imagings as disease progressed, and the imaging signatures correlated with histopathological features. Furthermore, compared with 18F-FDG imaging, the SUVmax values of 18F-FTTM at the aortic arch of ApoE−/− mice of high-fat feeding for 20 and 30 weeks were 43% and 53% higher than those of the control group, respectively. Conclusion: We innovatively apply a new type P2X7-targeted PET probe (18F-FTTM) to identify vulnerable atherosclerotic plaques, to detect the inflammatory response of atherosclerosis, and to provide a powerful non-invasive method for the diagnosis of atherosclerotic lesions and new drug screening for accurate treatment. [ABSTRACT FROM AUTHOR]

Ozanimod, a sphingosine 1-phosphate (S1P) receptor modulator, binds with high affinity selectively to S1P receptor subtypes 1 (S1P1) and 5 (S1P5), and is approved in multiple countries for treating adults with relapsing forms of multiple sclerosis (MS) or moderately to severely active ulcerative colitis (UC). Other S1P receptor modulators have been approved for the treatment of MS or are in clinical development for MS or UC, but it is unknown whether these compounds bind competitively with each other to S1P1 or S1P5. We developed a competitive radioligand binding assay using tritiated ozanimod and demonstrate full displacement of ozanimod by S1P (endogenous ligand), suggesting that ozanimod binds to the S1P1 and S1P5 orthosteric binding sites. S1P receptor modulators FTY720-p, siponimod, etrasimod, ponesimod, KRP-203-p, and amiselimod-p also completely displacing radiolabeled ozanimod; thus, on a macroscopic level, all bind to the same site. Molecular docking studies support these results and predict the binding of each molecule to the orthosteric site of the receptors, creating similar interactions within S1P1 and S1P5. The absolute free energy perturbation method further validated key proposed binding modes. Functional potency tightly aligned with binding affinities across S1P1 and S1P5 and all compounds elicited S1P1-mediated β-arrestin recruitment. Since all the S1P modulators included in this study display similar receptor pharmacology and compete for binding at the same site, they can be considered interchangeable with one another. The choice of any one particular agent should therefore be made on the basis of overall therapeutic profile, and patients can be offered the opportunity to switch S1P medications without the potential concern of additive S1P pharmacology. [ABSTRACT FROM AUTHOR]

Kappa-opioid receptor (KOR) antagonists are promising innovative therapeutics for the treatment of the central nervous system (CNS) disorders. The new scaffold opioid ligand, Compound A, was originally found as a mu-opioid receptor (MOR) antagonist but its binding/selectivity and activation profile at the KOR and delta-opioid receptor (DOR) remain elusive. In this study, we present an in vitro, in vivo and in silico characterization of Compound A by revealing this ligand as a KOR antagonist in vitro and in vivo. In the radioligand competitive binding assay, Compound A bound at the human KOR, albeit with moderate affinity, but with increased affinity than to the human MOR and without specific binding at the human DOR, thus displaying a preferential KOR selectivity profile. Following subcutaneous administration in mice, Compound A effectively reverse the antinociceptive effects of the prototypical KOR agonist, U50,488. In silico investigations were carried out to assess the structural determinants responsible for opioid receptor subtype selectivity of Compound A. Molecular docking, molecular dynamics simulations and dynamic pharmacophore (dynophore) generation revealed differences in the stabilization of the chlorophenyl moiety of Compound A within the opioid receptor binding pockets, rationalizing the experimentally determined binding affinity values. This new chemotype bears the potential for favorable ADMET properties and holds promise for chemical optimization toward the development of potential therapeutics. [ABSTRACT FROM AUTHOR]

Novel synthetic opioids continue to emerge on recreational drug markets worldwide. In response to legislative bans on fentanyl analogues, non-fentanyl structural templates, such as 2-benzylbenzimidazoles ('nitazenes'), are being exploited to create new μ-opioid receptor (MOR) agonists. Here, we pharmacologically characterize an emerging cyclic analogue of etonitazene, called N-pyrrolidino etonitazene (etonitazepyne), using in vitro and in vivo methods. A series of analytically confirmed fatalities is described to complement preclinical findings. Radioligand binding assays in rat brain tissue revealed that N-pyrrolidino etonitazene has high affinity for MOR (Ki = 4.09 nM) over δ-opioid (Ki = 959 nM) and κ-opioid (Ki = 980 nM) receptors. In a MOR-β-arrestin2 activation assay, N-pyrrolidino etonitazene displayed high potency (EC50 = 0.348 nM), similar to etonitazene (EC50 = 0.360 nM), and largely exceeding the potencies of fentanyl (EC50 = 14.9 nM) and morphine (EC50 = 290 nM). When administered s.c. to male Sprague Dawley rats, N-pyrrolidino etonitazene induced opioid-like antinociceptive, cataleptic, and thermic effects. Its potency in the hot plate test (ED50 = 0.0017 mg/kg) was tenfold and 2,000-fold greater than fentanyl (ED50 = 0.0209 mg/kg) and morphine (ED50 = 3.940 mg/kg), respectively. Twenty-one overdose fatalities associated with N-pyrrolidino etonitazene were found to contain low blood concentrations of the drug (median = 2.2 ng/mL), commonly in the context of polysubstance use. N-Pyrrolidino etonitazene was reported as a cause of death in at least two cases, demonstrating toxicity in humans. We demonstrate that N-pyrrolidino etonitazene is an extremely potent MOR agonist that is likely to present high risk to users. Continued vigilance is required to identify and characterize emergent 2-benzylbenzimidazoles, and other non-fentanyl opioids, as they appear in the marketplace. [ABSTRACT FROM AUTHOR]

N-Piperidinyl etonitazene ('etonitazepipne') represents a recent addition to the rapidly expanding class of 2-benzylbenzimidazole 'nitazene' opioids. Following its first identification in an online-sourced powder and in biological samples from a patient seeking help for detoxification, this report details its in-depth chemical analysis and pharmacological characterization. Analysis of the powder via different techniques (LC-HRMS, GC–MS, UHPLC-DAD, FT-IR) led to the unequivocal identification of N-piperidinyl etonitazene. Furthermore, we report the first activity-based detection and analytical identification of N-piperidinyl etonitazene in authentic samples. LC-HRMS analysis revealed concentrations of 1.21 ng/mL in serum and 0.51 ng/mL in urine, whereas molecular networking enabled the tentative identification of various (potentially active) urinary metabolites. In addition, we determined that the extent of opioid activity present in the patient's serum was equivalent to the in vitro opioid activity exerted by 2.5–10 ng/mL fentanyl or 10–25 ng/mL hydromorphone in serum. Radioligand binding assays in rat brain tissue revealed that the drug binds with high affinity (Ki = 14.3 nM) to the µ-opioid receptor (MOR). Using a MOR-β-arrestin2 activation assay, we found that N-piperidinyl etonitazene is highly potent (EC50 = 2.49 nM) and efficacious (Emax = 183% versus hydromorphone) in vitro. Pharmacodynamic evaluation in male Sprague Dawley rats showed that N-piperidinyl etonitazene induces opioid-like antinociceptive, cataleptic, and thermic effects, its potency in the hot plate assay (ED50 = 0.0205 mg/kg) being comparable to that of fentanyl (ED50 = 0.0209 mg/kg), and > 190 times higher than that of morphine (ED50 = 3.940 mg/kg). Taken together, our findings indicate that N-piperidinyl etonitazene is a potent opioid with the potential to cause harm in users. [ABSTRACT FROM AUTHOR]

Nondioxin-like polychlorinated biphenyls (NDL PCBs) activate ryanodine-sensitive Ca2+ channels (RyRs) and this activation has been associated with neurotoxicity in exposed animals. RyR-active congeners follow a distinct structure-activity relationship and a quantitative structure-activity relationship (QSAR) predicts that a large number of PCBs likely activate the receptor, which requires validation. Additionally, previous structural based conclusions have been established using receptor ligand binding assays but the impact of varying PCB structures on ion channel gating behavior is not understood. We used [3H]Ryanodine ([3H]Ry) binding to assess the RyR-activity of 14 previously untested PCB congeners evaluating the predictability of the QSAR. Congeners determined to display widely varying potency were then assayed with single channel voltage clamp analysis to assess direct influences on channel gating kinetics. The RyR-activity of individual PCBs assessed in in vitro assays followed the general pattern predicted by the QSAR but binding and lipid bilayer experiments demonstrated higher potency than predicted. Of the 49 congeners tested to date, tetra-ortho PCB 202 was found to be the most potent RyR-active congener increasing channel open probability at 200 pM. Shifting meta-substitutions to the para-position resulted in a > 100-fold reduction in potency as seen with PCB 197. Non-ortho PCB 11 was found to lack activity at the receptor supporting a minimum mono-ortho substitution for PCB RyR activity. These findings expand and support previous SAR assessments; where out of the 49 congeners tested to date 42 activate the receptor demonstrating that the RyR is a sensitive and common target of PCBs.

Design of ligands that provide receptor selectivity has emerged as a new paradigm for drug discovery of G protein-coupled receptors, and may, for certain families of receptors, only be achieved via identification of chemically diverse allosteric modulators. Here, the extracellular vestibule of the M2 muscarinic acetylcholine receptor (mAChR) is targeted for structure-based design of allosteric modulators. Accelerated molecular dynamics (aMD) simulations were performed to construct structural ensembles that account for the receptor flexibility. Compounds obtained from the National Cancer Institute (NCI) were docked to the receptor ensembles. Retrospective docking of known ligands showed that combining aMD simulations with Glide induced fit docking (IFD) provided much-improved enrichment factors, compared with the Glide virtual screening workflow. Glide IFD was thus applied in receptor ensemble docking, and 38 top-ranked NCI compounds were selected for experimental testing. In [(3)H]N-methylscopolamine radioligand dissociation assays, approximately half of the 38 lead compounds altered the radioligand dissociation rate, a hallmark of allosteric behavior. In further competition binding experiments, we identified 12 compounds with affinity of ≤30 μM. With final functional experiments on six selected compounds, we confirmed four of them as new negative allosteric modulators (NAMs) and one as positive allosteric modulator of agonist-mediated response at the M2 mAChR. Two of the NAMs showed subtype selectivity without significant effect at the M1 and M3 mAChRs. This study demonstrates an unprecedented successful structure-based approach to identify chemically diverse and selective GPCR allosteric modulators with outstanding potential for further structure-activity relationship studies.

The p75 neurotrophin receptor is important in multiple physiological actions including neuronal survival and neurite outgrowth during development, and after central nervous system injury. We have discovered a novel piperazine-derived compound, EVT901, which interferes with p75 neurotrophin receptor oligomerization through direct interaction with the first cysteine-rich domain of the extracellular region. Using ligand binding assays with cysteine-rich domains-fused p75 neurotrophin receptor, we confirmed that EVT901 interferes with oligomerization of full-length p75 neurotrophin receptor in a dose-dependent manner. Here we report that EVT901 reduces binding of pro-nerve growth factor to p75 neurotrophin receptor, blocks pro-nerve growth factor induced apoptosis in cells expressing p75 neurotrophin receptor, and enhances neurite outgrowth in vitro Furthermore, we demonstrate that EVT901 abrogates p75 neurotrophin receptor signalling by other ligands, such as prion peptide and amyloid-β. To test the efficacy of EVT901 in vivo, we evaluated the outcome in two models of traumatic brain injury. We generated controlled cortical impacts in adult rats. Using unbiased stereological analysis, we found that EVT901 delivered intravenously daily for 1 week after injury, reduced lesion size, protected cortical neurons and oligodendrocytes, and had a positive effect on neurological function. After lateral fluid percussion injury in adult rats, oral treatment with EVT901 reduced neuronal death in the hippocampus and thalamus, reduced long-term cognitive deficits, and reduced the occurrence of post-traumatic seizure activity. Together, these studies provide a new reagent for altering p75 neurotrophin receptor actions after injury and suggest that EVT901 may be useful in treatment of central nervous system trauma and other neurological disorders where p75 neurotrophin receptor signalling is affected.

BackgroundIndividuals who use methamphetamine chronically exhibit emotional and dopaminergic neurochemical deficits. Although the amygdala has an important role in emotion processing and receives dopaminergic innervation, little is known about how dopamine transmission in this region contributes to emotion regulation. This investigation aimed to evaluate emotion regulation in subjects who met DSM-IV criteria for methamphetamine dependence, and to test for a relationship between self-reports of difficulty in emotion regulation and D2-type dopamine receptor availability in the amygdala.MethodNinety-four methamphetamine-using and 102 healthy-control subjects completed the Difficulties in Emotion Regulation Scale (DERS); 33 of those who used methamphetamine completed the Addiction Severity Index (ASI). A subset of 27 methamphetamine-group and 20 control-group subjects completed positron emission tomography with [(18)F]fallypride to assay amygdala D2-type dopamine receptor availability, measured as binding potential (BPND).ResultsThe methamphetamine group scored higher than the control group on the DERS total score (p

The muscarinic acetylcholine receptor family is a highly sought-after target in drug and molecular imaging discovery efforts aimed at neurological disorders. Hampered by the structural similarity of the five subtypes' orthosteric binding pockets, these efforts largely failed to deliver subtype-selective ligands. Building on our recent successes with arecaidine-derived ligands targeting M1, herein we report the synthesis of a related series of 11 hydroxylated arecaidine esters. Their physicochemical property profiles, expressed in terms of their computationally calculated CNS MPO scores and HPLC-logD values, point towards blood–brain barrier permeability. By means of a competitive radioligand binding assay, the binding affinity values towards each of the individual human mAChR subtypes hM1–hM5 were determined. The most promising compound of this series 17b was shown to have a binding constant towards hM1 in the single-digit nanomolar region (5.5 nM). Similar to our previously reported arecaidine-derived esters, the entire series was shown to act as hM1R antagonists in a calcium flux assay. Overall, this study greatly expanded our understanding of this recurring scaffolds' structure–activity relationship and will guide the development towards highly selective mAChRs ligands. [ABSTRACT FROM AUTHOR]

The decriminalization and legalization of cannabis has paved the way for investigations into the potential of the use of phytocannabinoids (pCBs) as natural therapeutics for the treatment of human diseases. This growing interest has recently focused on rare (less abundant) pCBs that are non-psychotropic compounds, such as cannabigerol (CBG), cannabichromene (CBC), Δ9-tetrahydrocannabivarin (THCV) and cannabigerolic acid (CBGA). Notably, pCBs can act via the endocannabinoid system (ECS), which is involved in the regulation of key pathophysiological processes, and also in the skin. In this study, we used human keratinocytes (HaCaT cells) as an in vitro model that expresses all major ECS elements in order to systematically investigate the effects of CBG, CBC, THCV and CBGA. To this end, we analyzed the gene and protein expression of ECS components (receptors: CB1, CB2, GPR55, TRPV1 and PPARα/γ/δ; enzymes: NAPE-PLD, FAAH, DAGLα/β and MAGL) using qRT-PCR and Western blotting, along with assessments of their functionality using radioligand binding and activity assays. In addition, we quantified the content of endocannabinoid(-like) compounds (AEA, 2-AG, PEA, etc.) using UHPLC-MS/MS. Our results demonstrated that rare pCBs modulate the gene and protein expression of distinct ECS elements differently, as well as the content of endocannabinoid(-like) compounds. Notably, they all increased CB1/2 binding, TRPV1 channel stimulation and FAAH and MAGL catalytic activity. These unprecedented observations should be considered when exploring the therapeutic potential of cannabis extracts for the treatment of human skin diseases. [ABSTRACT FROM AUTHOR]

The μ-opioid receptors belong to the family of G protein-coupled receptors (GPCRs), and their activation triggers a cascade of intracellular relays with the final effect of analgesia. Classical agonists of this receptor, such as morphine, are the main targets in the treatment of both acute and chronic pain. However, the dangerous side effects, such as respiratory depression or addiction, significantly limit their widespread use. The allosteric centers of the receptors exhibit large structural diversity within particular types and even subtypes. Currently, a considerable interest is aroused by the modulation of μ-opioid receptors. The application of such a technique may result in a reduction in the dose or even discontinuation of classical opiates, thus eliminating the side effects typical of this class of drugs. Our aim is to obtain a series of 1-aryl-5,6(1H)dioxo-2,3-dihydroimidazo[1,2-a]imidazole derivatives and provide more information about their activity and selectivity on OP3 (MOP, human mu opioid receptor). The study was based on an observation that some carbonyl derivatives of 1-aryl-2-aminoimidazoline cooperate strongly with morphine or DAMGO in sub-threshold doses, producing similar results to those of normal active doses. To elucidate the possible mechanism of such enhancement, we performed a few in vitro functional tests (involving cAMP and β-arrestin recruitment) and a radioligand binding assay on CHO-K1 cells with the expression of the OP3 receptor. One of the compounds had no orthosteric affinity or intrinsic activity, but inhibited the efficiency of DAMGO. These results allow to conclude that this compound is a negative allosteric modulator (NAM) of the human μ-opioid receptor. [ABSTRACT FROM AUTHOR]

Rationale: Prolonged in vivo evaluation of PSMA tracers could improve tumor imaging and patient selection for 177Lu-PSMA-617 and 177Lu-PSMA-I&T. In this study, we present the radiolabeling method of PSMA-617 and PSMA-I&T with the long-lived positron emitter 89Zr to enable PET imaging up to 7 days post-injection. We compared the biodistribution of 89Zr-PSMA-617 and 89Zr-PSMA-I&T to those of 177Lu-PSMA-617 and 177Lu-PSMA-I&T, respectively, in a PSMA+ xenograft model. Moreover, we provide the first human 89Zr-PSMA-617 images. Materials and methods: PSMA ligands were labeled with 50-55 MBq [89Zr]ZrCl4 using a two-step labeling protocol. For biodistribution, BALB/c nude mice bearing PSMA+ and PSMA− xenografts received 0.6 µg (0.6–1 MBq) of 89Zr-PSMA-617, 89Zr-PSMA-I&T, 177Lu-PSMA-617, or 177Lu-PSMA-I&T intravenously. Ex vivo biodistribution and PET/SPECT imaging were performed up to 168 h post-injection. Dosimetry was performed from the biodistribution data. The patient received 90.5 MBq 89Zr-PSMA-617 followed by PET/CT imaging. Results: 89Zr-labeled PSMA ligands showed a comparable ex vivo biodistribution to its respective 177Lu-labeled counterparts with high tumor accumulation in the PSMA+ xenografts. However, using a dose estimation model for 177Lu, absorbed radiation dose in bone and kidneys differed among the 177Lu-PSMA and 89Zr-PSMA tracers. 89Zr-PSMA-617 PET in the first human patient showed high contrast of PSMA expressing tissues up to 48 h post-injection. Conclusion: PSMA-617 and PSMA-I&T were successfully labeled with 89Zr and demonstrated high uptake in PSMA+ xenografts, which enabled PET up to 168 h post-injection. The biodistribution of 89Zr-PSMA-I&T and 89Zr-PSMA-617 resembled that of 177Lu-PSMA-I&T and 177Lu-PSMA-617, respectively. The first patient 89Zr-PSMA-617 PET images were of high quality warranting further clinical investigation. [ABSTRACT FROM AUTHOR]

To probe the mechanism of dopamine receptors in drug addiction and look for potential new methods for treating this disease, we have designed and synthesized benzothiophene morpholine analogues that were considered as dopamine D3 receptor‐selective ligands. Radioligand binding assay was used to determine the binding affinity of target compounds. Members of this class have great selectivity and binding affinity in D3 receptor. In addition, the ability of these compounds to mitigate the symptoms of addiction from opioids was investigated in animal behavior patterns, and we have found that two compounds (18a and 18d) have good affinity in the D3R and exhibit the efficacy of anti‐drug addiction in morphine‐dependent mice induced by naloxone. [ABSTRACT FROM AUTHOR]

Objective: Transient receptor potential vanilloid subtype 1 (TRPV1) may play a significant role in the pathophysiology of the bladder. The present study investigated the effects of the herbal product, saw palmetto extract (SPE) on TRPV1‐mediated Ca2+ influx and specific [3H]resiniferatoxin ([3H]RTX) binding to TRPV1 in HEK293 cells expressing TRPV1 (HEK293VR11 cells). Methods: Ca2+ influx induced by and the direct binding activity of TRPV1 were measured using a method with Fura 2‐AM, a cytoplasmic calcium indicator, and a radioligand binding assay using a [3H]RTX, respectively. Results: SPE did not markedly affect Ca2+ influx in HEK293VR11 cells; however, it significantly inhibited capsaicin‐induced increases in Ca2+ influx in these cells. The specific binding of [3H]RTX in HEK293VR11 cells was saturable with Kd value of 120 ± 7 pM and Bmax of 1.07 ± 0.10 fmol/mg protein, and was inhibited by low concentrations of non‐labeled RTX with Ki of 60.1 ± 7.6 nM. These results confirmed the pharmacological specificity of specific binding sites of [3H]RTX to TRPV1 in HEK293VR11 cells. SPE inhibited the specific binding of [3H]RTX in a concentration‐dependent manner, with Ki of 24.2 ± 1.4 μg/mL. Conclusions: The present study demonstrated for the first time, that SPE inhibited capsaicin‐induced Ca2+ influx with binding to TRPV1 in HEL293VR11 cells. These results will contribute to a more detailed understanding of the pharmacological effects of SPE on urinary dysfunction. [ABSTRACT FROM AUTHOR]

The therapy of intoxication with distinct organophosphorus (OP) compounds is still limited today. Especially chemical warfare agents like tabun and soman as well as novichok intoxications are difficult to address using established oxime therapeutics. These neurotoxins inhibit acetylcholinesterase (AChE), a pivotal enzyme in the synaptic cleft. The following accumulation of acetylcholine in the synaptic cleft leads to a dysfunctional, desensitized state of nicotinic acetylcholine receptors (nAChR). Without adequate treatment, the resulting cholinergic crisis leads to death by respiratory arrest. Consequently, the research approach for new therapeutic options needs to be expanded. A promising option would be substances interacting directly with nAChRs. Therefore, screening methods for new drug candidates are needed, with affinity assays playing an important role. In the present work, a saturation and competition scintillation proximity assay (SPA) for binding studies at [ 3 H]epibatidine binding sites, conventionally classified as orthosteric binding sites of the muscle type nAChR was developed. This method offers several advantages over other assay technologies because no separation as well as washing steps are required to remove unbound ligands. Assay precision and solvent tolerance were validated according to the guidelines for validation of bioanalytical methods of the Food and Drug Administration (FDA) and European Medicines Agency (EMA). The newly developed binding assay was successfully implemented on an automated pipetting platform and is suitable for high-throughput-screening of receptor-ligand interactions at the nAChR. Furthermore, it allows to investigate/quantify competition of highly toxic agents such as nerve agents or structurally similar pesticides at the orthosteric binding site. Related to further pharmacological results, the affinity to [ 3 H]epibatidine binding sites can provide additional information on whether potential drug candidates would be suitable for treatment of nerve agent poisoning., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The determination of ligand-receptor binding affinities plays a key role in the development process of pharmaceuticals. While the classical radiochemical binding assay uses radioligands, fluorescence-based binding assays require fluorescent probes. Usually, radio- and fluorescence-labeled ligands are dissimilar in terms of structure and bioactivity, and can be used in either radiochemical or fluorescence-based assays. Aiming for a close comparison of both assay types, we synthesized tritiated fluorescent neurotensin receptor ligands ([ 3 H] 13 , [ 3 H] 18 ) and their nontritiated analogues ( 13 , 18 ). The labeled probes were studied in radiochemical and fluorescence-based (high-content imaging, flow cytometry, fluorescence anisotropy) binding assays. Equilibrium saturation binding yielded well-comparable ligand-receptor affinities, indicating that all these setups can be used for the screening of new drugs. In contrast, discrepancies were found in the kinetic behavior of the probes, which can be attributed to technical differences of the methods and require further studies with respect to the elucidation of the underlying mechanisms.

Purpose: Positron emission tomography (PET) studies with radioligands for 18-kDa translocator protein (TSPO) have been instrumental in increasing our understanding of the complex role neuroinflammation plays in disorders affecting the brain. However, (R)-[11C]PK11195, the first and most widely used TSPO radioligand has limitations, while the next-generation TSPO radioligands have suffered from high interindividual variability in binding due to a genetic polymorphism in the TSPO gene (rs6971). Herein, we present the biological evaluation of the two enantiomers of [18F]GE387, which we have previously shown to have low sensitivity to this polymorphism. Methods: Dynamic PET scans were conducted in male Wistar rats and female rhesus macaques to investigate the in vivo behaviour of (S)-[18F]GE387 and (R)-[18F]GE387. The specific binding of (S)-[18F]GE387 to TSPO was investigated by pre-treatment with (R)-PK11195. (S)-[18F]GE387 was further evaluated in a rat model of lipopolysaccharide (LPS)-induced neuroinflammation. Sensitivity to polymorphism of (S)-GE387 was evaluated in genotyped human brain tissue. Results: (S)-[18F]GE387 and (R)-[18F]GE387 entered the brain in both rats and rhesus macaques. (R)-PK11195 blocked the uptake of (S)-[18F]GE387 in healthy olfactory bulb and peripheral tissues constitutively expressing TSPO. A 2.7-fold higher uptake of (S)-[18F]GE387 was found in the inflamed striatum of LPS-treated rodents. In genotyped human brain tissue, (S)-GE387 was shown to bind similarly in low affinity binders (LABs) and high affinity binders (HABs) with a LAB to HAB ratio of 1.8. Conclusion: We established that (S)-[18F]GE387 has favourable kinetics in healthy rats and non-human primates and that it can distinguish inflamed from normal brain regions in the LPS model of neuroinflammation. Crucially, we have reconfirmed its low sensitivity to the TSPO polymorphism on genotyped human brain tissue. Based on these factors, we conclude that (S)-[18F]GE387 warrants further evaluation with studies on human subjects to assess its suitability as a TSPO PET radioligand for assessing neuroinflammation. [ABSTRACT FROM AUTHOR]

Purpose: This longitudinal study aims to evaluate the performance of 68 Ga-FAPI-04 and 18F-FDG and to profile the dynamic process of tumor metastasis in a preclinical 4T1 breast cancer model. Although both of these two radioligands are wildly used in clinic, no study was reported on their performance in the longitudinal monitoring of tumor metastasis. Also, no correlation between the expression level of fibroblast activation protein (FAP) and the development of tumor metastasis has been elucidated previously. In this study, we evaluated the performance of 68 Ga-FAPI-04 and 18F-FDG PET during the entire process of tumor metastasis, and their potential for the early diagnosis of tumor metastasis. We also clarified the correlation of uptakes as well as the signal-to-background (S/B) ratios between these two probes at different stages of tumor metastasis. Methods: Forty 4T1 metastatic breast cancer murine models were established using female BALB/c mice, followed by the longitudinal imaging with 68 Ga-FAPI-04 and 18F-FDG once a week for up to 6 weeks. In vitro hematoxylin and eosin (H&E) and immunochemistry (IHE) staining were performed to evaluate FAP expression on the metastatic lesions. Further statistical analysis was performed to evaluate the correlation of 68 Ga-FAPI-04 and 18F-FDG uptake (%ID/cc) at different stages of the metastasis. Results: 68 Ga-FPAI-04 holds an advantage over 18F-FDG with higher sensitivity at the early stage of tumor metastasis. However, with the progress of tumor metastasis, uptake of 68 Ga-FAPI-04 decreases and becomes less sensitive than 18F-FDG. There is also no direct correlation between uptake or S/B ratios of 68 Ga-FAPI-04 and 18F-FDG during this dynamic process. Conclusion: 68 Ga-FAPI-04 is more sensitive than 18F-FDG in detecting the early stage of tumor metastasis, but becomes less sensitive than 18F-FDG at the late stage of tumor metastasis. We envision this result would be meaningful for the explanation of the 68 Ga-FAPI-04 and 18F-FDG imaging both in the future clinic and preclinic studies. [ABSTRACT FROM AUTHOR]

Purpose: Recent research in last years in substance use disorders (SUD) synthesized a proinflammatory hypothesis of SUD based on reported pieces of evidence of non-neuronal central immune signalling pathways modulated by drug of abuse and that contribute to their pharmacodynamic actions. Positron emission tomography has been shown to be a precious imaging technique to study in vivo neurochemical processes involved in SUD and to highlight the central immune signalling actions of drugs of abuse. Methods: In this review, we investigate the contribution of the central immune system, with a particular focus on translocator protein 18 kDa (TSPO) imaging, associated with a series of drugs involved in substance use disorders (SUD) specifically alcohol, opioids, tobacco, methamphetamine, cocaine, and cannabis. Results: The large majority of preclinical and clinical studies presented in this review converges towards SUD modulation of the neuroimmune responses and TSPO expression and speculated a pivotal positioning in the pathogenesis of SUD. However, some contradictions concerning the same drug or between preclinical and clinical studies make it difficult to draw a clear picture about the significance of glial state in SUD. Discussion: Significant disparities in clinical and biological characteristics are present between investigated populations among studies. Heterogeneity in genetic factors and other clinical co-morbidities, difficult to be reproduced in animal models, may affect findings. On the other hand, technical aspects including study designs, radioligand limitations, or PET imaging quantification methods could impact the study results and should be considered to explain discrepancies in outcomes. Conclusion: The supposed neuroimmune component of SUD provides new therapeutic approaches in the prediction and treatment of SUD pointing to the central immune signalling. [ABSTRACT FROM AUTHOR]

Objectives: To examine the effects of vibegron, a selective β3‐adrenoceptor agonist, used to treat overactive bladder, on muscarinic receptors in the rat bladder, and to predict the occupancy levels of muscarinic receptors by vibegron in the bladders of humans orally administered a clinical dose. Methods: Muscarinic receptors in the rat bladder and other tissues were examined by a radioligand binding assay using [N‐methyl‐3H]scopolamine chloride. The occupancy levels of muscarinic receptors by vibegron in bladders of humans after its oral administration were predicted from the estimation of unbound concentrations in human plasma and urine in the literature. Results: Vibegron (0.1–100 μmol/L) inhibited specific [N‐methyl‐3H]scopolamine chloride binding in the bladder and other tissues of rats in a concentration‐dependent manner. The 50% inhibitory concentration value of vibegron in the bladder was approximately twofold higher than that in the heart, and approximately 315‐ and 3.5‐fold lower than those in the submaxillary gland and brain, respectively. Therefore, the binding affinity of vibegron for muscarinic receptors was higher in the heart and bladder than in the submaxillary gland and brain. By using the rat bladder receptor binding affinity, occupancy levels of muscarinic receptors in the human bladder were predicted to be 51–91% until 24 h after its oral administration at 50 mg of vibegron. Conclusions: This is the first study to suggest that vibegron binds to muscarinic receptors in the rat bladder and other tissues, with a potentially higher affinity for the M2 subtype than the M1 and M3 subtypes. These results might be clinically relevant for pharmacotherapy with vibegron for overactive bladder. [ABSTRACT FROM AUTHOR]

The type 1 cholecystokinin receptor (CCK1R) has multiple physiologic roles relating to nutrient homeostasis, including mediation of postcibal satiety. This effect has been central in efforts to develop agonists of this receptor as part of a program to manage and/or prevent obesity. While a number of small molecule CCK1R agonists have been developed, none have yet been approved for clinical use, based on inadequate efficacy, side effects, or the potential for toxicity. Understanding the molecular details of docking and mechanism of action of these ligands can be helpful in the rational refinement and enhancement of small molecule drug candidates. In the current work, we have defined the mechanism of binding and activity of two triazolobenzodiazepinones, CE-326597 and PF-04756956, which are reported to be full agonist ligands. To achieve this, we utilized receptor binding with a series of allosteric and orthosteric radioligands at structurally related CCK1R and CCK2R, as well as chimeric CCK1R/CCK2R constructs exchanging residues in the allosteric pocket, and assessment of biological activity. These triazolobenzodiazepinones docked within the intramembranous small molecule allosteric ligand pocket, with higher affinity binding to CCK2R than CCK1R, yet with biological activity exclusive to or greatly enhanced at CCK1R. These ligands exhibited cooperativity with benzodiazepine binding across the CCK1R homodimeric complex, resulting in their ability to inhibit only a fraction of the saturable binding of a benzodiazepine radioligand, unlike other small molecule antagonists and agonists of this receptor. This may contribute to the understanding of the unique short duration and reversible gallbladder contraction observed in vivo upon administration of these drugs.