Radioligand Binding Research Articles

Identification of a Novel Allosteric Site at the M5 Muscarinic Acetylcholine Receptor.

The M5 muscarinic acetylcholine receptor (mAChR) has emerged as an exciting therapeutic target for the treatment of addiction and behavioral disorders. This has been in part due to promising preclinical studies with the M5 mAChR selective negative allosteric modulator (NAM), ML375. The binding site of ML375 remains unknown, however, making it difficult to develop improved M5 mAChR selective modulators. To determine the possible location of the ML375 binding site, we used radioligand binding and functional assays to show that ML375 does not interact with the well-characterized "common" mAChR allosteric site located in the receptor's extracellular vestibule, nor a previously proposed second allosteric site recognized by the modulator, amiodarone. Molecular docking was used to predict potential allosteric sites within the transmembrane (TM) domain of the M5 mAChR. These predicted sites were assessed using M5-M2 mAChR receptor chimeras and further targeted with site-directed mutagenesis, which enabled the identification of a putative binding site for ML375 at the interface of TMs 2-4. Collectively, these results identify a third allosteric site at the M5 mAChR and highlight the ability of allosteric modulators to selectively target highly conserved proteins.

Pharmacological Characterization of the Imipridone Anticancer Drug ONC201 Reveals a Negative Allosteric Mechanism of Action at the D2 Dopamine Receptor.

ONC201 is a first-in-class imipridone compound that is in clinical trials for the treatment of high-grade gliomas and other advanced cancers. Recent studies identified that ONC201 antagonizes D2-like dopamine receptors at therapeutically relevant concentrations. In the current study, characterization of ONC201 using radioligand binding and multiple functional assays revealed that it was a full antagonist of the D2 and D3 receptors (D2R and D3R) with low micromolar potencies, similar to its potency for antiproliferative effects. Curve-shift experiments using D2R-mediated β-arrestin recruitment and cAMP assays revealed that ONC201 exhibited a mixed form of antagonism. An operational model of allostery was used to analyze these data, which suggested that the predominant modulatory effect of ONC201 was on dopamine efficacy with little to no effect on dopamine affinity. To investigate how ONC201 binds to the D2R, we employed scanning mutagenesis coupled with a D2R-mediated calcium efflux assay. Eight residues were identified as being important for ONC201's functional antagonism of the D2R. Mutation of these residues followed by assessing ONC201 antagonism in multiple signaling assays highlighted specific residues involved in ONC201 binding. Together with computational modeling and simulation studies, our results suggest that ONC201 interacts with the D2R in a bitopic manner where the imipridone core of the molecule protrudes into the orthosteric binding site, but does not compete with dopamine, whereas a secondary phenyl ring engages an allosteric binding pocket that may be associated with negative modulation of receptor activity. SIGNIFICANCE STATEMENT: ONC201 is a novel antagonist of the D2 dopamine receptor with demonstrated efficacy in the treatment of various cancers, especially high-grade glioma. This study demonstrates that ONC201 antagonizes the D2 receptor with novel bitopic and negative allosteric mechanisms of action, which may explain its high selectivity and some of its clinical anticancer properties that are distinct from other D2 receptor antagonists widely used for the treatment of schizophrenia and other neuropsychiatric disorders.

Functional and Anatomical Characterization of Corticotropin-Releasing Factor Receptor Subtypes of the Rat Spinal Cord Involved in Somatic Pain Relief

Corticotropin-releasing factor (CRF) orchestrates our body’s response to stressful stimuli. Pain is often stressful and counterbalanced by activation of CRF receptors along the nociceptive pathway, although the involvement of the CRF receptor subtypes 1 and/or 2 (CRF-R1 and CRF-R2, respectively) in CRF-induced analgesia remains controversial. Thus, the aim of the present study was to examine CRF-R1 and CRF-R2 expression within the spinal cord of rats with Freund’s complete adjuvant-induced unilateral inflammation of the hind paw using reverse transcriptase polymerase chain reaction, Western blot, radioligand binding, and immunofluorescence confocal analysis. Moreover, the antinociceptive effects of intrathecal (i.t.) CRF were measured by paw pressure algesiometer and their possible antagonism by selective antagonists for CRF-R1 and/or CRF-R2 as well as for opioid receptors. Our results demonstrated a preference for the expression of CRF-R2 over CRF-R1 mRNA, protein, binding sites and immunoreactivity in the dorsal horn of the rat spinal cord. Consistently, CRF as well as CRF-R2 agonists elicited potent dose-dependent antinociceptive effects which were antagonized by the i.t. CRF-R2 selective antagonist K41498, but not by the CRF-R1 selective antagonist NBI35965. In addition, i.t. applied opioid antagonist naloxone dose-dependently abolished the i.t. CRF- as well as CRF-R2 agonist-elicited inhibition of somatic pain. Importantly, double immunofluorescence confocal microscopy of the spinal dorsal horn showed CRF-R2 on enkephalin (ENK)-containing inhibitory interneurons in close opposition of incoming mu-opioid receptor-immunoreactive nociceptive neurons. CRF-R2 was, however, not seen on pre- or on postsynaptic sensory neurons of the spinal cord. Taken together, these findings suggest that i.t. CRF or CRF-R2 agonists inhibit somatic inflammatory pain predominantly through CRF-R2 receptors located on spinal enkephalinergic inhibitory interneurons which finally results in endogenous opioid-mediated pain inhibition.

Hyperthermia-induced seizures during neonatal period alter the functionality of A1 and A2A receptors in the cerebellum and evoke fine motor impairment and gait disturbances in adult rats

Febrile seizures (FS) are one of the most common types of convulsive disorder of early childhood and they can be classified into simple and prolonged depending on the duration. Nowadays, simple FS have a good prognosis but there is controversy about the outcome of prolonged FS. In a previous work using an animal model of prolonged FS, we showed that hiperthermia-induced seizures (HIS) evoked fine motor coordination impairment and gait disturbances in adolescent rats in a process in which seemed to be involved modulation of the cerebellar adenosinergic system. The aim of the present work was to verify whether the effect was maintained in adulthood. To this end, neonatal rats (PD 12) were exposed to HIS and after 48 days (PD 60) they were assayed on balance beam and footprint tests. Animals were sacrificed 53 days after HIS and adenosine A1 and A2A receptor signalling pathways were studied in cerebellar plasma membranes by using radioligand binding assays and by measuring the activities of 5´-nucleotidase and adenylyl cyclase. Results obtained revealed that adult rats exposed to HIS showed gait disturbances and motor impairments. Besides, animals exposed to hyperthermic insult showed an increase in adenosine A2A receptor functionality and 5´-nucleotidase activity. Surprisingly, the functionality of the adenosine A1 receptor resulted significantly changed causing stimulation instead of inhibition of adenylate cyclase activity. These results showed that the effect of prolonged FS at the early age also persist in adulthood suggesting on must pay attention to FS in children.

Novel High Affinity Sigma-1 Receptor Ligands from Minimal Ensemble Docking-Based Virtual Screening.

Sigma-1 receptor (S1R) is an intracellular, multi-functional, ligand operated protein that also acts as a chaperone. It is considered as a pluripotent drug target in several pathologies. The publication of agonist and antagonist bound receptor structures has paved the way for receptor-based in silico drug design. However, recent studies on this subject payed no attention to the structural differences of agonist and antagonist binding. In this work, we have developed a new ensemble docking-based virtual screening protocol utilizing both agonist and antagonist bound S1R structures. This protocol was used to screen our in-house compound library. The S1R binding affinities of the 40 highest ranked compounds were measured in competitive radioligand binding assays and the sigma-2 receptor (S2R) affinities of the best S1R binders were also determined. This way three novel high affinity S1R ligands were identified and one of them exhibited a notable S1R/S2R selectivity.

Neuroactive steroids, WIN-compounds and cholesterol share a common binding site on muscarinic acetylcholine receptors

Endogenous neurosteroids and their synthetic analogues-neuroactive steroids-have been found to bind to muscarinic acetylcholine receptors and allosterically modulate acetylcholine binding and function. Using radioligand binding experiments we investigated their binding mode. We show that neuroactive steroids bind to two binding sites on muscarinic receptors. Their affinity for the high-affinity binding site is about 100 nM. Their affinity for the low-affinity binding site is about 10 µM. The high-affinity binding occurs at the same site as binding of steroid-based WIN-compounds that is different from the common allosteric binding site for alcuronium or gallamine that is located between the second and third extracellular loop of the receptor. This binding site is also different from the allosteric binding site for the structurally related aminosteroid-based myorelaxants pancuronium and rapacuronium. Membrane cholesterol competes with neurosteroids/neuroactive steroids binding to both high- and low-affinity binding site, indicating that both sites are oriented towards the cell membrane..

Potential of nafimidone derivatives against co-morbidities of epilepsy: In vitro, in vivo, and in silico investigations.

Nafimidone is known for its clinical antiepileptic effects and alcohol derivatives of nafimidone were reported be potent anticonvulsants. These compounds are structurally similar to miconazole, which is known to inhibit cholinesterases, protect neurons, and ameliorate cognitive decline. Herein, we aimed to reveal the potential of three nafimidone alcohol esters (5g, 5i, and 5k), which were previously reported for their anticonvulsant effects, against co-morbidities of epilepsy such as inflammatory and neuropathic pain, cognitive and behavioral deficits, and neuron death, and understand their roles in related pathways such as γ-butyric acid type A (GABAA ) receptor and cholinesterases using in vitro, in vivo and in silico methods. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used for cytotoxicity evaluation, hippocampal slice culture assay for neuroprotection, formalin test for acute and inflammatory pain, sciatic ligation for neuropathic pain, Morris water maze and open field locomotor tasks for cognitive and behavioral deficits, radioligand binding for GABAA receptor affinity, spectrophotometric methods for cholinesterase inhibition in vitro, and molecular docking in silico. The compounds were non-toxic to fibroblast cells. 5 k was neuroprotective against kainic acid-induced neuron death. 5i reduced pain response of mice in both the acute and the inflammatory phases. 5i improved survival upon status epilepticus. The compounds showed no affinity to GABAA receptor but inhibited acetylcholinesterase, 5k also inhibited butyrylcholinesterase. The compounds were predicted to interact mainly with the peripheric anionic site of cholinesterase enzymes. The title compounds showed neuroprotective, analgesic, and cholinesterase inhibitory effects, thus they bear promise against certain co-morbidities of epilepsy with neurological insults.

In Vitro and In Vivo Evaluation of GSK-3 Radioligands in Alzheimer's Disease: Preliminary Evidence of Sex Differences.

Glycogen synthase kinase-3 (GSK-3) is a positron emission tomography (PET) imaging target with implications in the pathogenesis of Alzheimer's disease (AD). This preliminary study evaluates human AD and transgenic P301L mouse brain tissues using the GSK-3-targeting radiotracers [3H]PF-367 and [3H]OCM-44 in radioligand binding assays. A saturation analysis showed decreased GSK-3 density in female human AD compared to a normal healthy brain. Equivalence in density (B max), affinity (K d), and apparent affinity (K i) of both radiotracers was demonstrated to enable their interchangeability for in vitro evaluations of GSK-3 expression. An evaluation of P301L mouse brain by [3H]/[11C]OCM-44 delineated differences in the B max of GSK-3 between the control and transgenic mice within male subjects. PET imaging showed similar trends to those observed in vitro. Sex differences are revealed as a potential parameter to consider in the development of GSK-3-targeted diagnostics and therapeutics and could guide recruitment for clinical studies.

Design, synthesis and preliminary bioactivity evaluation of bitopic benzopyranomorpholine analogues as selective dopamine D3 receptor ligands as anti-drug addiction therapeutic agents

Three series of bitopic benzopyranomorpholine analogues were designed, synthesized, and evaluated as a novel class of selective ligands for the dopamine D3 receptor. Binding affinities of target compounds were determined using the method of radioligand binding assay. Most compounds demonstrated considerable binding affinities and selectivity for D3 receptor. Besides, the compounds were screened for their ability to alleviate withdrawal symptoms of opioid addiction in animal behavioral models. The results showed that compound 20h displayed nanomolar affinity for the D3R, and exhibited anti-drug addiction efficacy in the animal model of of naloxone-induced withdrawal symptoms in morphine-dependent mice.

Ten years follow up of first degree relatives of type 1 diabetes patients: presence of autoimmune biomarkers and the progression to diabetes in a retrospective cohort.

The aim of the study was to assess the autoimmunity in first degrees relatives (FDR) of patients with type 1 diabetes (T1DM) and the progression to T1DM after 10 years of follow up in the Brazilian population. Non-diabetic FDR of T1DM patients were interviewed and blood was drawn for autoantibodies measurement (GADA, IA-2A, IAA, ZnT8A). Serum samples were analyzed by standard radioligand binding assays performed at the Federal University of Rio de Janeiro (GADA, IAA and IA2A), and at the Skäne University Hospital, Sweden (ZnT8A). The FDR were interviewed by phone after 10 years to determine if they had developed T1DM. Descriptive statistical analysis was performed and results were described as means and standard deviation (SD). 81 individuals were analyzed. Thirteen subjects had positive autoantibodies associated with T1DM.10 were positive for 1 autoantibody and 3 subjects were positive for multiple autoantibodies (1 of them showed positivity for 2 autoantibodies - GADA, ZnT8A - and the other two were positive for 3 autoantibodies - GADA, IA2A, ZnT8A). The 3 subjects with multiple positive autoantibodies developed T1DM within 10 years. In Brazilian FDR of T1DM patients, the positivity for multiple autoantibodies indicate a greater chance of progression to T1DM, similar to observed in Caucasians. ZnT8A was helpful in the risk assessment for T1DM development.

Acute Lung Injury in Cholinergic-Deficient Mice Supports Anti-Inflammatory Role of α7 Nicotinic Acetylcholine Receptor.

(1) Background: The lung cholinergic pathway is important for controlling pulmonary inflammation in acute lung injury, a condition that is characterized by a sudden onset and intense inflammation. This study investigated changes in the expression levels of nicotinic and muscarinic acetylcholine receptors (nAChR and mAChR) in the lung during acute lung injury. (2) Methods: acute lung injury (ALI) was induced in wild-type and cholinergic-deficient (VAChT-KDHOM) mice using intratracheal lipopolysaccharide (LPS) instillation with or without concurrent treatment with nicotinic ligands. Bronchoalveolar lavage fluid was collected to evaluate markers of inflammation, and then the lung was removed and processed for isolation of membrane fraction and determination of acetylcholine receptors level using radioligand binding assays. (3) Results: LPS-induced increase in lung inflammatory markers (e.g., neutrophils and IL-1β) was significantly higher in VAChT-KDHOM than wild-type mice. In contrast, LPS treatment resulted in a significant increase in lung’s α7 nicotinic receptor level in wild-type, but not in VAChT-KDHOM mice. However, treatment with PNU 282987, a selective α7 nicotinic receptor agonist, restored VAChT-KDHOM mice’s ability to increase α7 nicotinic receptor levels in response to LPS-induced acute lung injury and reduced lung inflammation. LPS also increased muscarinic receptors level in VAChT-KDHOM mice, and PNU 282987 treatment reduced this response. (4) Conclusions: Our data indicate that the anti-inflammatory effects of the lung cholinergic system involve an increase in the level of α7 nicotinic receptors. Pharmacological agents that increase the expression or the function of lung α7 nicotinic receptors have potential clinical uses for treating acute lung injury.

A Food Supplement with Antioxidative Santa Herba Extract Modulates Energy Metabolism and Contributes to Weight Management.

The plant Santa herba (Eriodictyon californicum) contains high flavonoids, thus potentially exerting beneficial effects in context of obesity, often accompanied by inflammation and metabolic imbalance. The study assessed the impact of Santa herba on oxidative stress, energy metabolism, weight reduction, and eating behavior, combining in vitro models with clinical data. Santa herba binding of the adenosine receptor A2A (ADORA2A) was assessed using a radioligand binding assay. A Caenorhabditis elegans model was used to determine mobility boosting effects, and Santa herba oxygen radical absorbance capacity (ORAC) values were determined in comparison to antioxidative plants. Clinical data, that is, body weight and appetite-related parameters, were obtained from overweight and obese women receiving either Santa herba or placebo for 12 weeks. Results showed that Santa herba extract binds to ADORA2A, stimulates C. elegans motility (+7.5%) and locomotion, and yields high antioxidative capacities (ORAC: 819 trolox equivalent). Clinical data, obtained from 24 overweight and 25 obese women (mean: 47.5 years), demonstrated a reduced body weight (P = .042) and body fat (P = .044), and by trend reduced leptin levels (P = .065) in women with obesity after Santa herba consumption compared to placebo. In conclusion, Santa herba extract has energizing and antioxidative properties and may aid in weight management of people with obesity. ClinicalTrials.gov Identifier: NCT03853603.

Receptor Activity-Modifying Protein 2 (RAMP2) alters glucagon receptor trafficking in hepatocytes with functional effects on receptor signalling

ObjectivesReceptor Activity-Modifying Protein 2 (RAMP2) is a chaperone protein which allosterically binds to and interacts with the glucagon receptor (GCGR). The aims of this study were to investigate the effects of RAMP2 on GCGR trafficking and signalling in the liver, where glucagon (GCG) is important for carbohydrate and lipid metabolism. MethodsSubcellular localisation of GCGR in the presence and absence of RAMP2 was investigated using confocal microscopy, trafficking and radioligand binding assays in human embryonic kidney (HEK293T) and human hepatoma (Huh7) cells. Mouse embryonic fibroblasts (MEFs) lacking the Wiskott-Aldrich Syndrome protein and scar homologue (WASH) complex and the trafficking inhibitor monensin were used to investigate the effect of halted recycling of internalised proteins on GCGR subcellular localisation and signalling in the absence of RAMP2. NanoBiT complementation and cyclic AMP assays were used to study the functional effect of RAMP2 on the recruitment and activation of GCGR signalling mediators. Response to hepatic RAMP2 upregulation in lean and obese adult mice using a bespoke adeno-associated viral vector was also studied. ResultsGCGR is predominantly localised at the plasma membrane in the absence of RAMP2 and exhibits remarkably slow internalisation in response to agonist stimulation. Rapid intracellular accumulation of GCG-stimulated GCGR in cells lacking the WASH complex or in the presence of monensin indicates that activated GCGR undergoes continuous cycles of internalisation and recycling, despite apparent GCGR plasma membrane localisation up to 40 min post-stimulation. Co-expression of RAMP2 induces GCGR internalisation both basally and in response to agonist stimulation. The intracellular retention of GCGR in the presence of RAMP2 confers a bias away from β-arrestin-2 recruitment coupled with increased activation of Gαs proteins at endosomes. This is associated with increased short-term efficacy for glucagon-stimulated cAMP production, although long-term signalling is dampened by increased receptor lysosomal targeting for degradation. Despite these signalling effects, only a minor disturbance of carbohydrate metabolism was observed in mice with upregulated hepatic RAMP2. ConclusionsBy retaining GCGR intracellularly, RAMP2 alters the spatiotemporal pattern of GCGR signalling. Further exploration of the effects of RAMP2 on GCGR in vivo is warranted.

Identification of V6.51L as a selectivity hotspot in stereoselective A2B adenosine receptor antagonist recognition

The four adenosine receptors (ARs) A1AR, A2AAR, A2BAR, and A3AR are G protein-coupled receptors (GPCRs) for which an exceptional amount of experimental and structural data is available. Still, limited success has been achieved in getting new chemical modulators on the market. As such, there is a clear interest in the design of novel selective chemical entities for this family of receptors. In this work, we investigate the selective recognition of ISAM-140, a recently reported A2BAR reference antagonist. A combination of semipreparative chiral HPLC, circular dichroism and X-ray crystallography was used to separate and unequivocally assign the configuration of each enantiomer. Subsequently affinity evaluation for both A2A and A2B receptors demonstrate the stereospecific and selective recognition of (S)-ISAM140 to the A2BAR. The molecular modeling suggested that the structural determinants of this selectivity profile would be residue V2506.51 in A2BAR, which is a leucine in all other ARs including the closely related A2AAR. This was herein confirmed by radioligand binding assays and rigorous free energy perturbation (FEP) calculations performed on the L249V6.51 mutant A2AAR receptor. Taken together, this study provides further insights in the binding mode of these A2BAR antagonists, paving the way for future ligand optimization.

Feature-Based Molecular Networking for the Targeted Identification of Gq-Inhibiting FR900359 Derivatives.

Both the soil bacterium Chromobacterium vaccinii and the bacterial endosymbiont Candidatus Burkholderia crenata of the plant Ardisia crenata are producers of FR900359 (FR). This cyclic depsipeptide is a potent and selective Gq protein inhibitor used extensively to investigate the intracellular signaling of G protein coupled receptors (GPCRs). In this study, the metabolomes of both FR producers were investigated and compared using feature-based molecular networking (FBMN). As a result, 30 previously unknown FR derivatives were identified, one-third being unique to C. vaccinii. Guided by MS, a novel FR derivative, FR-6 (compound 1), was isolated, and its structure unambiguously established. In a whole-cell biosensing assay based on detection of dynamic mass redistribution (DMR) as readout for Gq inhibition, FR-6 suppressed Gq signaling with micromolar potency (pIC50 = 5.56). This functional activity was confirmed in radioligand binding assays (pKi = 7.50). This work demonstrates the power of molecular networking, guiding the way to a novel Gq-inhibiting FR derivative and underlining the potency of FR as a Gq inhibitor.

Lysophospholipid (S1P) receptors in GtoPdb v.2021.2

Sphingosine 1-phosphate (S1P) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Lysophospholipid receptors [89]) are activated by the endogenous lipid sphingosine 1-phosphate (S1P). Originally cloned as orphan members of the endothelial differentiation gene (edg) family [16, 112], the receptors are currently designated as S1P1R through S1P5R [69, 16, 112]. Their gene nomenclature has been codified as human S1PR1, S1PR2, etc. (HUGO Gene Nomenclature Committee, HGNC) and S1pr1, S1pr2, etc. for mice (Mouse Genome Informatics Database, MGI) to reflect species and receptor function. All S1P receptors have been knocked-out in mice constitutively and in some cases, conditionally. S1PRs, particularly S1P1, are expressed throughout all mammalian organ systems. Ligand delivery occurs via two known carriers (or "chaperones"): albumin and HDL-bound apolipoprotein M (ApoM), the latter of which elicits biased agonist signaling by S1P1 in multiple cell types [18, 49]. The five S1PRs, two chaperones, and active cellular metabolism have complicated analyses of receptor ligand binding in native systems. Signaling pathways and physiological roles have been characterized through radioligand binding in heterologous expression systems, targeted deletion of the different S1PRs, and most recently, mouse models that report in vivo S1P1R activation [94, 96]. A crystal structure of an S1P1-T4 fusion protein confirmed aspects of ligand binding, specificity, and receptor activation, determined previously through biochemical and genetic studies [65, 17]. fingolimod (FTY720), the first FDA-approved drug to target any of the lysophospholipid receptors, binds as a phosphorylated metabolite to four of the five S1PRs, and was the first oral therapy for multiple sclerosis (MS) [33]. siponimod and ozanimod that target S1P1 and S1P5 are also FDA approved for the treatment of various MS forms [16, 112]. The mechanisms of action of fingolimod and other S1PR-modulating drugs now in development include binding S1PRs in multiple organ systems, e.g., immune and nervous systems, although the precise nature of their receptor interactions requires clarification [129, 35, 59, 60].

Modulation of Adenosine Receptors by Hops and Xanthohumol in Cell Cultures.

Adenosine receptors (ARs) have been involved in neurodegenerative diseases such as Alzheimer disease, where oxidative stress contributes to neurodegeneration and cell death. Therefore, there is increasing interest in developing antioxidative strategies to avoid or reduce neurodegeneration. We have previously described that different beer extracts modulate ARs and protect glioma and neuroblastoma cells from oxidative stress. The present work aimed to analyze the possible protective effect of hops (Humulus lupulus L.), a major component of beer, and xanthohumol on cell death elicited by oxidative stress and their modulation of ARs in rat C6 glioma and human SH-SY5Y neuroblastoma cells. Different extraction methods were employed in two hops varieties (Nugget and Columbus). Cell viability was determined by the XTT method in cells exposed to these hops extracts and xanthohumol. ARs were analyzed by radioligand binding and real-time PCR assays. Hops extract reverted the cell death observed under oxidative stress and modulated adenosine A1 and A2 receptors in both cell types. Xanthohumol was unable to revert the effect of oxidative stress in cell viability but it also modulated ARs similarly to hops. Therefore, healthy effects of beer described previously could be due, at least in part, to their content of hops and the modulation of ARs.

SPECT/CT Imaging, Biodistribution and Radiation Dosimetry of a 177Lu-DOTA-Integrin αvβ6 Cystine Knot Peptide in a Pancreatic Cancer Xenograft Model.

IntroductionPancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant neoplasms, as many cases go undetected until they reach an advanced stage. Integrin αvβ6 is a cell surface receptor overexpressed in PDAC. Consequently, it may serve as a target for the development of probes for imaging diagnosis and radioligand therapy. Engineered cystine knottin peptides specific for integrin αvβ6 have recently been developed showing high affinity and stability. This study aimed to evaluate an integrin αvβ6-specific knottin molecular probe containing the therapeutic radionuclide 177Lu for targeting of PDAC.MethodsThe expression of integrin αvβ6 in PDAC cell lines BxPC-3 and Capan-2 was analyzed using RT-qPCR and immunofluorescence. In vitro competition and saturation radioligand binding assays were performed to calculate the binding affinity of the DOTA-coupled tracer loaded with and without lutetium to BxPC-3 and Capan-2 cell lines as well as the maximum number of binding sites in these cell lines. To evaluate tracer accumulation in the tumor and organs, SPECT/CT, biodistribution and dosimetry projections were carried out using a Capan-2 xenograft tumor mouse model.ResultsRT-qPCR and immunofluorescence results showed high expression of integrin αvβ6 in BxPC-3 and Capan-2 cells. A competition binding assay revealed high affinity of the tracer with IC50 values of 1.69 nM and 9.46 nM for BxPC-3 and Capan-2, respectively. SPECT/CT and biodistribution analysis of the conjugate 177Lu-DOTA-integrin αvβ6 knottin demonstrated accumulation in Capan-2 xenograft tumors (3.13 ± 0.63%IA/g at day 1 post injection) with kidney uptake at 19.2 ± 2.5 %IA/g, declining much more rapidly than in tumors.Conclusion 177Lu-DOTA-integrin αvβ6 knottin was found to be a high-affinity tracer for PDAC tumors with considerable tumor accumulation and moderate, rapidly declining kidney uptake. These promising results warrant a preclinical treatment study to establish therapeutic efficacy.

N,N-Dimethyltryptamine attenuates spreading depolarization and restrains neurodegeneration by sigma-1 receptor activation in the ischemic rat brain

Dimethyltryptamine (DMT), an endogenous ligand of sigma-1 receptors (Sig-1Rs), acts against systemic hypoxia, but whether DMT may prevent cerebral ischemic injury is unexplored. Here global forebrain ischemia was created in anesthetized rats and aggravated with the induction of spreading depolarizations (SDs) and subsequent short hypoxia before reperfusion. Drugs (DMT, the selective Sig-1R agonist PRE-084, the Sig-1R antagonist NE-100, or the serotonin receptor antagonist asenapine) were administered intravenously alone or in combination while physiological variables and local field potential from the cerebral cortex was recorded. Neuroprotection and the cellular localization of Sig-1R were evaluated with immunocytochemistry. Plasma and brain DMT content was measured by 2D-LC-HRMS/MS. The affinity of drugs for cerebral Sig-1R was evaluated with a radioligand binding assay. Both DMT and PRE-084 mitigated SDs, counteracted with NE-100. Further, DMT attenuated SD when co-administered with asenapine, compared to asenapine alone. DMT reduced the number of apoptotic and ferroptotic cells and supported astrocyte survival. The binding affinity of DMT to Sig-1R matched previously reported values. Sig-1Rs were associated with the perinuclear cytoplasm of neurons, astrocytes and microglia, and with glial processes. According to these data, DMT may be considered as adjuvant pharmacological therapy in the management of acute cerebral ischemia.

Fluoroethylnormemantine, a Novel NMDA Receptor Antagonist, for the Prevention and Treatment of Stress-Induced Maladaptive Behavior

BackgroundMajor depressive disorder is a common, recurrent illness. Recent studies have implicated the NMDA receptor in the pathophysiology of major depressive disorder. (R,S)-ketamine, an NMDA receptor antagonist, is an effective antidepressant but has numerous side effects. Here, we characterized a novel NMDA receptor antagonist, fluoroethylnormemantine (FENM), to determine its effectiveness as a prophylactic and/or antidepressant against stress-induced maladaptive behavior. MethodsSaline, memantine (10 mg/kg), (R,S)-ketamine (30 mg/kg), or FENM (10, 20, or 30 mg/kg) was administered before or after contextual fear conditioning in 129S6/SvEv mice. Drug efficacy was assayed using various behavioral tests. Protein expression in the hippocampus was quantified with immunohistochemistry or Western blotting. In vitro radioligand binding was used to assay drug binding affinity. Patch clamp electrophysiology was used to determine the effect of drug administration on glutamatergic activity in ventral hippocampal cornu ammonis 3 (vCA3) 1 week after injection. ResultsGiven after stress, FENM decreased behavioral despair and reduced perseverative behavior. When administered after re-exposure, FENM facilitated extinction learning. As a prophylactic, FENM attenuated learned fear and decreased stress-induced behavioral despair. FENM was behaviorally effective in both male and female mice. (R,S)-ketamine, but not FENM, increased expression of c-fos in vCA3. Both (R,S)-ketamine and FENM attenuated large-amplitude AMPA receptor–mediated bursts in vCA3, indicating a common neurobiological mechanism for further study. ConclusionsOur results indicate that FENM is a novel drug that is efficacious when administered at various times before or after stress. Future work will further characterize FENM’s mechanism of action with the goal of clinical development.