Receptor Antagonist Binding Research Articles

Impact of cancer-associated mutations in CC chemokine receptor 2 on receptor function and antagonism

CC chemokine receptor 2 (CCR2), a G protein-coupled receptor, plays a role in many cancer-related processes such as metastasis formation and immunosuppression. Since ∼ 20 % of human cancers contain mutations in G protein-coupled receptors, ten cancer-associated CCR2 mutants obtained from the Genome Data Commons were investigated for their effect on receptor functionality and antagonist binding. Mutations were selected based on either their vicinity to CCR2′s orthosteric or allosteric binding sites or their presence in conserved amino acid motifs. One of the mutant receptors, namely S101P2.63 with a mutation near the orthosteric binding site, did not express on the cell surface. All other studied mutants showed a decrease in or a lack of G protein activation in response to the main endogenous CCR2 ligand CCL2, but no change in potency was observed. Furthermore, INCB3344 and LUF7482 were chosen as representative orthosteric and allosteric antagonists, respectively. No change in potency was observed in a functional assay, but mutations located at F1163.28 impacted orthosteric antagonist binding significantly, while allosteric antagonist binding was abolished for L134Q3.46 and D137N3.49 mutants. As CC chemokine receptor 2 is an attractive drug target in cancer, the negative effect of these mutations on receptor functionality and drugability should be considered in the drug discovery process.

Development of a Translational Model to Assess the Impact of Opioid Overdose and Naloxone Dosing on Respiratory Depression and Cardiac Arrest.

In response to a surge of deaths from synthetic opioid overdoses, there have been increased efforts to distribute naloxone products in community settings. Prior research has assessed the effectiveness of naloxone in the hospital setting; however, it is challenging to assess naloxone dosing regimens in the community/first-responder setting, including reversal of respiratory depression effects of fentanyl and its derivatives (fentanyls). Here, we describe the development and validation of a mechanistic model that combines opioid mu receptor binding kinetics, opioid agonist and antagonist pharmacokinetics, and human respiratory and circulatory physiology, to evaluate naloxone dosing to reverse respiratory depression. Validation supports our model, which can quantitatively predict displacement of opioids by naloxone from opioid mu receptors in vitro, hypoxia-induced cardiac arrest in vivo, and opioid-induced respiratory depression in humans from different fentanyls. After validation, overdose simulations were performed with fentanyl and carfentanil followed by administration of different intramuscular naloxone products. Carfentanil induced more cardiac arrest events and was more difficult to reverse than fentanyl. Opioid receptor binding data indicated that carfentanil has substantially slower dissociation kinetics from the opioid receptor compared with nine other fentanyls tested, which likely contributes to the difficulty in reversing carfentanil. Administration of the same dose of naloxone intramuscularly from two different naloxone products with different formulations resulted in differences in the number of virtual patients experiencing cardiac arrest. This work provides a robust framework to evaluate dosing regimens of opioid receptor antagonists to reverse opioid-induced respiratory depression, including those caused by newly emerging synthetic opioids.

Opioid antagonism mitigates antipsychotic-associated weight gain: focus on olanzapine.

The endogenous opioid system affects metabolism, including weight regulation. Evidence from preclinical and clinical studies provides a rationale for targeting this system to mitigate weight-related side effects of antipsychotics. This review describes the role of the opioid system in regulating weight and metabolism, examines the effects of opioid receptor antagonism on those functions, and explores the use of opioid antagonists to mitigate antipsychotic-associated weight gain and/or metabolic effects. A PubMed literature search was conducted to identify representative opioid antagonists and associated preclinical and clinical studies examining their potential for the regulation of weight and metabolism. The mu opioid receptor (MOR), delta opioid receptor (DOR), and kappa opioid receptor (KOR) types have overlapping but distinct patterns of central and peripheral expression, and each contributes to the regulation of body weight and metabolism. Three representative opioid antagonists (eg, naltrexone, samidorphan, and LY255582) were identified for illustration. These opioid antagonists differed in their receptor binding and pharmacokinetic profiles, including oral bioavailability, systemic clearance, and half-life, and were associated with varying effects on food intake, energy utilization, and metabolic dysregulation. Preclinical and clinical data suggest that antagonism of the endogenous opioid system is a mechanism to address antipsychotic-associated weight gain and metabolic dysregulation. However, evidence suggests that the differing roles of MOR, DOR, and KOR in metabolism, together with the differences in receptor binding, pharmacokinetic, and functional activity profiles of the opioid receptor antagonists discussed in this review, likely contribute to their differential pharmacodynamic effects and clinical outcomes observed regarding antipsychotic-associated weight gain.

Modulation of Interleukin-1 and -18 Mediated Injury in Donation after Circulatory Death Mouse Hearts

BackgroundDonation after circulatory death donors (DCD) can expand the donor pool for heart transplantation, which primarily depends on brain death donors. Ischemia and reperfusion injury are inherent to the DCD process. We hypothesize that pharmacologic inhibition of interleukin-1 (IL-1) and/or IL-18 is protective to DCD hearts. Materials and methodsFollowing clinical protocol, in-situ ischemia time in control beating-heart donor (CBD) and DCD groups was less than 5 and 40 min, respectively. Wild type (WT) C57Bl6/j, IL-1 receptor type I knockout (IL-1RI-KO), and IL-18 KO mice were used. Hearts were reanimated for 90 min on a Langendorff system with Krebs-Henseleit buffer at 37°C, to assess physiologic parameters. Recombinant IL-1 receptor antagonist (IL-1Ra) and/or IL-18 binding protein (IL-18BP) were added to the Krebs-Henseleit buffer to inhibit IL-1 and/or the IL-18 signaling, respectively. ResultsDeveloped pressure and ± dP/dt were significantly impaired in the DCD-WT group compared to CBD-WT (P ≤ 0.05). Troponin release was higher in DCD-WT groups. Functional parameters were preserved, and troponin release was significantly less in the DCD knockout groups. Heart function was improved in DCD groups treated with IL-1Ra or IL-18BP compared to the DCD-WT group. ConclusionsHeart function was significantly impaired in the DCD-WT group compared to CBD-WT. Genetic deletion or pharmacologic blockade of IL-1 or IL-18 was protective to DCD hearts.

Facial erythema after the treatment of dupilumab in SLE patient

BackgroundDupilumab is a receptor antagonist binding to the alpha subunit of the interleukin-4 receptor. Through binding to it, dupilumab inhibits signaling of both IL-4 and IL-13, the representative Th2 biomarkers. Recently, in addition to the treatment effects for atopic dermatitis (AD), there is an emerging adverse event as facial erythema.Case presentationA twenty-seven-year-old female patient developed erythema and desquamation on the face and neck after dupilumab administration. She had AD on her arms, legs, and trunk before the treatment but there was no atopic clinical feature in her face and neck. With the treatment of dupilumab, her skin lesions of the body have improved from the beginning of the treatment. In the patch test, including dupilumab, there was no specific finding other than the 1+ response to neomycin on day 2. In the intradermal test to dupilumab, a positive result was observed 15 min later, but negative both days 1 and 2. The blood examination showed an elevation of both ANA as 1:80 and anti-phospholipid antibodies (Anti-cardiolipin IgM, IgG, and Anti- beta 2 GPI IgG). She was diagnosed with Systemic lupus erythematosus (SLE) based on diagnostic criteria by a rheumatologist.ConclusionDupilumab is an emerging therapeutic agent for AD, and treatment cases are increasing in Korea. However, there are several adverse events during the treatment of dupilumab. Herein, we report the unexpected adverse event during the treatment of dupilumab in SLE patients.

Randomized multicenter open-label trial of GT0918 (proxalutamide) in patients with mCRPC that progressed on abiraterone or enzalutamide.

e17578 Background: Both abiraterone and enzalutamide have been approved for the treatment of mCRPC. GT0918 (proxalutamide) is a novel second-generation androgen receptor (AR) antagonist binding directly to the androgen receptor, impairing nuclear translocation and decreasing AR protein expression. This phase II portion of the Ph I/II study is being conducted to further evaluate safety and tolerability of GT0918 at 400 mg or 500 mg daily dosing. Methods: All eligible mCRPC pts with progressive disease based on rising prostate-specific antigen (PSA) or/and imaging while on either abiraterone or enzalutamide, but not both. One line of prior chemotherapy was allowed. Pts (n = 60) will be randomized to GT0918 either 400 mg or 500 mg po once daily for 6 months. The primary endpoint is safety and tolerability. Secondary endpoints are PSA reduction at 12 weeks, % radiographic progression at 6-month, time to imaging progression, and exploratory biomarker CTC & cf-DNA/RNA. PSA is assessed every 4 wks and tumor imaging is done every 12 wks. Results: 56 pts were screened June 2019 to January 15, 2020 and 40 pts were randomized to receive GT0918 either 400 mg or 500 mg po daily. 22 pts (55%) had enzalutamide as prior therapy and 18 pts (45%) had abiraterone as prior therapy. 17 pts (43%) had one or more lines of chemotherapy. The median age was 72 years (range 53-89) and median baseline PSA was 162 ng/mL (0.3-4195). 10 pts had 1 cycle or less of study drug due to rapid PSA progression, AEs and/or withdrawal of consent. The most common treatment-related AEs were fatigue (48%), loss of appetite/anorexia (25%), nausea (20%), lower leg extremity edema (18%), constipation (15%), weight loss (13%), vomiting (10%), and headache (8%). 8 pts (6 in 500 mg vs. 2 in 400 mg) required dose reduction due to AE. No seizures have been observed. As Jan 30, 2020, 3 pts (8%) and 8 pts (20%) had PSA reduction at 12 weeks and 4 weeks, respectively, but no 50% PSA reduction was observed. 2 pts (5%) received study drug treatment over 24 weeks and 10 pts (25%) received study drug over 16 weeks based on stable disease (SD) on imaging. Conclusions: GT0918 administrated orally once a day is well tolerated and first 40-pts taking GT0918 show some anti-tumor activities. Dose of 400 mg po daily is recommended for GT 0918 for further clinical trials. Clinical trial information: NCT03899467 .

Quartz Dust Exposure Affects NLRP3 Inflammasome Activation and Plasma Levels of IL-18 and IL-1Ra in Iron Foundry Workers.

Purpose To study the association between inhalation of particulate matter or quartz in Swedish iron foundries and the effects on NLRP3 inflammasome activation. Methods Particle exposure measurements were performed during an eight-hour work day for 85 foundry workers at three Swedish iron foundries. Personal sampling was used for measurement of respirable quartz and dust and stationary measurements to obtain exposure measurements for inhalable dust and PM10. The NLRP3 inflammasome markers, interleukin- (IL-) 1β and IL-18, and inhibitors IL-1 receptor antagonist (IL-1Ra) and IL-18 binding protein (IL-18BP) were measured in plasma. Inflammasome activation was measured by caspase-1 enzymatic activity in monocytes in whole blood by flow cytometry, and expression of inflammasome-related genes was quantified using real-time PCR. Multiple linear regression analysis was used to investigate associations between PM exposures and inflammatory markers. Sex, age, smoking, current infection, BMI, and single nucleotide polymorphism in the inflammasome regulating genes CARD8 (C10X) and NLRP3 (Q705K) were included as covariates. Results The average exposure levels of respirable dust and quartz were 0.85 and 0.052 mg/m3, respectively. A significant exposure-response was found for respirable dust and IL-18 and for inhalable dust and IL-1Ra. Whole blood, drawn from study participants, was stimulated ex vivo with inflammasome priming stimuli LPS or Pam3CSK4, resulting in a 47% and 49% increase in caspase-1 enzymatic activity in monocytes. This increase in caspase-1 activity was significantly attenuated in the higher exposure groups for most PM exposure measures. Conclusions The results indicate that exposure levels of PM in the iron foundry environment can affect the NLRP3 inflammasome and systemic inflammation.

Distribution and properties of cardiac and pulmonary β-adrenergic receptors in corn snakes (Pantherophis guttatus) and Boa constrictor (Boa constrictor)

The aim of the present study was to characterize β-adrenergic receptors in the snake heart and lung of corn and Boa constrictor snakes. The β-adrenergic receptor binding sites were studied in purified heart and lung membranes using the specific β-adrenergic receptor antagonist [125J]-iodocyanopindolol (ICYP) and subtypes using selective β1-adrenergic receptor antagonist CGP-20712A and selective β2-adrenergic receptor antagonist ICI-118.551. A saturable and specific β-adrenergic receptor binding site was detected in cardiac membranes with maximal receptor density (Bmax) of 43.99 ± 3.86 fmol/mg protein (corn snake) and 58.07 ± 2.88 fmol/mg protein (Boa constrictor) as well as KD of 24.21 ± 7.38 pM (corn snake) and 21.48 ± 3.85 pM (Boa constrictor) and in lung membranes (Bmax fmol/mg protein: 55.95 ± 16.28 (corn snake) and 107.00 ± 14.21 (Boa constrictor); KD pM: 71.25 ± 21.92 (corn snake) and 55.04 ± 18.68 (Boa constrictor)). Competition-binding studies showed β-adrenergic receptors with low affinities to the β2-selective adrenergic receptor antagonist and high affinity binding to β1-selective adrenergic receptor antagonist in both heart and lung tissues of both snake species, suggesting the presence of high population of the post-synaptic β1-adrenergic receptor subtype. It seems that the presence of the predominant β1-subtype also in lung tissues may indicate the importance of the vascular system in the snake lung.

Identification of avian vasotocin receptor subtype-specific antagonists involved in the stress response of the chicken, Gallus gallus

Vasotocin 1a and 1b receptors (V1aR and V1bR) have been shown to play important roles in the neuroendocrine regulation of stress responses via the anterior pituitary (AP) of birds. To identify effective subtype-specific antagonists for the chicken V1aR (cV1aR) and cV1bR, potential antagonists to the mammalian V1R were screened against the cV1aR and cV1bR 3D structural models by molecular docking analysis with determination of binding pocket/amino acid residues involved in the interaction. The antagonistic effects of the selected ligands were examined by measuring pro-opiomelanocortin (POMC) heteronuclear RNA (hnPOMC) levels following the in vitro stress administration to primary chicken AP cells. Results of in silico analysis showed that the Manning compound and several other antagonists were bound to cV1bR with higher affinity than the natural agonist, arginine vasotocin (AVT). Similarities and differences in the antagonist-receptor binding interface with receptors were characterized for each ligand. Non-peptide mammalian V1bR antagonists, SSR-149415 and L-368899, were shown to be effective and had an additive effect in blocking POMC hnRNA expression in pituitary cell culture studies. SR-49059 antagonized the effect(s) of AVT/CRH on the downregulation of the cV1aR and the upregulation of the cCRH-R2 expression but not the cV1bR and cCRH-R1. The Manning compound antagonized the downregulation of cV1aR, cV1bR and cCRH-R1 and the upregulation of cCRH-R2 expression. The specificity of antagonists apparently resulted from unique differences in the interacting residues and their binding affinities. Collectively, these results provide valuable leads for future development of novel compounds capable of blocking or attenuating the AP stress response of avian species and perhaps other non-mammalian vertebrates as well.

Kinetic operational models of agonism for G-protein-coupled receptors

The application of kinetics to research and therapeutic development of G-protein-coupled receptors has become increasingly valuable. Pharmacological models provide the foundation of pharmacology, providing concepts and measurable parameters such as efficacy and potency that have underlain decades of successful drug discovery. Currently there are few pharmacological models that incorporate kinetic activity in such a way as to yield experimentally-accessible drug parameters. In this study, a kinetic model of pharmacological response was developed that provides a kinetic descriptor of efficacy (the transduction rate constant, kτ) and allows measurement of receptor-ligand binding kinetics from functional data. The model assumes: (1) receptor interacts with a precursor of the response (“Transduction potential”) and converts it to the response. (2) The response can decay. Familiar response vs time plots emerge, depending on whether transduction potential is depleted and/or response decays. These are the straight line, the “association” exponential curve, and the rise-and-fall curve. Convenient, familiar methods are described for measuring the model parameters and files are provided for the curve-fitting program Prism (GraphPad Software) that can be used as a guide. The efficacy parameter kτ is straightforward to measure and accounts for receptor reserve; all that is required is measurement of response over time at a maximally-stimulating concentration of agonist. The modular nature of the model framework allows it to be extended. Here this is done to incorporate antagonist-receptor binding kinetics and slow agonist-receptor equilibration. In principle, the modular framework can incorporate other cellular processes, such as receptor desensitization. The kinetic response model described here can be applied to measure kinetic pharmacological parameters than can be used to advance the understanding of GPCR pharmacology and optimize new and improved therapeutics.

GPCRs: Glucagon receptor antagonist binding site identified.

GPCRs: Glucagon receptor antagonist binding site identified.

Activation of NLRP3 inflammasome enhances the proliferation and migration of A549 lung cancer cells.

Lung cancer is the leading cause of cancer death, and it is widely accepted that chronic inflammation is an important risk for the development of lung cancer. Now, it is recognized that the nucleotide-binding and oligomerization domain (NOD) like receptors (NLRs)-containing inflammasomes are involved in cancer-related inflammation. This study was designed to investigate the effects of NLR family pyrin domain containing protein 3 (NLRP3) inflammasome on the proliferation and migration of lung adenocarcinoma cell line A549. Using 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, scratch assay, and Transwell migration assay, we showed that activation of the NLRP3 inflammasome by LPS+ATP enhanced the proliferation and migration of A549 cells. Western blot analysis showed that activation of phosphorylation of Akt, ERK1/2, CREB and the expression of Snail increased, while the expression of E-cadherin decreased after the activation of NLRP3 inflammasome. Moreover, these effects were inhibited by the following treatments: i) downregulating the expression of NLRP3 by short hairpin RNA (shRNA) interference, ii) inhibiting the activation of NLRP3 inflammasome with a caspase-1 inhibitor, iii) blocking the interleukin-1β (IL-1β) and IL-18 signal transduction with IL-1 receptor antagonist (IL-1Ra) and IL-18 binding protein (IL-18BP). Collectively, these results indicate that NLRP3 inflammasome plays a vital role in regulating the proliferation and migration of A549 cells and it might be a potential target for the treatment of lung cancer.

Changes in cholinergic and glutamatergic markers in the striatum from a sub-set of subjects with schizophrenia

Background/objectivesHaving separated a sub-group of people with schizophrenia based on a marked loss of cortical [3H]pirenzepine binding (MRDS); we wished to determine if MRDS had lower levels of [3H]pirenzepine and other muscarinic receptor antagonist binding to the striatum and if this was due to loss of pre- or post-synaptic neurons or glia measured using surrogate markers (25kilodalton synaptosomal-associated protein (SNAP 25), postsynaptic density protein 95 (PSD 95), glial fibrillary acidic protein (GFAP) 41/43) of cell number. Methods[3H]pirenzepine, [3H]AF-DX 384 and [3H]4-DAMP binding to the striatum from 37 subjects with schizophrenia (19 MRDS) and 20 controls as well as SNAP 25, PSD 95 and GFAP 41/43 in crude particulate membrane were measured. Results[3H]pirenzepine and [3H]AF-DX 384 binding to the striatum were significantly lower in schizophrenia due to lower binding of both radioligands in the striatum from MRDS. Levels of PSD 95 were higher in schizophrenia, predominantly due to higher levels in MRDS. ConclusionsOur data suggest muscarinic M1 ([3H]pirenzepine) and M2 and/or M4 receptors ([3H]AF-DX 384) are lower in the striatum from MRDS which could mediate inappropriate adaption to internal and external cues which, in turn, would affect motivation, cognition and motor control. Increased levels of PSD 95 could indicate increased post-synaptic boutons or changes in NMDA receptor-mediated signalling in MRDS.

Macitentan inhibits the transforming growth factor-β profibrotic action, blocking the signaling mediated by the ETR/TβRI complex in systemic sclerosis dermal fibroblasts.

IntroductionSystemic sclerosis (SSc) is a complex and not fully understood autoimmune disease associated with fibrosis of multiple organs. The main effector cells, the myofibroblasts, are collagen-producing cells derived from the activation of resting fibroblasts. This process is regulated by a complex repertoire of profibrotic cytokines, and among them transforming growth factor beta (TGF-β) and endothelin-1 (ET-1) play a major role. In this paper we show that TGF-β and ET-1 receptors co-operate in myofibroblast activation, and macitentan, an ET-1 receptor antagonist binding ET-1 receptors, might interfere with both TGF-β and ET-1 pathways, preventing myofibroblast differentiation.MethodsFibroblasts isolated from healthy controls and SSc patients were treated with TGF-β and ET-1 and successively analyzed for alpha smooth muscle actin (α-SMA) and collagen (Col1A1) expression and for the Sma and Mad Related (SMAD) phosphorylation. We further tested the ability of macitentan to interfere with these process. Furthermore, we silenced ET-1 and endothelin-1 receptor A expression and evaluated the formation of an ET-1/TGF-β receptor complex by immunoprecitation assay.ResultsWe showed myofibroblast activation in SSc fibroblasts assessing the expression of α-SMA and Col1A1, after stimulation with TGF-β and ET-1. Macitentan interfered with both ET-1- and TGF-β-induced fibroblast activation. To explain this unexpected inhibitory effect of macitentan on TGF-β activity, we silenced ET-1 expression on SSc fibroblasts and co-immunoprecipitated these two receptors, showing the formation of an ET-1/TGF-β receptor complex.ConclusionsDuring SSc, ET-1 produced by activated endothelia contributes to myofibroblast activation using TGF-β machinery via an ET-1/TGF-β receptor complex. Macitentan interferes with the profibrotic action of TGF-β, blocking the ET-1 receptor portion of the ET-1/TGF-β receptor complex.

Localization of CGRP receptor components and receptor binding sites in rhesus monkey brainstem: A detailed study using in situ hybridization, immunofluorescence, and autoradiography.

Functional imaging studies have revealed that certain brainstem areas are activated during migraine attacks. The neuropeptide calcitonin gene-related peptide (CGRP) is associated with activation of the trigeminovascular system and transmission of nociceptive information and plays a key role in migraine pathophysiology. Therefore, to elucidate the role of CGRP, it is critical to identify the regions within the brainstem that process CGRP signaling. In situ hybridization and immunofluorescence were performed to detect mRNA expression and define cellular localization of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), respectively. To define CGRP receptor binding sites, in vitro autoradiography was performed with [(3)H]MK-3207 (a CGRP receptor antagonist). CLR and RAMP1 mRNA and protein expression were detected in the pineal gland, medial mammillary nucleus, median eminence, infundibular stem, periaqueductal gray, area postrema, pontine raphe nucleus, gracile nucleus, spinal trigeminal nucleus, and spinal cord. RAMP1 mRNA expression was also detected in the posterior hypothalamic area, trochlear nucleus, dorsal raphe nucleus, medial lemniscus, pontine nuclei, vagus nerve, inferior olive, abducens nucleus, and motor trigeminal nucleus; protein coexpression of CLR and RAMP1 was observed in these areas via immunofluorescence. [(3)H]MK-3207 showed high binding densities concordant with mRNA and protein expression. The present study suggests that several regions in the brainstem may be involved in CGRP signaling. Interestingly, we found receptor expression and antagonist binding in some areas that are not protected by the blood-brain barrier, which suggests that drugs inhibiting CGRP signaling may not be able to penetrate the central nervous system to antagonize receptors in these brain regions.

The predicted ensemble of low-energy conformations of human somatostatin receptor subtype 5 and the binding of antagonists.

Human somatostatin receptor subtype 5 (hSSTR5) regulates cell proliferation and hormone secretion. However, the identification of effective therapeutic small-molecule ligands is impeded because experimental structures are not available for any SSTR subtypes. Here, we predict the ensemble of low-energy 3D structures of hSSTR5 using a modified GPCR Ensemble of Structures in Membrane BiLayer Environment (GEnSeMBLE) complete sampling computational method. We find that this conformational ensemble displays most interhelical interactions conserved in class A G protein-coupled receptors (GPCRs) plus seven additional interactions (e.g., Y2.43-D3.49, T3.38-S4.53, K5.64-Y3.51) likely conserved among SSTRs. We then predicted the binding sites for a series of five known antagonists, leading to predicted binding energies consistent with experimental results reported in the literature. Molecular dynamics (MD) simulation of 50 ns in explicit water and lipid retained the predicted ligand-bound structure and formed new interaction patterns (e.g. R3.50-T6.34) consistent with the inactive μ-opioid receptor X-ray structure. We suggest more than six mutations for experimental validation of our prediction. The final predicted receptor conformations and antagonist binding sites provide valuable insights for designing new small-molecule drugs targeting SSTRs.

Ligand-based homology modelling of the human CB2 receptor SR144528 antagonist binding site: a computational approach to explore the 1,5-diaryl pyrazole scaffold

SR144528 docking mode into the LBHM of the human CB2 receptor antagonist binding site.

Quantitation of In Vitro α-1 Adrenergic Receptor Antagonist Binding Capacity to Biologic Melanin Using Tandem Mass Spectrometry

Purpose: The purpose of this study was to develop methods to allow evaluation of the binding characteristics for a series of α-1 antagonists to biologically-derived melanin.Methods: Fresh bovine globes were used to obtain iridal and choroid/retinal pigment epithelial (CRPE) derived melanin. Binding characteristics of chloroquine, tamsulosin and doxazosin were then evaluated in vitro using tandem mass spectroscopy.Results: Tandem mass spectrometry-based assays were developed for three α-1 antagonists that provided linear assay ranges which spanned (minimally) 0.01–10 µg/mL, while exhibiting excellent inter-assay precision and accuracy. When applied to the evaluation of binding characteristics for iridal melanin, mean chloroquine and tamsulosin fractions were found to be 41.9 ± 14.2 pmoles mg−1 and 25.34 ± 6.186 pmoles mg−1, respectively. Mean iridal doxazosin binding was found to be 6.36 ± 2.19 pmoles mg−1. Interestingly, mean levels of tamsulosin, but not doxazosin found bound to choroid/CRPE derived melanin approached that of chloroquine (27.91 µg/mL, 25.68 µg/mL and 5.94 µg/mL for chloroquine, tamsulosin and doxazosin, respectively). One way ANOVA for binding affinity for chloroquine, tamsulosin and doxazosin was statistically significant for both iridal and CRPE-derived melanin (p = 0.0012 and 0.0023), respectively. A Bonferroni post-hoc analysis demonstrated a statistically significant difference in the amount of binding between tamsulosin, doxazosin and chloroquine to iridal but not CRPE derived melanin (p < 0.05).Conclusions: Tamsulosin appears to demonstrate melanin binding affinity which approaches chloroquine and exceeds doxazosin for both iridal and CRPE-derived bovine melanin.

Multiparametric Homogeneous Method for Identification of Ligand Binding to G Protein-Coupled Receptors: Receptor–Ligand Binding and β-Arrestin Assay

Two homogeneous assay systems have been combined to provide a new cell-based functional assay. The assay can be used to identify ligand binding to β(2)-adrenergic receptors, but also the downstream response can be determined in the same assay. Both the quenching resonance energy transfer (QRET) and the DiscoveRx PathHunter assay formats allow the use of intact cells. The homogeneous QRET technique is a single-label approach based on nonspecific quenching of the time-resolved luminescence, enabling agonist and antagonist receptor binding measurements. The commercial PathHunter assay is in turn based on enzyme fragment complementation, which can be detected on the basis of chemiluminescence signal. In the PathHunter technology the enzyme complementation is recorded immediately downstream of agonist-induced receptor activation. The new multiparametric detection technology combines these two assay methods enabling the identification of agonist, and antagonist binding to the receptor, and the agonist-induced response. Using the QRET and the PathHunter methods a panel of β(2)-adrenergic receptor ligands (epinephrine, terbutaline, metaproterenol, salmeterol, propranolol, alprenolol, bisoprolol, ICI 118,551, and bucindolol) was tested to prove the assay performance. The signal-to-background ratio for tested ligands ranged from 5 to 11 and from 6 to 18 with QRET and PathHunter, respectively. Combined homogeneous assay technique can provide an informative method for screening purposes and an efficient way to monitor receptor-ligand interaction, thus separating agonist from antagonist.

IL-37: a new anti-inflammatory cytokine of the IL-1 family

The IL-1 family of cytokines encompasses eleven proteins that each share a similar β-barrel structure and bind to Ig-like receptors. Some of the IL-1-like cytokines have been well characterised, and play key roles in the development and regulation of inflammation. Indeed, IL-1α (IL-1F1), IL-1β (IL-1F2), and IL-18 (IL-1F4) are well-known inflammatory cytokines active in the initiation of the inflammatory reaction and in driving Th1 and Th17 inflammatory responses. In contrast, IL-1 receptor antagonist (IL-1Ra, IL-1F3) and the receptor antagonist binding to IL-1Rrp2 (IL-36Ra, IL-1F5) reduce inflammation by blocking the binding of the agonist receptor ligands. In the case of IL-37 (IL-1F7), of which five different splice variants have been described, less is known of its function, and identification of the components of a heterodimeric receptor complex remains unclear. Some studies suggest that IL-37 binds to the α chain of the IL-18 receptor in a non-competitive fashion, and this may explain some of the disparate biological effects that have been reported for mice deficient in the IL-18R. The biological properties of IL-37 are mainly those of down-regulating inflammation, as assessed in models where human IL-37 is expressed in mice. In this review, an overview of the role of IL-37 in the regulation of inflammation is presented. The finding that IL-37 also locates to the nucleus, as do IL-1α and IL-33, for receptor-independent organ/tissue-specific regulation of inflammation is also reviewed.