1,721,014 research outputs found
Novel ligands and assays for nociceptin/orphanin FQ and classical opioid receptors
No full textThe aim of the present study was twofold: pharmacologically characterize novel ligands and set-up and validate novel in vitro assays for nociceptin/orphanin FQ (N/OFQ) peptide (NOP) and classical opioid receptors. NOP and opioid receptors are 7TM receptors coupled with inhibitory G proteins; receptor activation leads to the inhibition of cAMP formation and calcium currents, and opening of potassium channels. Via these cellular inhibitory mechanisms, the N/OFQ – NOP receptor and classical opioid systems regulate a variety of biological functions both in the central nervous system and in the periphery.
The calcium mobilization assay has been and still is broadly used as primary screening for novel molecules in academic and industrial in vitro pharmacology laboratories. The use of chimeric G proteins allows to extend the calcium mobilization assay to virtually all types of G protein coupled receptors. This approach was previously used in our laboratories for characterizing NOP receptor ligands. In the frame of the present study, the calcium mobilization assay has been extended and validated for classical opioid receptors using a panel of standard opioid receptor agonists and antagonist. This test was used for investigating the pharmacological profile of novel opioid ligands including a series of morphine and oxymorphone analogues and novel cyclic endomorphin-2 derivatives. Calcium mobilization studies together with classical in vitro assays such as receptor binding, [35S]GTPγS binding and bioassays with isolated organs were applied to novel NOP receptor ligands including i) 3 different series of spiroxatrine derivatives; ii) the antagonist NiK-21273; iii) [X5]N/OFQ(1-13)-NH2 derivatives; iv) three tetrabranched derivatives of N/OFQ generated with an innovative chemical approach named peptide welding technology.
Recent data demonstrated that biased agonists, i.e. receptor ligands able to select which signaling pathways become activated upon binding to the receptor, may display advantages over unbiased ligands. In particular, in the field of opioids, G-protein (vs arrestin) biased agonists for the mu receptor displayed an increased therapeutic index associated to reduced tolerance liability. No data are yet available about biased agonism in the NOP receptor field. Therefore a novel bioluminescence resonance energy transfer (BRET) based assay was set-up for the NOP receptor. This method that allows to study both NOP/G-protein and NOP/β-arrestin interactions has been validated using a large panel of NOP ligands encompassing full and partial agonist as well as antagonist activity. The comparison of data achieved investigating NOP/G-protein and NOP/β-arrestin interaction allowed us to perform the very first study of biased agonism in the NOP receptor field.
In summary the studies performed in the frame of my PhD project extend our knowledge on the pharmacological profile of NOP and classical opioid receptors, provided to the scientific community novel compounds, pharmacologically characterized in detail, to be used as research tools and possibly as drug prototypes, and made available novel pharmacological assays useful for selecting fully innovative drugs such NOP receptor biased agonists
GPCR in Adipose Tissue Function—Focus on Lipolysis
Full text linkAdipose tissue can be divided anatomically, histologically, and functionally into two major entities white and brown adipose tissues (WAT and BAT, respectively). WAT is the primary energy depot, storing most of the bioavailable triacylglycerol molecules of the body, whereas BAT is designed for dissipating energy in the form of heat, a process also known as non-shivering thermogenesis as a defense against a cold environment. Importantly, BAT-dependent energy dissipation directly correlates with cardiometabolic health and has been postulated as an intriguing target for anti-obesity therapies. In general, adipose tissue (AT) lipid content is defined by lipid uptake and lipogenesis on one side, and, on the other side, it is defined by the breakdown of lipids and the release of fatty acids by lipolysis. The equilibrium between lipogenesis and lipolysis is important for adipocyte and general metabolic homeostasis. Overloading adipocytes with lipids causes cell stress, leading to the recruitment of immune cells and adipose tissue inflammation, which can affect the whole organism (metaflammation). The most important consequence of energy and lipid overload is obesity and associated pathophysiologies, including insulin resistance, type 2 diabetes, and cardiovascular disease. The fate of lipolysis products (fatty acids and glycerol) largely differs between AT: WAT releases fatty acids into the blood to deliver energy to other tissues (e.g., muscle). Activation of BAT, instead, liberates fatty acids that are used within brown adipocyte mitochondria for thermogenesis. The enzymes involved in lipolysis are tightly regulated by the second messenger cyclic adenosine monophosphate (cAMP), which is activated or inhibited by G protein-coupled receptors (GPCRs) that interact with heterotrimeric G proteins (G proteins). Thus, GPCRs are the upstream regulators of the equilibrium between lipogenesis and lipolysis. Moreover, GPCRs are of special pharmacological interest because about one third of the approved drugs target GPCRs. Here, we will discuss the effects of some of most studied as well as “novel” GPCRs and their ligands. We will review different facets of in vitro, ex vivo, and in vivo studies, obtained with both pharmacological and genetic approaches. Finally, we will report some possible therapeutic strategies to treat obesity employing GPCRs as primary target
Functional Selectivity Does Not Predict Antinociceptive/Locomotor Impairing Potencies of NOP Receptor Agonists
Full text linkNociceptin/orphanin FQ controls several functions, including pain transmission, via stimulation of the N/OFQ peptide (NOP) receptor. Here we tested the hypothesis that NOP biased agonism may be instrumental for identifying innovative analgesics. In vitro experiments were performed with the dynamic mass redistribution label free assay and the NOP non-peptide agonists Ro 65-6570, AT-403 and MCOPPB. In vivo studies were performed in wild type and β-arrestin 2 knockout mice using the formalin, rotarod and locomotor activity tests. In vitro all compounds mimicked the effects of N/OFQ behaving as potent NOP full agonists. In vivo Ro 65-6570 demonstrated a slightly higher therapeutic index (antinociceptive vs. motor impairment effects) in knockout mice. However, all NOP agonists displayed very similar therapeutic index in normal mice despite significant differences in G protein biased agonism. In conclusion the different ability of inducing G protein vs. β-arrestin 2 recruitment of a NOP agonist cannot be applied to predict its antinociceptive vs. motor impairment properties
A Multi-Angle Approach to Predict Peptide-GPCR Complexes: The N/OFQ-NOP System as a Successful AlphaFold Application Case Study
No full textWith nearly 700 structures solved and a growing number of customized structure prediction algorithms being developed at a fast pace, G protein-coupled receptors (GPCRs) are an optimal test case for validating new approaches for the prediction of receptor active state and ligand bioactive conformation complexes. In this study, we leveraged the availability of hundreds of peptide GPCRs in the active state and both classical homology and artificial intelligence (AI) based protein modeling combined with docking and AI-based peptide structure prediction approaches to predict the nociceptin/orphanin FQ-NOP receptor active state complex (N/OFQ-NOPa). The In Silico generated hypotheses were validated via the design, synthesis, and pharmacological characterization of novel linear N/OFQ(1-13)-NH2 analogues, leading to the discovery of a novel antagonist (3B; pK(B) = 6.63) bearing a single ring-constrained residue in place of the Gly(2)-Gly(3) motif of the N/OFQ message sequence (FGGF). While the experimental validation was ongoing, the availability of the Cryo-EM structure of the predicted complex enabled us to unambiguously validate the generated hypotheses. To the best of our knowledge, this is the first example of a peptide-GPCR complex predicted with atomistic accuracy (full complex C alpha RMSD < 1.0 & Aring;) and of the N/OFQ message moiety being successfully modified with a rigid scaffold
Probing non-peptide agonists binding at the human nociceptin/orphanin FQ receptor: a molecular modelling study
No full textThe N/OFQ-NOP receptor is a fascinating peptidergic system with the potential to be exploited for the development of analgesic drugs devoid of side effects associated with classical opioid signalling modulation. To date, up to four X-ray and cryo-EM structures of the NOP receptor in complex with the endogenous peptide agonist N/OFQ and three small molecule antagonists have been solved and released. Despite the available structural information, the details of selective small molecule agonist binding to the NOP receptor in the active state remain elusive. In this study, by leveraging the available structural information and using N/OFQ(1-13)-NH2 as a reference compound, we developed a computational protocol based on docking followed by short molecular dynamics (MD) simulations that can suggest small molecule agonist binding modes at the NOP receptor that are reproducible and stable over time in the solvated membrane-embedded receptor active state and in agreement with known structure-activity relationship (SAR) data
In vitro and in vivo pharmacological characterization of Pronetupitant, a prodrug of the neurokinin 1 receptor antagonist Netupitant
No full texttThe aim of the present study was to investigate the pharmacological activity of Pronetupitant, a novelcompound designed to act as prodrug of the NK1antagonist Netupitant. In receptor binding experi-ments Pronetupitant displayed high selectivity for the NK1receptor. In a calcium mobilization assayperformed on CHONK1cells Pronetupitant (100 nM, 15 min preincubation) behaved as an NK1antago-nist more potent than Netupitant (pKB8.72 and 7.54, respectively). In the guinea pig ileum bioassayPronetupitant antagonized the contractile effect of SP showing a similar potency as Netupitant (pKB≈ 9).Similar results were obtained with 5 min preincubation time while at 2 min only Pronetupitant pro-duced significant effects. In vivo in mice the intrathecal injection of 0.1 nmol SP elicited the typicalscratching, biting and licking (SBL) nociceptive response. This effect of SP was dose dependently(0.1–10 mg/kg) antagonized by Pronetupitant given intravenously 2 h before the peptide. Superimpo-sable results were obtained using Netupitant. Pharmacokinetic studies performed in rats demonstratethat Pronetupitant, after i.v. administration, is quickly (few minutes) and completely converted toNetupitant. Collectively the present results indicated that Pronetupitant acts in vitro as selective NK1antagonist more potent than Netupitant. However based on the short half-life measured for Pronetupi-tant in rats, the in vivo action of Pronetupitant can be entirely interpreted as due to its conversion to Netupitant
Synthesis, structural characterization and pharmacological evaluation of spiroxatrine analogs as potential nociceptin/orphanin FQ receptor ligands
No full textThe nociceptin/Orphanin FQ (N/OFQ) peptide (NOP) receptor is a G protein-coupled receptor with a high degree of structural homology (~ 60%) to the classical opioid receptors μ, δ and κ. The interaction between NOPand its endogenous agonist N/OFQ plays a key role in pain transmission, among other biological functions. Therefore, this system opened a new option for the treatment of acute and chronic pain possibly by generating drugs with a lower side effect profile.The confirmed affinity towards NOP receptor of the α2 adrenergic and 5-HT1A partial agonist spiroxatrine (Ki= 127 nM) has led us to the synthesis of a series of novel and optimized analogs based upon the spiropiperidinecore, in order to perform preliminarySAR studies
Beta-arrestin 2 rather than G protein efficacy determines the anxiolytic- versus antidepressant-like effects of nociceptin/orphanin FQ receptor ligands
No full textBackground and purpose Nociceptin/orphanin FQ (N/OFQ) receptor (NOP) agonists produce anxiolytic-like effects in rodents while antagonists promote antidepressant-like effects. The aim of this study was to investigate the effect on anxiety and depression of NOP receptor partial agonists such as the peptides [F/G]N/OFQ(1-13)NH2 and UFP-113 and the non-peptide AT-090. Experimental approach In vitro AT-090, UFP-113, and [F/G]N/OFQ(1-13)NH2 were tested for their ability to promote NOP/G-protein and NOP/β-arrestin 2 interaction, using a bioluminescence resonance energy transfer assay. In vivo, they were tested in mice in the elevated plus maze (EPM) and in the forced swim (FST) tests. NOP partial agonists effects were systematically compared to those of full agonists (N/OFQ and Ro 65-6570) and antagonists (UFP-101 and SB-612111). Key results In vitro, AT-090, UFP-113, and [F/G]N/OFQ(1-13)NH2 promoted NOP/G protein interaction, with maximal effects lower than those evoked by N/OFQ and Ro 65-6570. AT-090 behaved as a NOP partial agonist also in inducing β-arrestin 2 recruitment, while UFP-113 and [F/G]N/OFQ(1-13)NH2 were inactive in this assay. In vivo, AT-090 induced anxiolytic-like effects in the EPM but was inactive in the FST. Opposite results were obtained with UFP-113 and [F/G]N/OFQ(1-13)NH2. Conclusions and implications NOP ligands producing similar effects on NOP/G protein interaction (partial agonism) but showing different effects on β-arrestin 2 recruitment (partial agonism vs antagonism) elicited different actions on anxiety and mood. These results suggest that the action of a NOP ligand on emotional states is better predicted based on its β-arrestin 2 rather than G-protein efficacy
Pharmacological characterization of naloxegol: In vitro and in vivo studies
No full textOpioid-induced constipation is the most prevalent adverse effect of opioid drugs. Peripherally acting mu opioid receptor antagonists (PAMORAs), including naloxegol, are indicated for the treatment of opioid-induced constipation. The aim of this study was the in vitro and in vivo pharmacological characterization of naloxegol in comparison with naloxone. In vitro experiments were performed to measure calcium mobilization in cells coexpressing opioid receptors and chimeric G proteins and mu receptor interaction with G protein and β-arrestin 2 using bioluminescence resonance energy transfer. In vivo experiments were performed in mice to measure pain threshold using the tail withdrawal assay and colonic transit using the bead expulsion assay. In vitro, naloxegol behaved as a selective and competitive mu receptor antagonist similarly to naloxone, being 3-10-fold less potent. In vivo, naloxone was effective in blocking fentanyl actions when given subcutaneously (sc), but not per os (po). In contrast, naloxegol elicited very similar effects with sc or po administration counteracting in a dose dependent manner the constipating effects of fentanyl without interfering with the fentanyl mediated analgesia. Thus, a useful PAMORA action could be obtained with naloxegol both after po and sc administration
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