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Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Pain remains a key therapeutic area with intensive efforts directed toward finding effective and safer analgesics in light of the ongoing opioid crisis. Amongst the neurotransmitter systems involved in pain perception and modulation, the mu-opioid receptor MOR , a G protein-coupled receptor, represents one of the most important targets for achieving effective pain relief. Most clinically used opioid analgesics are agonists to the MOR, but they can also cause severe side effects. Medicinal plants represent important sources of new drug candidates, with morphine and its semisynthetic analogues as well-known examples as analgesic drugs. In this study, combining in silico pharmacophore-based virtual screening and docking and pharmacological in vitro binding and functional assays, and behavioral tests approaches, we report on the discovery of two naturally occurring plant alkaloids, corydine and corydaline, as new MOR agonists that produce antinociceptive effects in mice after subcutaneous administration via a MOR-dependent mechanism. Thus, these new scaffolds represent valuable starting points for future chemical optimization towards the development of novel opioid analgesics, which may exhibit improved therapeutic profiles. Naturally occurring opioid alkaloids, such as morphine Fig. Over several decades, new opioids with diverse scaffolds were synthesized, pharmacologically evaluated and clinically used as the most effective class of analgesic drugs 2 , 3 , 4 , 5. However, all currently available opioid analgesics share a similar spectrum of undesirable side effects, including respiratory depression, constipation, sedation, nausea and analgesic tolerance 5 , 6. Additionally, the potential for addiction and abuse of opioids has seriously hindered their clinical application, with a huge rise in opioid misuse and overdose deaths resulting in an ongoing and rapidly emerging opioid epidemic worldwide 7 , 8. Currently, intensive research focuses on finding new, innovative medications and technologies to treat opioid addiction, together with the discovery of safe, effective, non-addictive drugs to manage chronic pain 9 , 10 , 11 , Opioids produce their pharmacological effects through the activation of opioid receptors, which include three main types, mu MOR , delta DOR and kappa KOR 13 , 14 , of which the MOR type is the primary target of most clinically used opioid analgesics 3 , 5. Opioid receptors share high homology and belong to the superfamily of seven transmembrane-spanning G protein-coupled receptors GPCRs. The detailed structural information of the MOR available nowadays 15 , 16 , 17 , as well as the emerging concept of biased agonism to the MOR 5 , 12 , 18 , provide innovative research directions that not only aid to understand MOR-mediated signaling and its pharmacology, but also offer novel opportunities for the discovery of new opioid therapeutics Further, an important source of new drug candidates is represented by natural product medicines, having a long history of use in the treatment and prevention of many human diseases 20 , Natural products and their derivatives account for about half of approved drugs Morphine Fig. In the search for ligands with new chemotypes and further understanding the mechanism by which known ligands i. We have previously reported on a virtual screening campaign that led to the identification of novel chemotypes that displayed MOR antagonism in vitro and in vivo In this study, we generated a collection of virtual screening protocols based on different in silico methods, such as pharmacophore- and shape-based modelling and docking. After theoretical validation, we prospectively applied these protocols to a library of synthetic compounds, and the MOR activity of three virtual screening hits could be confirmed experimentally. Structural analogues of one of these validated hits were reported as natural products isolated from different Berberis species This prompted us to apply the computational models to an in-house library containing, beyond others, also Berberis constituents. In the present study, by combining molecular modeling and pharmacological approaches, we report on the discovery of two plant-derived alkaloids, corydine 1 and corydaline 2 Fig. We have previously reported the generation, validation, and prospective application of a set of MOR agonist and antagonist pharmacophore models Whereas the three agonist models mapped mainly MOR agonists during theoretical validation, the antagonist models proved to have little discriminative power i. In this study, we have used this set of MOR agonist and antagonist pharmacophore models to screen a small in-house library of naturally occurring alkaloids and synthetic analogues. As none of the molecules matched any of the models when mapping of all features was required, the number of omitted features was increased to one, which means that compounds are also recognized as potentially active compounds if they miss one of the model features. Using these settings, we retrieved 15 virtual hits. The central role of Asp and Tyr of peptides, morphinans ligands, and other chemotypes for binding to the MOR is well recognized 22 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , Further, both of these interactions appeared to be critical for ligand binding in our previous study Mapping of these features was therefore chosen as requirement for virtual hits in order to be subjected to experimental testing. Based on the current results, we have selected seven natural products, corydine 1 , corydaline 2 , bulbocapnine 3 , thalictricavine 4 , bernumidine 6 , intebrimine 7 and capnosinine 8 , and one natural product analogue, 2- 2,3-dimethoxybenzyl -6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline 9 Figs. They were mapped by one compounds 2 — 4 and 6 , two compounds 1 , 7 , and 9 or even three pharmacophore models compound 8 Table 1. Figure 4 shows corydine 1 and corydaline 2 aligned to the pharmacophore models pm-ag-lig-model-1 and pm-ag-4dkl-model, respectively. Due to the structural similarity, berberine 5 was also added to the list of test compounds. Exemplary virtual hit compounds mapping MOR agonist pharmacophore models. Out of the nine compounds selected for biological testing, eight compounds 1 — 8 are natural products, whereas 9 is a synthetic compound, structurally related to the natural product intebrimine 7 Figs. A synopsis of their origin and known bioactivities is presented in the Supporting Information. The initial biological screening was performed using a competitive radioligand binding assay to the human MOR with the eight natural products 1 — 8 and the synthetic compound 9. Therefore, they were selected for further investigations of their MOR activities. Corydaline 2 displayed a binding affinity as K i value to the MOR about 2-times higher than that on corydine 1 , although it was much lower than the MOR affinity of morphine Table 2. In vitro activity profiles of corydine 1 and corydaline 2 to the human MOR. The assay was performed according to earlier described procedures As shown in Fig. Corydine 1 displayed about 3-times greater potency compared to corydaline 2 , while being times less potent as an agonist than the reference MOR agonist DAMGO Table 3. While the G protein-mediated signaling is linked to beneficial effects i. The concept of biased agonism or functional selectivity was introduced as a means to separate desirable and adverse drug responses 8 , 35 , and the in vivo relevance of this phenomenon has attained much attention in the past years 18 , Targeting biased agonism to the MOR has gained significance for drug discovery over the recent years, where G protein-biased MOR agonists may deliver the desired analgesia without liability for unwanted side effects 27 , 36 , 37 , 38 , The MOR agonist activity of corydine 1 and corydaline 2 was further evaluated in vivo in a mouse model of chemical sensitivity, the writhing assay, a widely used model of visceral pain This test involves intraperitoneal i. Both compounds showed antinociceptive effects in mice after subcutaneous s. Compared to morphine 0. The antiwrithing response of corydine 1 and corydaline 2 was antagonized by the MOR antagonist naltrexone, demonstrating a MOR-mediate mechanism of action Fig. Antinociceptive effects of corydine 1 , corydaline 2 , and the reference MOR ligand morphine in the acetic acid-induced writhing assay in mice after s. Mice received s. Experimentally, we established that corydine 1 displays a more potent MOR activation despite lower binding affinity compared to corydaline 2 Tables 2 and 3. In our experience, corydine 1 shows a highly unusual profile. To investigate whether we could find a potential structural explanation for this observation, we have generated in silico binding models to the MOR for the two compounds. As these water molecules appear to have a functional role, we included them in the structural modelling. Both corydine 1 and corydaline 2 Fig. To account for these differences and allow for some structural adaptions, we decided to employ the induced fit docking procedure in Maestro Analysis of the proposed binding modes suggests that the observed differences in receptor activation could be due to alterations of the water network mediating interactions between the agonists and the MOR in the active conformation. In our model, corydine 1 is involved in a similar water network as BU72 Fig. On the other hand, corydaline 2 may require an additional water molecule to maintain this water network Fig. Notably, this additional water molecule occupies a similar position as the BUOH group in the crystal structure Fig. Predicted binding modes of corydine 1 and corydaline 2 to the MOR. In addition, interaction with Lys is lost. Among other targets, multiple dopamine receptor subtypes were predicted, in line with previous reports by Ma et al. Furthermore, to rule out that the observed in vivo effects were mediated by potential active metabolites, we subjected metabolites of corydaline 2 16 — 22 described in Ji et al. Similar to the parent compounds, metabolites were also projected to have activity at the dopamine receptors along other targets. A detailed summary of investigated metabolites and predicted targets is presented in Figure S2 and Table S3. Noteworthy, MOR was not suggested as target for neither parent compounds nor metabolites, indicating that corydine 1 and corydaline 2 indeed represent novel chemical scaffolds to this receptor. In the present study, combining in silico pharmacophore-based virtual screening and docking and pharmacological in vitro and in vivo assays approaches, we report on the discovery of two natural products, corydine 1 and corydaline 2 , as new MOR agonists that produce antinociceptive effects in mice after s. Among the neurotransmitter systems involved in pain perception and modulation, the opioid system, particularly the MOR, is one of the most important 5. Most clinically available opioid analgesics are agonists to the MOR that are highly effective in relieving pain, but they also have severe side effects, including abuse and misuse liability 5 , 6 , 7 , 8. Medicinal plants are tremendous sources of new drug candidates 20 , During the past decades, there has been a renewed interest in natural product research due to the drawback of alternative drug discovery methods to deliver lead compounds in key therapeutic areas, such as pain. Natural products are a robust source of unique structural scaffolds. The study of psychoactive natural products had a continuous influence on the understanding of their function in the central nervous system 23 , 49 , Evidence on the neuropsychiatric effects of natural agonists to the KOR in humans comes from experience with salvinorin A, the main active psychotropic molecule in Salvia divinorum Chemical derivatization and modification of psychoactive natural products have provided and continues to offer innovative scientific and therapeutic discoveries. The progress in medicinal chemistry, drug discovery technologies and significant advances in structural biology of GPCRs by means of modern methodological and powerful computational systems 26 , 27 , 49 , 52 plays an essential role in such discoveries. We have previously reported the generation, validation, and application of a set of MOR agonist and antagonist pharmacophore models By using this collection of models for pharmacophore-based virtual screening, corydine 1 and corydaline 2 were identified as active ligands to the MOR, albeit they interact with the MOR relatively weakly. It is commonly recognized that hits identified in a virtual screening campaign often display weaker activity than the compounds the models were based on 26 , Our docking study to the MOR revealed that both compounds share several essential receptor-ligand interactions, including the salt bridge with Asp and hydrogen bond formation with Tyr water-mediated in the case of corydaline 2 , analogous to BU72 , as two residues recognized as key interaction sites for ligand small molecules and peptides binding to the MOR 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , However, there are also receptor-ligand interaction pattern dissimilarities. The experimental differences in receptor activation, with corydine 1 being more potent than corydaline 2 , are possibly due to alterations of the water network mediating interactions between the agonists and the MOR in the active conformation. Corydaline 2 may require an additional water molecule to maintain the water network, and we hypothesize that it is therefore less potent in activating the receptor. Corydine 1 and corydaline 2 are two naturally occurring alkaloids in different Corydalis and Berberis species 45 , 55 , 56 , 57 , 58 , 59 , 60 that are used as medicinal plants to treat pain spastic pain, abdominal pain, or pain due to injury and other human ailments 61 , 62 , 63 , Plant extracts and isolated alkaloids, mostly corydaline 2 , were reported to produce antinociceptive effects in rodents 59 , 65 , 66 , 67 , 68 , although no mechanism of action was associated to the observed pain inhibitory effects so far. Further, pharmacokinetic studies demonstrated that corydaline 2 can effectively cross the blood—brain barrier in rats 69 , with O -demethylation and hydroxylation as the major metabolic pathways in human liver In vitro investigations, using cell-based functional assays, established corydaline 2 to bind to the dopamine D1 receptor with antagonist activity 45 , 46 , whereas no molecular target was attributed to the biological effects of corydine 1. Our in silico profiling study also revealed that multiple dopamine receptor subtypes have been prioritized by SEA 44 for both compounds as well as for metabolities of corydine 1 and corydaline 2. In the present study, we show that corydine 1 and corydaline 2 bind to the MOR and are full agonists to the receptor, and produce MOR-mediated antinociceptive effects in a mouse model of visceral pain writhing assay after s. Besides analgesia, MOR agonists are well-known to induce other physiological and behavioral responses 5 , 6. While generally, no major alterations in locomotor activity and no sedation were observed in animals at the tested doses of corydine 1 and corydaline 2 , studies on side effects profiling may be of future interest. Altogether, our findings indicate that the applied MOR pharmacophore models and virtual screening workflows have a clear potential for the discovery of novel bioactive molecules to the MOR. The new chemotypes, corydine 1 and corydaline 2 as natural products, showed MOR biased agonist properties, thus representing valuable starting points for further chemical optimization toward the development of novel opioid analgesics with potentially reduced side effects. A conformational database was generated for the in house compounds using Omega 2. A maximum number of conformers were calculated per molecule. For pharmacophore based virtual screening with LigandScout 3. Default settings were used except that the maximum number of omitted features was increased to 1. A longer stretch of the N-terminus is resolved in this structure compared to e. This N-terminal section is unlikely to participate in binding of the structurally unrelated alkaloids 1 and 2 , but may render parts of the binding site inaccessible. Therefore, residues 52—63 were deleted. All water molecules except , , , , , , , and were removed. The structure was then prepared with the Protein Preparation Wizard 74 in Maestro release —4 Briefly, bond orders were assigned, hydrogens added, selenomethionines were converted to methionines, missing side chains and loops were added, termini were capped, het states were generated, H-bonds assignment was refined, and a restrained minimization was conducted. The prepared structure was then used for induced fit docking of BU72 as a control, and corydine 1 and corydaline 2. The co-crystallized ligand BU72 was used to define the docking site and the default induced fit docking settings were applied. For results validation, corydaline 2 was also submitted, and the predicted metabolites were compared to the experimentally identified ones reported in Ji et al. Corydine 1 , corydaline 2 , bulbocapnine 3 and thalictricavine 4 were isolated from Corydalis cava as previously described The natural products 6 — 8 were commercially acquired. Bernumidine 6 was obtained from Pharmeks Moscow, Russia. Cell culture media and supplements were obtained from Sigma-Aldrich Chemicals St. Louis, MO. Naltrexone hydrochloride was purchased from Siegfried Ltd Zofingen, Switzerland. All other chemicals were of analytical grade and obtained from standard commercial sources. For in vitro assays, morphine and U69, were prepared as 1 mM stocks in water. Compounds 1 — 9 and naltrindole were prepared as 1 mM stocks in 0. Stock solutions were further diluted to working concentrations in the appropriate medium. In vitro binding assays were conducted on human opioid receptors stably transfected into CHO cells according to the published procedures Protein content of cell membrane preparations was determined by the method of Bradford using bovine serum albumin as the standard The inhibitory constant K i values were calculated from the competition binding curves by nonlinear regression analysis and the Cheng-Prusoff eqaution All experiments were performed in duplicate, and repeated at least three times with independently prepared samples. All experiments were performed in duplicate and repeated at least three times with independently prepared samples. All animal studies were conducted in accordance with ethical guidelines and animal welfare standards according to Austrian regulations for animal research and were approved by the Committee of Animal Care of the Austrian Federal Ministry of Science and Research. Test compounds or vehicle were administered by s. Writhing was induced in mice by intraperitoneal i. Drugs or control vehicle were s. Each mouse was placed in individual transparent Plexiglas chambers, and the number of writhes was counted during a 10 min observation period. Each experimental group included five to six animals. Experimental data were analyzed and graphically processed using the GraphPad Prism 5. Devereaux, A. DARK classics in chemical neuroscience: Morphine. ACS Chem. The chemical and pharmacological importance of morphine analogues. Acta Physiol. PubMed Google Scholar. Spetea, M. Armenian, P. Fentanyl, fentanyl analogs and novel synthetic opioids: A comprehensive review. Neuropharmacology , — Pasternak, G. Emerging insights into mu opioid pharmacology. Benyamin, R. Opioid complications and side effects. Pain Physician 11 , S—S Seth, P. Overdose deaths involving opioids, cocaine, and psychostimulants—United States, — MMWR Morb. Volkow, N. Prevention and treatment of opioid misuse and addiction: A review. JAMA Psychiatry 76 , — Baumann, M. Pharmacological research as a key component in mitigating the opioid overdose crisis. Trends Pharmacol. Skolnick, P. The opioid epidemic: Crisis and solutions. Ehrlich, A. Current strategies toward safer mu opioid receptor drugs for pain management. Expert Opin. Targets 23 , — Turnaturi, R. Progress in the development of more effective and safer analgesics for pain management. Stein, C. Opioid receptors. Corder, G. Endogenous and exogenous opioids in pain. Manglik, A. Crystal structure of the mu-opioid receptor bound to a morphinan antagonist. Nature , — Huang, W. Koehl, A. Grim, T. Toward directing opioid receptor signaling to refine opioid therapeutics. Psychiatry 87 , 15—21 Valentino, R. Untangling the complexity of opioid receptor function. Neuropsychopharmacology 43 , — Li, J. Drug discovery and natural products: End of an era or an endless frontier?. Science , — Newman, D. Kruegel, A. Synthetic and receptor signaling explorations of the Mitragyna alkaloids: Mitragynine as an atypical molecular framework for opioid receptor modulators. The medicinal chemistry and neuropharmacology of kratom: A preliminary discussion of a promising medicinal plant and analysis of its potential for abuse. ACS Cent. Cui, X. Ligand interaction, binding site and G protein activation of the mu opioid receptor. Kaserer, T. Structure-based discovery of opioid analgesics with reduced side effects. Noha, S. Sutcliffe, K. Wtorek, K. Endomorphin-2 analogs containing modified tyrosines: Biological and theoretical investigation of the influence on conformation and pharmacological profile. Obeng, S. Ellis, C. Surratt, C. Mu opiate receptor Charged transmembrane domain amino acids are critical for agonist recognition and intrinsic activity. Mansour, A. Reiter, E. Zhou, L. Functional selectivity of GPCR signaling in animals. CAS Google Scholar. DeWire, S. Schmid, C. Bias factor and therapeutic window correlate to predict safer opioid analgesics. Cell , — Martin, C. Biodegradable amphipathic peptide hydrogels as extended-release system for opioid peptides. Kenakin, T. Signaling bias in new drug discovery: Detection, quentification and therapeutic impact. Drug Discov. Le Bars, D. Animal models of nociception. Keiser, M. Relating protein pharmacology by ligand chemistry. Ma, Z. Isoquinoline alkaloids isolated from Corydalis yanhusuo and their binding affinities at the dopamine D1 receptor. Molecules 13 , — Wu, L. Identification of alkaloids from Corydalis yanhusuo W. Wang as dopamine D1 receptor antagonists by using CRE-luciferase reporter gene assay. Molecules 23 , PubMed Central Google Scholar. Ji, H. In vitro metabolism of corydaline in human liver microsomes and hepatocytes using liquid chromatography-ion trap mass spectrometry. Rankovic, Z. McClatchey, W. Ethnobotany as a pharmacological research tool and recent developments in CNS-active natural products from ethnobotanical sources. Prevatt-Smith, K. New therapeutic potential for psychoactive natural products. Di Marzo, V. Endocannabinoids: Synthesis and degradation. Bermudez, M. Strategies for the discovery of biased GPCR ligands. Today 24 , — Scior, T. Recognizing pitfalls in virtual screening: A critical review. Model 52 , — Dumitrascuta, M. Molecules 25 , Karimov, A. Berberis alkaloids. Structure of turcberine. Khim Prirodn Soedin 1 , 77—81 Google Scholar. Adsersen, A. Acetylcholinesterase and butyrylcholinesterase inhibitory compounds from Corydalis cava Schweigg. Wang, X. Preparative isolation of alkaloids from Dactylicapnos scandens using pH-zone-refining counter-current chromatography by changing the length of the separation column. B Analyt. Life Sci. Han, J. In vivo disease control efficacy of isoquinoline alkaloids isolated from Corydalis ternata against wheat leaf rust and pepper anthracnose. Wang, C. Screening of antinociceptive components in Corydalis yanhusuo W. Ruiz, A. Flavonols, alkaloids, and antioxidant capacity of edible wild berberis species from Patagonia. Food Chem. Wang, J. Future Med. China Pharmacopoeia Committee. Blumenthal, M. The complete German E monographs-therapeutic guide to herbal medicines. Sezik, E. Traditional medicine in Turkey II. Folk medicine in Kastamonu. Chang, H. Study on chemical constituents of Dicranostigma leptopodium Maxim. Yaoxue Tongbao. Qiu, Z. Comparative study between Rhizoma Corydalis processing with vinegar and cleansing Rhizoma Corydalis in anti-inflammatory effect and analgesic effect. Li, R. Comparative study for pharmacological action of Corydalis Rhizoma before and after processing. Tang, W. Chinese drugs of plant origin: Chemistry, pharmacology, and use in traditional and modern medicine — Springer, Berlin, Preclinical pharmacokinetics, tissue distribution and excretion studies of a potential analgesics—corydaline using an ultra performance liquid chromatography-tandem mass spectrometry. OMEGA 2. Hawkins, P. Conformer generation with OMEGA: Algorithm and validation using high quality structures from the protein databank and cambridge structural database. Model 50 , — Wolber, G. LigandScout: 3-D pharmacophores derived from protein-bound ligands and their use as virtual screening filters. Model 45 , — Inte:Ligand GmbH LigandScout 3. Vienna: Inte:Ligand GmbH. Madhavi Sastry, G. Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. Aided Mol. Sturm, S. Analysis of central European Corydalis species by nonaqueous capillary electrophoresis—electrospray ion trap mass spectrometry. A , 41—50 Bradford, M. A Rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Cheng, Y. Relationship between the inhibition constant k1 and the concentration of inhibitor which causes 50 per cent inhibition i50 of an enzymatic reaction. Download references. We thank Inte:Ligand and OpenEye for providing academic licenses for their programs. You can also search for this author in PubMed Google Scholar. All authors have given approval to the final version of the manuscript. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Reprints and permissions. Identification and characterization of plant-derived alkaloids, corydine and corydaline, as novel mu opioid receptor agonists. Sci Rep 10 , Download citation. Received : 10 June Accepted : 27 July Published : 14 August Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma. Skip to main content Thank you for visiting nature. Download PDF. Abstract Pain remains a key therapeutic area with intensive efforts directed toward finding effective and safer analgesics in light of the ongoing opioid crisis. Site selective C—H functionalization of Mitragyna alkaloids reveals a molecular switch for tuning opioid receptor signaling efficacy Article Open access 22 June Chemical composition and biological effects of kratom Mitragyna speciosa : In vitro studies with implications for efficacy and drug interactions Article Open access 05 November Introduction Naturally occurring opioid alkaloids, such as morphine Fig. Figure 1. Full size image. Figure 2. Chemical structures of corydine 1 and corydaline 2. Results Molecular modeling and virtual screening We have previously reported the generation, validation, and prospective application of a set of MOR agonist and antagonist pharmacophore models Figure 3. Chemical structures of compounds 3 — 9. Table 1 MOR pharmacophore models mapping selected test compounds 1—9. Full size table. Figure 4. Figure 5. Figure 6. Table 2 In vitro binding affinities of corydine 1 and corydaline 2 to the MOR. Table 3 In vitro functional activities of corydine 1 and corydaline 2 to the MOR. Figure 7. Figure 8. Discussion and conclusions In the present study, combining in silico pharmacophore-based virtual screening and docking and pharmacological in vitro and in vivo assays approaches, we report on the discovery of two natural products, corydine 1 and corydaline 2 , as new MOR agonists that produce antinociceptive effects in mice after s. Materials and methods In silico methods. Virtual screening A conformational database was generated for the in house compounds using Omega 2. Compounds, chemicals and reagents Corydine 1 , corydaline 2 , bulbocapnine 3 and thalictricavine 4 were isolated from Corydalis cava as previously described Competitive radioligand binding assays In vitro binding assays were conducted on human opioid receptors stably transfected into CHO cells according to the published procedures Acetic acid-induced writhing test Writhing was induced in mice by intraperitoneal i. Data and statistical analysis Experimental data were analyzed and graphically processed using the GraphPad Prism 5. References Devereaux, A. Google Scholar Adsersen, A. Google Scholar Chang, H. Google Scholar Qiu, Z. Google Scholar Li, R. Google Scholar Wang, J. Google Scholar Bradford, M. Acknowledgements We thank Inte:Ligand and OpenEye for providing academic licenses for their programs. View author publications. Ethics declarations Competing interests The authors declare no competing interests. Additional information Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary information. Supplementary file1 PDF kb. About this article. Cite this article Kaserer, T. Copy to clipboard. This article is cited by N-substituted tetrahydro-beta-carboline as mu-opioid receptors ligands: in silico study; molecular docking, ADMET and molecular dynamics approach Waleed A. Alananzeh Mohammed N. Al-qattan Mohd N. Publish with us For authors Language editing services Submit manuscript. Search Search articles by subject, keyword or author. Show results from All journals This journal. Advanced search. Close banner Close. Email address Sign up. Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research.

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