Evaluation of [Cys(ATTO 488)8]Dermorphin-NH2 as a novel tool for the study of μ-opioid peptide receptors.

Autor: Giakomidi D; Department of Cardiovascular Sciences (Anaesthesia, Critical Care and Pain Management), University of Leicester, Leicester Royal Infirmary, Leicester, United Kingdom., Bird MF; Department of Cardiovascular Sciences (Anaesthesia, Critical Care and Pain Management), University of Leicester, Leicester Royal Infirmary, Leicester, United Kingdom., McDonald J; Department of Cardiovascular Sciences (Anaesthesia, Critical Care and Pain Management), University of Leicester, Leicester Royal Infirmary, Leicester, United Kingdom., Marzola E; Department of Chemical and Pharmaceutical Sciences and LTTA, University of Ferrara, Ferrara, Italy., Guerrini R; Department of Chemical and Pharmaceutical Sciences and LTTA, University of Ferrara, Ferrara, Italy., Chanoch S; Department of Cardiovascular Sciences (Anaesthesia, Critical Care and Pain Management), University of Leicester, Leicester Royal Infirmary, Leicester, United Kingdom., Sabu N; Department of Cardiovascular Sciences (Anaesthesia, Critical Care and Pain Management), University of Leicester, Leicester Royal Infirmary, Leicester, United Kingdom., Horley B; Department of Cardiovascular Sciences (Anaesthesia, Critical Care and Pain Management), University of Leicester, Leicester Royal Infirmary, Leicester, United Kingdom., Calo G; Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy., Lambert DG; Department of Cardiovascular Sciences (Anaesthesia, Critical Care and Pain Management), University of Leicester, Leicester Royal Infirmary, Leicester, United Kingdom.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2021 Apr 23; Vol. 16 (4), pp. e0250011. Date of Electronic Publication: 2021 Apr 23 (Print Publication: 2021).
DOI: 10.1371/journal.pone.0250011
Abstrakt: The μ-opioid peptide (MOP) receptor is a member of the opioid receptor family and an important clinical target for analgesia. Measuring MOP receptor location and tracking its turnover traditionally used radiolabels or antibodies with attendant problems of utility of radiolabels in whole cells and poor antibody selectivity. To address these issues we have synthesized and characterised a novel ATTO488 based fluorescent Dermorphin analogue; [Cys(ATTO 488)8]Dermorphin-NH2 (DermATTO488). We initially assessed the binding profile of DermATTO488 in HEK cells expressing human MOP and CHO cells expressing human MOP, δ-opioid peptide (DOP), κ-opioid peptide (KOP) and Nociceptin/Orphanin FQ peptide (NOP) receptors using radioligand binding. Functional activity of the conjugated peptide was assessed by measuring (i) the ability of the ligand to engage G-protein by measuring the ability to stimulate GTPγ[35S] binding and (ii) the ability to stimulate phosphorylation of ERK1/2. Receptor location was visualised using confocal scanning laser microscopy. Dermorphin and DermATTO488 bound to HEKMOP (pKi: 8.29 and 7.00; p<0.05), CHOMOP (pKi: 9.26 and 8.12; p<0.05) and CHODOP (pKi: 7.03 and 7.16; p>0.05). Both ligands were inactive at KOP and NOP. Dermorphin and DermATTO488 stimulated the binding of GTPγ[35S] with similar pEC50 (7.84 and 7.62; p>0.05) and Emax (1.52 and 1.34fold p>0.05) values. Moreover, Dermorphin and DermATTO488 produced a monophasic stimulation of ERK1/2 phosphorylation peaking at 5mins (6.98 and 7.64-fold; p>0.05). Finally, in confocal microscopy DermATTO488 bound to recombinant MOP receptors on CHO and HEK cells in a concentration dependent manner that could be blocked by pre-incubation with unlabelled Dermorphin or Naloxone. Collectively, addition to ATTO488 to Dermorphin produced a ligand not dissimilar to Dermorphin; with ~10fold selectivity over DOP. This new ligand DermATTO488 retained functional activity and could be used to visualise MOP receptor location.
Competing Interests: In the past DGL has held consultancy with Grunenthal (this does not alter our adherence to PLOS ONE policies on sharing data and materials) and is a non-executive director of the SME-CRO; Cellomatics. None of the remaining authors have conflicts to declare.
Databáze: MEDLINE
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