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

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posted on 2021-08-25, 09:35 authored by Despina Giakomidi, Mark F Bird, John McDonald, Erika Marzola, Remo Guerrini, Serena Chanoch, Nidhuna Sabu, Barbara Horley, Girolamo Calo, David G Lambert
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.

Funding

DG Lambert British Journal of Anaesthesia (major funding) NO grant number - small funder, funded in 2019 NO role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript DG Lambert British Heart Foundation (minor funding) www.bhf.org Grant number: PG/18/54/33896. NO role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

History

Citation

Giakomidi D, Bird MF, McDonald J, Marzola E, Guerrini R, Chanoch S, et al. (2021) Evaluation of [Cys(ATTO 488)8]Dermorphin-NH2 as a novel tool for the study of μ-opioid peptide receptors. PLoS ONE 16(4): e0250011. https://doi.org/10.1371/journal.pone.0250011

Author affiliation

Department of Cardiovascular Sciences

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  • VoR (Version of Record)

Published in

PLoS One

Volume

16

Issue

4

Pagination

e0250011

Publisher

Public Library of Science

eissn

1932-6203

Acceptance date

2021-03-29

Copyright date

2020

Available date

2021-08-25

Language

eng

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