Evaluation of [Cys(ATTO 488)(8)]Dermorphin-NH(2) as a novel tool for the study of μ-opioid peptide receptors

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-NH(2) (Derm(ATTO488)). We initially assessed the binding profile of Derm(ATTO488) 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[(35)S] binding and (ii) the ability to stimulate phosphorylation of ERK1/2. Receptor location was visualised using confocal scanning laser microscopy. Dermorphin and Derm(ATTO488) bound to HEK(MOP) (pK(i): 8.29 and 7.00; p<0.05), CHO(MOP) (pK(i): 9.26 and 8.12; p<0.05) and CHO(DOP) (pK(i): 7.03 and 7.16; p>0.05). Both ligands were inactive at KOP and NOP. Dermorphin and Derm(ATTO488) stimulated the binding of GTPγ[(35)S] with similar pEC(50) (7.84 and 7.62; p>0.05) and E(max) (1.52 and 1.34fold p>0.05) values. Moreover, Dermorphin and Derm(ATTO488) produced a monophasic stimulation of ERK1/2 phosphorylation peaking at 5mins (6.98 and 7.64-fold; p>0.05). Finally, in confocal microscopy Derm(ATTO488) 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 Derm(ATTO488) retained functional activity and could be used to visualise MOP receptor location.