Department of Cardiovascular Sciences (Anaesthesia, Critical Care and Pain Management), University of Leicester, Leicester Royal Infirmary, Leicester, United Kingdom.
Department of Chemical and Pharmaceutical Sciences and LTTA, University of Ferrara, Ferrara, Italy.
PLoS One. 2021 Apr 23;16(4):e0250011. doi: 10.1371/journal.pone.0250011. eCollection 2021.
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.
μ-阿片肽（MOP）受体是阿片受体家族的成员，也是镇痛的重要临床靶点。传统上，测量 MOP 受体的位置并跟踪其周转率需要使用放射性标记物或抗体，但放射性标记物在全细胞中的应用存在问题，抗体的选择性也很差。为了解决这些问题，我们合成并表征了一种新型基于 ATTO488 的荧光德玛芬类似物；[Cys（ATTO488）8]德玛芬-NH2（DermATTO488）。我们最初使用放射性配体结合评估了 DermATTO488 在表达人 MOP 的 HEK 细胞和表达人 MOP、δ-阿片肽（DOP）、κ-阿片肽（KOP）和孤啡肽/孤啡肽 FQ 肽（NOP）受体的 CHO 细胞中的结合谱。通过测量（i）配体通过刺激 GTPγ[35S]结合来结合 G 蛋白的能力，以及（ii）刺激 ERK1/2 磷酸化的能力，评估了共轭肽的功能活性。使用共焦扫描激光显微镜观察受体位置。德玛芬和 DermATTO488 与 HEKMOP（pKi：8.29 和 7.00；p<0.05）、CHOMOP（pKi：9.26 和 8.12；p<0.05）和 CHODOP（pKi：7.03 和 7.16；p>0.05）结合。两种配体在 KOP 和 NOP 上均无活性。德玛芬和 DermATTO488 以相似的 pEC50（7.84 和 7.62；p>0.05）和 Emax（1.52 和 1.34 倍 p>0.05）值刺激 GTPγ[35S]结合。此外，德玛芬和 DermATTO488 在 5 分钟时产生 ERK1/2 磷酸化的单相刺激，峰值为 6.98 和 7.64 倍（p>0.05）。最后，在共焦显微镜下，DermATTO488 以浓度依赖的方式与 CHO 和 HEK 细胞上的重组 MOP 受体结合，这种结合可以通过用未标记的德玛芬或纳洛酮预孵育来阻断。总之，在德玛芬上添加 ATTO488 产生了一种与德玛芬相似的配体；对 DOP 的选择性约为 10 倍。这种新的配体 DermATTO488 保留了功能活性，可用于观察 MOP 受体的位置。
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