DataSheet1_Lipidated Calcitonin Gene-Related Peptide (CGRP) Peptide Antagonists Retain CGRP Receptor Activity and Attenuate CGRP Action In Vivo.docx

Signaling through calcitonin gene-related peptide (CGRP) receptors is associated with pain, migraine, and energy expenditure. Small molecule and monoclonal antibody CGRP receptor antagonists that block endogenous CGRP action are in clinical use as anti-migraine therapies. By comparison, the potential utility of peptide antagonists has received less attention due to suboptimal pharmacokinetic properties. Lipidation is an established strategy to increase peptide half-life in vivo. This study aimed to explore the feasibility of developing lipidated CGRP peptide antagonists that retain receptor antagonist activity in vitro and attenuate endogenous CGRP action in vivo. CGRP peptide analogues based on the archetypal CGRP receptor antagonist, CGRP8-37, were palmitoylated at the N-terminus, position 24, and near the C-terminus at position 35. The antagonist activities of the lipidated peptide analogues were tested in vitro using transfected Cos-7 cells expressing either the human or mouse CGRP receptor, amylin subtype 1 (AMY1) receptor, adrenomedullin (AM) receptors, or calcitonin receptor. Antagonist activities were also evaluated in SK-N-MC cells that endogenously express the human CGRP receptor. Lipidated peptides were then tested for their ability to antagonize endogenous CGRP action in vivo using a capsaicin-induced dermal vasodilation (CIDV) model in C57/BL6J mice. All lipidated peptides except for the C-terminally modified analogue retained potent antagonist activity compared to CGRP8-37 towards the CGRP receptor. The lipidated peptides also retained, and sometimes gained, antagonist activities at AMY1, AM1 and AM2 receptors. Several lipidated peptides produced robust inhibition of CIDV in mice. This study demonstrates that selected lipidated peptide antagonists based on αCGRP8-37 retain potent antagonist activity at the CGRP receptor and are capable of inhibition of endogenous CGRP action in vivo. These findings suggest that lipidation can be applied to peptide antagonists, such as αCGRP8-37 and are a potential strategy for antagonizing CGRP action.

降钙素基因相关肽（calcitonin gene-related peptide，CGRP）受体信号通路与疼痛、偏头痛及能量消耗密切相关。能够阻断内源性CGRP作用的小分子及单克隆抗体类CGRP受体拮抗剂，已作为抗偏头痛疗法投入临床使用。相较而言，肽类拮抗剂的应用潜力因药代动力学特性欠佳而未受到足够重视。脂质化是经证实可延长肽类在体半衰期的有效策略。本研究旨在探索开发脂质化CGRP肽类拮抗剂的可行性，这类拮抗剂需在体外保留受体拮抗活性，并在体内减弱内源性CGRP的作用。以经典CGRP受体拮抗剂CGRP8-37为母核的CGRP肽类似物，分别在N末端、第24位氨基酸位点以及C末端附近的第35位氨基酸位点进行了棕榈酰化修饰。随后，采用转染了人源或鼠源CGRP受体、淀粉素亚型1（amylin subtype 1，AMY1）受体、肾上腺髓质素（adrenomedullin，AM）受体或降钙素受体的Cos-7细胞，在体外检测各脂质化肽类似物的拮抗活性；同时在内源表达人源CGRP受体的SK-N-MC细胞中，同样评估了其拮抗活性。之后，利用C57/BL6J小鼠的辣椒素诱导皮肤血管扩张（capsaicin-induced dermal vasodilation，CIDV）模型，在体内测试脂质化肽类拮抗内源性CGRP作用的能力。实验结果显示，除C末端修饰的肽类似物外，其余所有脂质化肽类对CGRP受体的拮抗活性均与未修饰的CGRP8-37相当甚至更强。脂质化肽类对AMY1、AM1及AM2受体同样保留了拮抗活性，部分甚至有所提升。多款脂质化肽类可在小鼠体内对CIDV反应产生显著抑制作用。本研究证实，基于αCGRP8-37筛选得到的脂质化肽类拮抗剂，能够在CGRP受体上保留强效拮抗活性，并可在体内抑制内源性CGRP的作用。上述发现表明，脂质化策略可应用于αCGRP8-37这类肽类拮抗剂的开发，是拮抗CGRP作用的潜在可行方案。