Design and Evolution of a Macrocyclic Peptide Inhibitor of the Sonic Hedgehog/Patched Interaction.
Design and Evolution of a Macrocyclic Peptide Inhibitor of the Sonic Hedgehog/Patched Interaction.
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DOI：
10.1021/jacs.7b06087
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发表时间：
2017-09-13
影响因子：
15
通讯作者：
Fasan R
中科院分区：
文献类型：
--
作者：
Owens AE;de Paola I;Hansen WA;Liu YW;Khare SD;Fasan R
Hedgehog（Hh）信号通路在胚胎发育过程中起着核心作用，其异常激活与多种人类癌症的发生和进展有关。鉴定Hedgehog途径的化学调节剂的主要努力已经产生了GPCR样Smoothened受体的几种拮抗剂。相比之下，Sonic Hedgehog/Patched相互作用的有效抑制剂，配体诱导的该信号通路激活中的最上游事件，一直难以捉摸。为了解决这一缺口，基于刺猬相互作用蛋白（HHIP）的Shh结合环设计了一个遗传编码的环肽，并通过筛选大肠杆菌中产生的大环肽文库进行多轮亲和力成熟。大肠杆菌细胞。使用这种方法，获得了优化的大环肽抑制剂（HL 2-m5），其以170 nM的KD结合Shh，这对应于与母体分子相比120倍的亲和力改善。重要的是，HL 2-m5能够有效抑制Shh介导的Hedgehog信号传导和活细胞中Gli控制的基因转录（IC 50 = 250 nM），提供了迄今为止报道的Sonic Hedgehog/Patched相互作用的最有效抑制剂。这种Hedgehog通路的一流大环肽调节剂有望为研究和靶向癌症和其他病理学中的配体依赖性Hedgehog通路激活提供有价值的探针。这项工作还介绍了一个一般的策略，为蛋白质-蛋白质相互作用的环肽抑制剂的发展。
The Hedgehog (Hh) signaling pathway plays a central role during embryonic development and its aberrant activation has been implicated in the development and progression of several human cancers. Major efforts toward the identification of chemical modulators of the Hedgehog pathway has yielded several antagonists of the GPCR-like Smoothened receptor. In contrast, potent inhibitors of the Sonic Hedgehog/Patched interaction, the most upstream event in ligand-induced activation of this signaling pathway, have been elusive. To address this gap, a genetically encoded cyclic peptide was designed based on the Shh-binding loop of Hedgehog-Interacting Protein (HHIP) and subjected to multiple rounds of affinity maturation through the screening of macrocyclic peptide libraries produced in E. coli cells. Using this approach, an optimized macrocyclic peptide inhibitor (HL2-m5) was obtained that binds Shh with a KD of 170 nM, which corresponds to a 120-fold affinity improvement compared to the parent molecule. Importantly, HL2-m5 is able to effectively suppress Shh-mediated Hedgehog signaling and Gli-controlled gene transcription in living cells (IC50 = 250 nM), providing the most potent inhibitor of the Sonic Hedgehog/Patched interaction reported to date. This first-in-class macrocyclic peptide modulator of the Hedgehog pathway is expected to provide a valuable probe for investigating and targeting ligand-dependent Hedgehog pathway activation in cancer and other pathologies. This work also introduces a general strategy for the development of cyclopeptide inhibitors of protein-protein interactions.
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