Human rho-associated coiled-coil forming kinases (ROCKs) ROCK-I and ROCK-II have been documented as attractive therapeutic targets for cerebrovascular diseases. Although ROCK-I and ROCK-II share a high degree of structural conservation and are both present in classic rho/ROCK signaling pathway, their downstream substrates and pathological functions may be quite different. Selective targeting of the two kinase isoforms with traditional small-molecule inhibitors is a great challenge due to their surprisingly high homology in kinase domain (~90%) and the full identity in kinase active site (100%). Here, instead of developing small-molecule drugs to selectively target the adenosine triphosphate (ATP) site of two isoforms, we attempt to design peptide agents to selectively disrupt the homo-dimerization event of ROCK kinases through their dimerization domains which have a relatively low conservation (~60%). Three helical peptides H1, H2, and H3 are split from the kinase dimerization domain, from which the isolated H2 peptide is found to have the best capability to rebind at the dimerization interface. A simulated annealing (SA) iteration method is used to improve the H2 peptide selectivity between ROCK-I and ROCK-II. The method accepts moderate degradation in peptide affinity in order to maximize the affinity difference between peptide binding to the two isoforms. Consequently, hundreds of parallel SA runs yielded six promising peptide candidates with ROCK-I over ROCK-II (I over II [IoII]) calculated selectivity and four promising peptide candidates with ROCK-II over ROCK-I (II over I [IIoI]) calculated selectivity. Subsequent anisotropy assays confirm that the selectivity values range between 13.2-fold and 83.9-fold for IoII peptides, and between 5.8-fold and 21.2-fold for IIoI peptides, which are considerably increased relative to wild-type H2 peptide (2.6-fold for IoII and 2.0-fold for IIoI). The molecular origin of the designed peptide selectivity is also analyzed at structural level; it is revealed that the peptide residues can be classified into conserved, non-conserved, and others, in which the non-conserved residues play a crucial role in defining peptide selectivity, while conserved residues confer stability to kinase-peptide binding.

中文翻译：

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脑血管疾病中 ROCK-I 和 ROCK-II 激酶同种型之间二聚化破坏肽选择性的合理设计和改进。

人类 Rho 相关卷曲螺旋形成激酶 (ROCK) ROCK-I 和 ROCK-II 已被证明是脑血管疾病的有吸引力的治疗靶点。虽然 ROCK-I 和 ROCK-II 具有高度的结构保守性，都存在于经典的 rho/ROCK 信号通路中，但它们的下游底物和病理功能可能有很大不同。用传统的小分子抑制剂选择性靶向这两种激酶同工型是一个巨大的挑战，因为它们在激酶结构域中具有惊人的高度同源性 (~90%) 和激酶活性位点的完全同一性 (100%)。在这里，不是开发小分子药物来选择性靶向两种亚型的三磷酸腺苷 (ATP) 位点，我们尝试设计肽试剂，通过其具有相对较低保守性 (~60%) 的二聚化结构域来选择性破坏 ROCK 激酶的同源二聚化事件。三个螺旋肽 H1、H2 和 H3 从激酶二聚化结构域中分离出来，从中发现分离的 H2 肽在二聚化界面具有最佳的重新结合能力。模拟退火 (SA) 迭代方法用于提高 ROCK-I 和 ROCK-II 之间的 H2 肽选择性。该方法接受肽亲和力的适度降解，以最大化肽与两种同种型结合之间的亲和力差异。最后，数百个并行 SA 运行产生了六个有希望的候选肽，其中 ROCK-I 超过 ROCK-II（I 超过 II [IoII]）计算选择性和四个有希望的候选肽，ROCK-II 超过 ROCK-I（II 超过 I [IIoI]）计算选择性。随后的各向异性分析证实，IoII 肽的选择性值介于 13.2 倍和 83.9 倍之间，IoI 肽的选择性值介于 5.8 倍和 21.2 倍之间，相对于野生型 H2 肽而言，选择性值显着增加（对于 IoII 肽为 2.6 倍）。 IoII 和 IIoI 的 2.0 倍）。设计的肽选择性的分子起源也在结构水平上进行了分析；结果表明，肽残基可以分为保守的、非保守的和其他的，其中非保守的残基在定义肽选择性方面起着至关重要的作用，