1 Department of Biology and Biochemistry, University of Houston, Houston, TX 77024, USA.
2 Texas Medical Center, Texas Heart Institute, Houston, TX 77024, USA.
Exp Biol Med (Maywood). 2019 Aug;244(11):940-951. doi: 10.1177/1535370219849581. Epub 2019 May 27.
Rho-activated kinases (ROCKs) regulate many cellular functions such as proliferation, migration, and smooth muscle contractility, but they are also associated with pathogenesis of many human diseases such as heart failure and hypertension. We used phage display libraries to identify inhibitory polypeptides that bind to the ROCK1 catalytic domain, but do not compete with the ATP-binding pocket, by screening in the presence of high ATP concentrations (1 mM). Peptide7, a promising ROCK inhibitory peptide for both ROCK isoforms, measured at 1.45 ± 0.28 µM for ROCK1 (1–553) and 5.15 ± 1.15 µM for ROCK2. Peptide7 reduced cellular migration in wound healing assays. The binding epitope on ROCK1 was mapped to the flexible activation loop within the catalytic domain. Peptide alanine scanning mutants helped identify critical amino acids to generate optimized Peptide22. This compact ROCK inhibitor facilitated vascular relaxation, blocked neovascularization of endothelial cells, and inhibited MLC phosphatase phosphorylation. Our novel ROCK peptide inhibitors may provide potential treatment of hypertension and PAH progression.
Rho-activated kinases, known as ROCK(s), are significant signaling components in cells that lead alterations in cellular function. The central role of ROCK in smooth muscle cellular homeostasis makes it an important therapeutic target. Small molecule kinase inhibitors target enzyme active site competing for ATP binding. Although effective, ATP binding active sites are similar among very different kinases, and many small molecule inhibitors suffer from non-specific inactivation which as therapeutics can lead to substantial side effects. Here, we designed experiments to identify ROCK inhibitors that do not target ATP binding, rather develop peptides that inhibit ROCK in the presence of ATP. We identified a peptide that binds the activation loop of the enzyme and effectively inhibits activity. This will allow a development of a new class of drugs with exquisite specificity for the ROCK kinases and potentially revolutionize treatment of high blood pressure, cardiac hypertrophy, and many more diseases.
Rho 激活激酶（ROCK）调节许多细胞功能，如增殖、迁移和平滑肌收缩，但它们也与许多人类疾病的发病机制有关，如心力衰竭和高血压。我们使用噬菌体展示文库，通过在高 ATP 浓度（1mM）下筛选，来鉴定与 ROCK1 催化结构域结合但不与 ATP 结合口袋竞争的抑制性多肽。肽 7 是一种对两种 ROCK 同工型都有前途的 ROCK 抑制肽，对 ROCK1（1-553）的测定值为 1.45±0.28µM，对 ROCK2 的测定值为 5.15±1.15µM。肽 7 减少了划痕愈合试验中的细胞迁移。ROCK1 上的结合表位映射到催化结构域内的柔性激活环。肽丙氨酸扫描突变有助于确定产生优化肽 22 的关键氨基酸。这种紧凑的 ROCK 抑制剂促进血管舒张，阻断内皮细胞的新生血管形成，并抑制 MLC 磷酸酶磷酸化。我们的新型 ROCK 肽抑制剂可能为高血压和 PAH 进展提供潜在的治疗方法。
Rho 激活激酶，称为 ROCK（s），是导致细胞功能改变的细胞中重要的信号成分。ROCK 在平滑肌细胞稳态中的核心作用使其成为一个重要的治疗靶点。小分子激酶抑制剂靶向酶的活性位点，与 ATP 竞争结合。虽然有效，但 ATP 结合活性位点在非常不同的激酶中相似，许多小分子抑制剂因非特异性失活而受到影响，这在治疗中可能导致严重的副作用。在这里，我们设计了实验来鉴定不针对 ATP 结合的 ROCK 抑制剂，而是开发在 ATP 存在下抑制 ROCK 的肽。我们鉴定出一种与酶的激活环结合并有效抑制其活性的肽。这将允许开发一类具有对 ROCK 激酶极高特异性的新药，并可能彻底改变高血压、心脏肥大和许多其他疾病的治疗方法。
