Chemical Inhibitors of Myosin Function

肌球蛋白功能的化学抑制剂

• 批准号: 7158542
• 负责人: JAMES R. SELLERS
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7158542
• 关键词: Acanthamoeba; Dictyostelium; actins; binding sites; cell cycle; cell growth regulation; cell motility; computer simulation; cytotoxicity; drug screening /evaluation; enzyme activity; enzyme inhibitors; heterocyclic compounds; inhibitor /antagonist; membrane transport proteins; muscle function; muscle pharmacology; myosins; pharmacokinetics; photobiology; photochemistry; protein isoforms; protein structure function; small molecule

Blebbistatin is a small molecule inhibitor discovered in a screen for inhibitors of nonmuscle myosin IIA. We have examined the specificity and potency of the drug by assaying its effects on the actin-activated MgATPase assay of diverse members of the myosin superfamily. Blebbistatin potently inhibits several striated muscle myosins as well as vertebrate nonmuscle myosin IIA and IIB with IC50 values ranging from 0.5 to 5 microM. Interestingly, smooth muscle, which is highly homologous to vertebrate nonmuscle myosin, is only poorly inhibited (IC50=80 microM). The drug potently inhibits Dictyostelium myosin II, but poorly inhibits Acanthamoeba myosin II. Blebbistatin did not inhibit representative myosin superfamily members from classes I, V, and X. In cells, blebbistatin has been shown to inhibit contraction of the cytokinetic ring. Blebbistatin has some photochemical properties that may affect its behavior in cells. In particular, we have found that exposure to light at wavelengths below 488 nm rapidly inactivates the inhibitory action of blebbistatin using the in vitro motility of myosin as an assay. In addition, the inhibition of cytokinetic ring contraction can be reversed by exposure of the cells to blue light. This property may be useful in locally reversing the action of blebbistatin treatment in a cell. However, caution should be exercised as free radicals may be produced upon irradiation of blebbistatin that could result in cell damage. We conducted a detailed investigation of blebbistatin?s mechanism of inhibition. Blebbistatin does not compete with nucleotide binding to the skeletal muscle myosin subfragment-1. The inhibitor preferentially binds to the ATPase intermediate with ADP and phosphate bound at the active site, and it slows down phosphate release. Blebbistatin interferes neither with binding of myosin to actin nor with ATP-induced actomyosin dissociation. Instead, it blocks the myosin heads in a product complex with low actin affinity. Blind docking molecular simulations indicate that the productive blebbistatin-binding site of the myosin head is within the aqueous cavity between the nucleotide pocket and the cleft of the actin-binding interface. The property that blebbistatin blocks myosin II in an actin-detached state makes the compound useful both in muscle physiology and in exploring the cellular function of cytoplasmic myosin II isoforms, whereas the stabilization of a specific myosin intermediate confers a great potential in structural studies.

Blebbistatin是在筛选非肌肉肌球蛋白IIA抑制剂时发现的一种小分子抑制剂。我们已经检查了药物的特异性和效力，通过测定其对肌球蛋白超家族的不同成员的肌动蛋白激活的MgATPase测定的影响。Blebbistatin有效抑制几种横纹肌肌球蛋白以及脊椎动物非肌肉肌球蛋白IIA和IIB，IC 50值范围为0.5至5 μ M。有趣的是，与脊椎动物非肌肉肌球蛋白高度同源的平滑肌仅受到很差的抑制（IC 50 =80 μ M）。该药物有效抑制网囊藻肌球蛋白II，但对阿米巴肌球蛋白II的抑制作用较差。Blebbistatin不抑制I类、V类和X类肌球蛋白超家族成员。在细胞中，已显示blebbistatin抑制细胞动力学环的收缩。Blebbistatin具有一些光化学性质，可能会影响其在细胞中的行为。特别地，我们已经发现，暴露于波长低于488 nm的光下，使用肌球蛋白的体外运动性作为测定，使blebbistatin的抑制作用迅速失活。此外，细胞动力学环收缩的抑制可以通过将细胞暴露于蓝光来逆转。该性质可用于局部逆转细胞中的blebbistatin治疗的作用。但是，应谨慎操作，因为照射blebbistatin后可能产生自由基，可能导致细胞损伤。 我们对blebbistatin进行了详细的调查？的抑制机制。Blebbistatin不与核苷酸竞争结合骨骼肌肌球蛋白亚片段-1。该抑制剂优先与ATP酶中间体结合，ADP和磷酸盐结合在活性位点，并减缓磷酸盐释放。Blebbistatin既不干扰肌球蛋白与肌动蛋白的结合，也不干扰ATP诱导的肌动球蛋白解离。相反，它阻断了低肌动蛋白亲和力产物复合物中的肌球蛋白头。盲对接分子模拟表明，生产blebbistatin结合位点的肌球蛋白头是在水性腔之间的核苷酸口袋和肌动蛋白结合界面的裂缝。blebbistatin块肌球蛋白II在肌动蛋白分离状态的属性，使该化合物在肌肉生理学和探索细胞质肌球蛋白II亚型的细胞功能，而一个特定的肌球蛋白中间体的稳定赋予结构研究的巨大潜力。