Chemical Inhibitors of Myosin Function

肌球蛋白功能的化学抑制剂

• 批准号: 6967012
• 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/6967012
• 关键词: actins; cell cycle; cell motility; enzyme activity; enzyme inhibitors; inhibitor /antagonist; membrane transport proteins; myosins; photochemistry; protein structure function

Blebbistatin was discovered in a screen for chemical inhibitors of nonmuscle myosin IIA (NMIIA) ATPase activity. It inhibits blebbing and cytokinesis in Xenopus and human cells. We tested its activity against a number of other conventional and unconventional myosin isoforms. Blebbistatin inhibited the actin-activated MgATPase activity of NMIIA heavy meromyosin (HMM), NMIIB (HMM), smooth muscle HMM, and several striated muscle HMMs. The amount of blebbistatin required for half maximal inhibition of the actin-activated MgATPase is ranged from 0.4 micromolar for rabbit skeletal muscle HMM to 80 micromolar for smooth muscle HMM. Blebbistatin, even at 100 micromolar, did not effectively inhibit the activity of rat myo1b, Acanthamoeba myosin IC, myosin-V and myoxin-X. Blebbistatin completely inhibited the movement of rhodamine-phalloidin labeled F-actin by NMIIA and skeletal muscle HMM. This effect was reversible upon wash out of blebbistatin. The inhibitory activity of blebbistatin was destroyed by illumination with blue light (488 nm). This property could be useful in studying the reversibility of blebbistatin inhibition of contractile processes in cells and for synchronizing contractile events, but care must be taken since the inactivation of blebbistatin appears to be associated with the release of free radicals. We have characterized the kinetic mechanism for the blebbistatin inhibition of myosin. The main step that is affected in the release of phosphate which means that the steady-state product in the presence of blebbistatin will bind weakly to actin. We have characterized the inhibitory properties of several derivatives of blebbistatin in hopes of finding a derivative with higher affinity or one that does not inactivate in the presence of blue light. In addition, we are searching for molecules that will discriminate between the various nonmuscle myosin II isoforms. The preliminary data suggests that discrimination may be possible, but so far the affinity of the derivatives are weaker than that of the original blebbistatin. A nitro derivative has been prepared that does not photoinactivate.

Blebbistatin是在筛选非肌肉肌球蛋白IIA（NMIIA）ATP酶活性的化学抑制剂时发现的。它抑制非洲爪蟾和人类细胞的起泡和胞质分裂。我们测试了它对其他一些常规和非常规肌球蛋白亚型的活性。Blebbistatin可抑制NMIIA重酶解肌球蛋白（HMM）、NMIIB（HMM）、平滑肌HMM和几种横纹肌HHT的肌动蛋白激活的MgATPase活性。对于肌动蛋白激活的MgATP酶的半数最大抑制所需的blebbistatin的量范围为从兔骨骼肌HMM的0.4微摩尔到平滑肌HMM的80微摩尔。即使在100微摩尔浓度下，Blebbistatin也不能有效抑制大鼠myo 1b、阿米巴肌球蛋白IC、肌球蛋白-V和肌氧素-X的活性。Blebbistatin完全抑制NMIIA和骨骼肌HMM对罗丹明-鬼笔环肽标记的F-actin的运动。这种作用在blebbistatin洗脱后是可逆的。用蓝光（488 nm）照射可破坏blebbistatin的抑制活性。这种性质可能是有用的，在研究的可逆性blebbistatin抑制收缩过程中的细胞和同步收缩事件，但必须小心，因为blebbistatin的失活似乎与自由基的释放。我们已经表征了blebbistatin抑制肌球蛋白的动力学机制。在磷酸盐释放中受影响的主要步骤，这意味着在blebbistatin存在下的稳态产物将与肌动蛋白弱结合。我们已经表征了几种blebbistatin衍生物的抑制特性，希望找到一种具有更高亲和力的衍生物或在蓝光存在下不发光的衍生物。此外，我们正在寻找能够区分各种非肌肉肌球蛋白II亚型的分子。初步数据表明，区分可能是可能的，但到目前为止，衍生物的亲和力弱于原始blebbistatin。已经制备了一种硝基衍生物，它不会光致变色。