Chemical Inhibitors of Myosin Function

在筛选非肌肉肌球蛋白IIA(NMIIA)ATPase活性的化学抑制剂中发现了blbbistatin。它能抑制非洲爪哇和人类细胞的起泡和胞质分裂。我们测试了它与其他一些常规和非常规肌球蛋白亚型的活性。Blebbistatin抑制NMIIA重肌球蛋白(HMM)、NMIIB(HMM)、平滑肌HMM和几种横纹肌HMM的肌动蛋白激活的镁ATPase活性。肌动蛋白激活的镁ATPase的一半最大抑制所需的博尔比丁的量从兔骨骼肌HMM的0.4微摩尔到平滑肌HMM的80微摩尔不等。Blebbistatin，即使在100微摩尔时，也不能有效地抑制大鼠Myo1b、棘阿米巴肌球蛋白IC、肌球蛋白-V和肌毒素-X的活性。灯盏花素完全抑制NMIIA和骨骼肌HMM标记的罗丹明-鬼臼毒素标记的F-肌动蛋白的运动。这种作用在被Blebbistatin洗掉后是可逆的。用蓝光(488 Nm)照射后，其抑制活性被破坏。这一性质可用于研究Blebbistatin抑制细胞收缩过程的可逆性和同步收缩事件，但必须小心，因为Blebbistatin的失活似乎与自由基的释放有关。我们已经表征了Blebbistatin抑制肌球蛋白的动力学机制。影响磷酸盐释放的主要步骤，这意味着在博尔比斯坦存在的情况下，稳态产物将与肌动蛋白结合得很弱。我们已经表征了几种灯泡抑素衍生物的抑制特性，希望找到一种亲和力更高的衍生物，或者在蓝光存在下不会失活的衍生物。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，这种歧视可能是可能的，但到目前为止，这些衍生物的亲和力弱于原始的灯盏花素。已经制备了一种不会光灭活的硝基衍生物。

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.