Chemical Inhibitors of Myosin Function

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。我们通过分析其对肌球蛋白超家族不同成员的肌动蛋白激活的镁ATPase测定的影响，检验了该药物的特异性和有效性。Blebbistatin能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，其IC50值从0.5微米到5微米。有趣的是，与脊椎动物非肌肉肌球蛋白高度同源的平滑肌，其抑制作用很差(IC50=80微米)。该药物有效地抑制了Dictyostoma myosin II，但对棘阿米巴myosin II的抑制很差。blebistatin不抑制I、V和X类肌球蛋白超家族的代表性成员。在细胞中，blebistatin已被证明抑制细胞动态环的收缩。Blebbistatin具有一些光化学性质，可能会影响其在细胞中的行为。特别是，我们发现暴露在波长低于488 nm的光中可以迅速钝化Blebbistatin的抑制作用，并使用肌球蛋白的体外运动作为检测手段。此外，细胞动态环收缩的抑制可以通过将细胞暴露在蓝光下而逆转。这一性质可用于局部逆转细胞内博莱菌素治疗的作用。然而，应谨慎行事，因为照射灯盏花素可能会产生自由基，从而可能导致细胞损伤。 我们对起泡抑素？S的抑制机制进行了详细的探讨。Blebbistatin不与与骨骼肌肌球蛋白亚段1结合的核苷酸竞争。该抑制剂优先结合在活性部位结合了ADP和磷酸盐的ATPase中间体，从而减缓磷酸盐的释放。Blebbistatin既不干扰肌球蛋白与肌动蛋白的结合，也不干扰ATP诱导的肌球蛋白解离。相反，它会阻止肌动蛋白亲和力较低的产物复合体中的肌球蛋白头部。盲法对接分子模拟表明，肌球蛋白头部有效的blbbistatin结合部位位于核苷酸口袋和肌动蛋白结合界面裂隙之间的水腔内。Blebbistatin在肌动蛋白分离的状态下阻断肌球蛋白II的特性使该化合物在肌肉生理学和探索细胞质肌球蛋白II亚型的细胞功能方面都很有用，而特定肌球蛋白中间体的稳定在结构研究中赋予了巨大的潜力。

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.