Azidoblebbistatin, a photoreactive myosin inhibitor
Azidoblebbistatin, a photoreactive myosin inhibitor
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DOI：
10.1073/pnas.1202786109
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发表时间：
2012-06-12
影响因子：
11.1
通讯作者：
Malnasi-Csizmadia, Andras
中科院分区：
文献类型：
--
作者：
Kepiro, Miklos;Varkuti, Boglarka H.;Malnasi-Csizmadia, Andras
光反应性化合物是生命科学中的重要工具，其允许配体和靶标的精确定时共价交联。我们采用独特的合成方法合成了应用最广泛的肌球蛋白抑制剂blebbistatin的衍生物azidoblebistatin。在没有UV照射的情况下，azidoblebbistatin表现出与blebbistatin相同的抑制特性。使用UV照射，azidoblebbistatin可以共价交联到肌球蛋白，这大大增强了其在体外和体内的有效性。光交联还消除了与blebbistatin相对低的肌球蛋白亲和力和水溶性相关的限制。用于光交联的波长对细胞和组织无毒，这在体内测试中具有很大的优势。由于交联导致抑制剂与肌球蛋白的不可逆缔合，并且照射消除了未结合的抑制剂分子的残余活性，azidoblebbistatin具有很大的潜力，成为肌动球蛋白收缩性的结构研究和肌球蛋白II的细胞和生理功能的研究中的高效工具。我们使用azidoblebbistatin在盘基网柄藻中鉴定了以前未知的抑制剂的低亲和力靶点（EC 50>= 50 μ M），而发现最强的相互作用物是肌球蛋白II（EC 50 = 5 μ M）。我们的研究结果表明，azidoblebbistatin，和潜在的其他叠氮药物，可以成为非常有用的工具，用于识别强和弱结合的细胞靶点和确定在体内条件下的表观结合亲和力。
Photoreactive compounds are important tools in life sciences that allow precisely timed covalent crosslinking of ligands and targets. Using a unique technique we have synthesized azidoblebbistatin, which is a derivative of blebbistatin, the most widely used myosin inhibitor. Without UV irradiation azidoblebbistatin exhibits identical inhibitory properties to those of blebbistatin. Using UV irradiation, azidoblebbistatin can be covalently crosslinked to myosin, which greatly enhances its in vitro and in vivo effectiveness. Photo-crosslinking also eliminates limitations associated with the relatively low myosin affinity and water solubility of blebbistatin. The wavelength used for photo-crosslinking is not toxic for cells and tissues, which confers a great advantage in in vivo tests. Because the crosslink results in an irreversible association of the inhibitor to myosin and the irradiation eliminates the residual activity of unbound inhibitor molecules, azidoblebbistatin has a great potential to become a highly effective tool in both structural studies of actomyosin contractility and the investigation of cellular and physiological functions of myosin II. We used azidoblebbistatin to identify previously unknown low-affinity targets of the inhibitor (EC50 >= 50 mu M) in Dictyostelium discoideum, while the strongest interactant was found to be myosin II (EC50 = 5 mu M). Our results demonstrate that azidoblebbistatin, and potentially other azidated drugs, can become highly useful tools for the identification of strong-and weak-binding cellular targets and the determination of the apparent binding affinities in in vivo conditions.