Peterson Jeffrey R, Lebensohn Andres M, Pelish Henry E, Kirschner Marc W
Division of Basic Sciences, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, Pennsylvania 19111, USA.
Chem Biol. 2006 Apr;13(4):443-52. doi: 10.1016/j.chembiol.2006.02.009.
Identification of small-molecule targets remains an important challenge for chemical genetics. We report an approach for target identification and protein discovery based on functional suppression of chemical inhibition in vitro. We discovered pirl1, an inhibitor of actin assembly, in a screen conducted with cytoplasmic extracts. Pirl1 was used to partially inhibit actin assembly in the same assay, and concentrated biochemical fractions of cytoplasmic extracts were added to find activities that suppressed pirl1 inhibition. Two activities were detected, separately purified, and identified as Arp2/3 complex and Cdc42/RhoGDI complex, both known regulators of actin assembly. We show that pirl1 directly inhibits activation of Cdc42/RhoGDI, but that Arp2/3 complex represents a downstream suppressor. This work introduces a general method for using low-micromolar chemical inhibitors to identify both inhibitor targets and other components of a signaling pathway.
小分子靶点的鉴定仍然是化学生物学面临的一项重要挑战。我们报告了一种基于体外化学抑制功能抑制的靶点鉴定和蛋白质发现方法。我们在使用细胞质提取物进行的筛选中发现了肌动蛋白组装抑制剂pirl1。在同一实验中，使用pirl1部分抑制肌动蛋白组装，并添加细胞质提取物的浓缩生化组分以寻找抑制pirl1抑制作用的活性。检测到两种活性，分别进行纯化，并鉴定为Arp2/3复合物和Cdc42/RhoGDI复合物，二者均为已知的肌动蛋白组装调节因子。我们表明，pirl1直接抑制Cdc42/RhoGDI的激活，但Arp2/3复合物是下游抑制因子。这项工作引入了一种通用方法，即使用低微摩尔浓度的化学抑制剂来鉴定抑制剂靶点和信号通路的其他组分。
