Fedorov Oleg, Huber Kilian, Eisenreich Andreas, Filippakopoulos Panagis, King Oliver, Bullock Alex N, Szklarczyk Damian, Jensen Lars J, Fabbro Doriano, Trappe Jörg, Rauch Ursula, Bracher Franz, Knapp Stefan
University of Oxford, Nuffield Department of Clinical Medicine, Structural Genomics Consortium, Old Road Campus Research Building, Oxford OX37DQ, UK.
Chem Biol. 2011 Jan 28;18(1):67-76. doi: 10.1016/j.chembiol.2010.11.009.
There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile KH-CB19, a potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Cocrystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix αC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19 effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue factor isoforms flTF (full-length TF) and asHTF (alternatively spliced human TF).
人们越来越认识到蛋白激酶在控制可变剪接中的重要性。为了确定潜在的调控机制，需要高选择性抑制剂。在此，我们报告二氯吲哚基烯胺腈KH-CB19的发现和特性，它是细胞周期蛋白依赖性激酶样激酶亚型1和4（CLK1/CLK4）的一种有效且高度特异性的抑制剂。KH-CB19与CLK1和CLK3的共晶体结构揭示了一种非ATP模拟结合模式、αC螺旋和磷酸结合环的构象变化以及与激酶铰链区的卤素键合。KH-CB19有效抑制细胞中SR（丝氨酸/精氨酸）蛋白的磷酸化，与其预期的作用机制一致。CLK1/CLK4的化学抑制产生了一种独特的剪接因子去磷酸化模式，并且在低纳摩尔浓度下对两种组织因子亚型全长TF（flTF）和可变剪接的人类TF（asHTF）的剪接有深远影响。
