Oleg Fedorov 1 , 7 , Kilian Huber 2 , 7 , Andreas Eisenreich 3 , Panagis Filippakopoulos 1 , Oliver King 1 , Alex N. Bullock 1 , Damian Szklarczyk 4 , Lars J. Jensen 4 , Doriano Fabbro 5 , Jörg Trappe 5 , Ursula Rauch 3 , Franz Bracher 2 , ∗ , Stefan Knapp 1 , 6 , ∗∗
28 January 2011
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).
► We report a highly selective nanomolar inhibitor KH-CB19 for the kinases CLK1/4 and DYRK1 ► KH-CB19 cocrystal structures revealed an ATP competitive but not ATP mimetic binding mode ► KH-CB19 formed halogen bonds with the kinase hinge region ► KH-CB19 led to dephosphorylation of SR proteins and effected splicing of TF isoforms in cells