1. Specific CLK Inhibitors from a Novel Chemotype for Regulation of Alternative Splicing
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Panagis Filippakopoulos, Ursula Rauch, Oliver N. King, Kilian Huber, Stefan Knapp, Damian Szklarczyk, Alex N. Bullock, Franz Bracher, Oleg Fedorov, Jörg Trappe, Doriano Fabbro, Andreas Eisenreich, and Lars Juhl Jensen
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Models, Molecular ,Clinical Biochemistry ,RNA-binding protein ,Biology ,Protein Serine-Threonine Kinases ,chemistry ,01 natural sciences ,Biochemistry ,Heterocyclic Compounds, 2-Ring ,Substrate Specificity ,Thromboplastin ,CLK1 ,03 medical and health sciences ,Splicing factor ,Protein splicing ,Catalytic Domain ,CLK3 ,Drug Discovery ,Nitriles ,Humans ,RNA, Messenger ,Phosphorylation ,Molecular Biology ,Protein Kinase Inhibitors ,antagonists and inhibitors ,030304 developmental biology ,0303 health sciences ,010405 organic chemistry ,Kinase ,Alternative splicing ,Endothelial Cells ,RNA-Binding Proteins ,General Medicine ,Protein-Tyrosine Kinases ,Protein-Serine-Threonine Kinases ,3. Good health ,0104 chemical sciences ,Alternative Splicing ,drug effects ,RNA splicing ,Molecular Medicine ,pharmacology ,metabolism ,chemical synthesis - Abstract
SummaryThere 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).
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