title: Specific CLK inhibitors from a novel chemotype for regulation of alternative splicing.
abstract: 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).
authors.affiliation: University of Oxford, Nuffield Department of Clinical Medicine, Structural Genomics Consortium, Old Road Campus Research Building, Oxford OX37DQ, UK.
authors.forename: Oleg
authors.initials: O
authors.lastname: Fedorov
authors.forename: Kilian
authors.initials: K
authors.lastname: Huber
authors.forename: Andreas
authors.initials: A
authors.lastname: Eisenreich
authors.forename: Panagis
authors.initials: P
authors.lastname: Filippakopoulos
authors.forename: Oliver
authors.initials: O
authors.lastname: King
authors.forename: Alex N
authors.initials: AN
authors.lastname: Bullock
authors.forename: Damian
authors.initials: D
authors.lastname: Szklarczyk
authors.forename: Lars J
authors.initials: LJ
authors.lastname: Jensen
authors.forename: Doriano
authors.initials: D
authors.lastname: Fabbro
authors.forename: Jörg
authors.initials: J
authors.lastname: Trappe
authors.forename: Ursula
authors.initials: U
authors.lastname: Rauch
authors.forename: Franz
authors.initials: F
authors.lastname: Bracher
authors.forename: Stefan
authors.initials: S
authors.lastname: Knapp
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects*; D020134:Catalytic Domain; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006574:Heterocyclic Compounds, 2-Ring / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D009570:Nitriles / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013925:Thromboplastin / Q000235:genetics
chemical_list: D006574:Heterocyclic Compounds, 2-Ring; D009570:Nitriles; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C547711:enaminonitrile; D013925:Thromboplastin; C066780:Clk dual-specificity kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases