Specific CLK Inhibitors From a Novel Chemotype for Regulation of Alternative Splicing

Chem Biol. 2011 Jan 28;18(1):67-76. doi: 10.1016/j.chembiol.2010.11.009.

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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing / drug effects*
  • Catalytic Domain
  • Endothelial Cells / drug effects
  • Endothelial Cells / metabolism
  • Heterocyclic Compounds, 2-Ring / chemical synthesis
  • Heterocyclic Compounds, 2-Ring / chemistry
  • Heterocyclic Compounds, 2-Ring / pharmacology
  • Humans
  • Models, Molecular
  • Nitriles / chemical synthesis
  • Nitriles / chemistry
  • Nitriles / pharmacology
  • Phosphorylation / drug effects
  • Protein Kinase Inhibitors / chemical synthesis
  • Protein Kinase Inhibitors / chemistry*
  • Protein Kinase Inhibitors / pharmacology*
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors*
  • Protein-Serine-Threonine Kinases / chemistry
  • Protein-Serine-Threonine Kinases / metabolism
  • Protein-Tyrosine Kinases / antagonists & inhibitors*
  • Protein-Tyrosine Kinases / chemistry
  • Protein-Tyrosine Kinases / metabolism
  • RNA, Messenger / genetics
  • RNA-Binding Proteins / metabolism
  • Substrate Specificity
  • Thromboplastin / genetics

Substances

  • Heterocyclic Compounds, 2-Ring
  • Nitriles
  • Protein Kinase Inhibitors
  • RNA, Messenger
  • RNA-Binding Proteins
  • enaminonitrile
  • Thromboplastin
  • Clk dual-specificity kinases
  • Protein-Tyrosine Kinases
  • Protein-Serine-Threonine Kinases