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).
Highlights
There are about 23,000 protein-coding genes in the human genome
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 full-length TF (flTF) and asHTF
We reported a novel class of potent and selective class III histone deacetylase inhibitors, which are structural hybrids between a common kinase inhibitor scaffold and the b-carboline alkaloid bauerine C (Figure 1A), having a unique 7,8-dichloro substitution pattern (Huber et al, 2010a)
Summary
There are about 23,000 protein-coding genes in the human genome. Alternative splicing leads to the expression of several protein isoforms with different and sometimes antagonistic functions (Pajares et al, 2007). Notable examples include pro- and antiapoptotic isoforms of Bcl-2 family members (Akgul et al, 2004) and pro- and antiangiogenic forms of VEGFA (Harper and Bates, 2008). This plasticity plays a fundamental role in tissue development and the cellular response to external stimuli, for example in the control of blood clotting (Eisenreich et al, 2009) and insulin action (Jiang et al, 2009). The deregulation of alternative splicing has been linked to numerous human pathologies (Ward and Cooper, 2010)
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