A triple exon-skipping luciferase reporter assay identifies a new CLK inhibitor pharmacophore

Bioorg Med Chem Lett. 2017 Feb 1;27(3):406-412. doi: 10.1016/j.bmcl.2016.12.056. Epub 2016 Dec 24.

Abstract

The splicing of pre-mRNA is a critical process in normal cells and is deregulated in cancer. Compounds that modulate this process have recently been shown to target a specific vulnerability in tumors. We have developed a novel cell-based assay that specifically activates luciferase in cells exposed to SF3B1 targeted compounds, such as sudemycin D6. This assay was used to screen a combined collection of approved drugs and bioactive compounds. This screening approach identified several active hits, the most potent of which were CGP-74514A and aminopurvalanol A, both have been reported to be cyclin-dependent kinases (CDKs) inhibitors. We found that these compounds, and their analogs, show significant cdc2-like kinase (CLK) inhibition and clear structure-activity relationships (SAR) at CLKs. We prepared a set of analogs and were able to 'dial out' the CDK activity and simultaneously developed CLK inhibitors with low nanomolar activity. Thus, we have demonstrated the utility of our exon-skipping assay and identified new molecules that exhibit potency and selectivity for CLK, as well as some structurally related dual CLK/CDK inhibitors.

Keywords: Anti-tumor agents; Cdc-like kinase (CLK) inhibitors; High throughput screening; Modulators of pre-mRNA splicing.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 2-Aminopurine / analogs & derivatives
  • 2-Aminopurine / chemistry
  • 2-Aminopurine / metabolism
  • Adenine / analogs & derivatives
  • Adenine / chemistry
  • Adenine / metabolism
  • Binding Sites
  • Cyclin-Dependent Kinases / antagonists & inhibitors*
  • Cyclin-Dependent Kinases / genetics
  • Cyclin-Dependent Kinases / metabolism
  • Exons
  • Genes, Reporter
  • High-Throughput Screening Assays
  • Humans
  • Inhibitory Concentration 50
  • Luciferases / genetics
  • Molecular Dynamics Simulation
  • Protein Binding
  • Protein Kinase Inhibitors / chemistry*
  • Protein Kinase Inhibitors / metabolism
  • Protein Structure, Tertiary
  • RNA Splicing
  • Structure-Activity Relationship

Substances

  • N(2)-(2-aminocyclohexyl)-N(6)-(3-chlorophenyl)-9-ethyl-9H-purine-2,6-diamine
  • Protein Kinase Inhibitors
  • aminopurvalanol A
  • 2-Aminopurine
  • Luciferases
  • Cyclin-Dependent Kinases
  • Adenine