Fluorescence probe for lysophospholipase C/NPP6 activity and a potent NPP6 inhibitor

J Am Chem Soc. 2011 Aug 10;133(31):12021-30. doi: 10.1021/ja201028t. Epub 2011 Jul 18.


Nucleotide pyrophosphatases/phosphodiesterases (NPPs) are ubiquitous membrane-associated or secreted ectoenzymes that have a role in regulating extracellular nucleotide and phospholipid metabolism. Among the members of the NPP family, NPP1 and -3 act on nucleotides such as ATP, while NPP2, -6, and -7 act on phospholipids such as lysophosphatidylcholine and sphingomyelin. NPP6, a recently characterized NPP family member, is a choline-specific glycerophosphodiester phosphodiesterase, but its functions remain to be analyzed, partly due to the lack of highly sensitive activity assay systems and practical inhibitors. Here we report synthesis of novel NPP6 fluorescence probes, TG-mPC and its analogues TG-mPC(3)C, TG-mPC(5)C, TG-mPENE, TG-mPEA, TG-mPhos, TG-mPA, TG-mPMe, and TG-mPPr. Among the seven NPPs, only NPP6 hydrolyzed TG-mPC, TG-mPC(3)C, and TG-mPENE. TG-mPC was hydrolyzed in the cell lysate from NPP6-transfected cells, but not control cells, showing that it is suitable for use in cell-based NPP6 assays. We also examined the usefulness of TG-mPC as a fluorescence imaging probe. We further applied TG-mPC to carry out high-throughput NPP6 inhibitor screening and found several NPP6-selective inhibitors in a library of about 80,000 compounds. Through structure-activity relationship (SAR) analysis, we identified a potent and selective NPP6 inhibitor with an IC(50) value of 0.21 μM. Our NPP6-selective fluorescence probe, TG-mPC, and the inhibitor are expected to be useful to elucidate the biological function of NPP6.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Dose-Response Relationship, Drug
  • Enzyme Activation / drug effects
  • Enzyme Inhibitors / chemical synthesis
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology*
  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / pharmacology*
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Lysophospholipase / antagonists & inhibitors*
  • Lysophospholipase / chemistry
  • Lysophospholipase / metabolism*
  • Mice
  • Molecular Structure
  • NIH 3T3 Cells
  • Phosphoric Diester Hydrolases / chemistry
  • Phosphoric Diester Hydrolases / metabolism*
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Structure-Activity Relationship


  • Enzyme Inhibitors
  • Fluorescent Dyes
  • Recombinant Proteins
  • Lysophospholipase
  • Phosphoric Diester Hydrolases
  • ENPP6 protein, human