Development of an ENPP1 Fluorescence Probe for Inhibitor Screening, Cellular Imaging, and Prognostic Assessment of Malignant Breast Cancer

J Med Chem. 2019 Oct 24;62(20):9254-9269. doi: 10.1021/acs.jmedchem.9b01213. Epub 2019 Oct 3.


Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is a type II transmembrane glycoprotein that is involved in bone metabolism and insulin resistance, hydrolyzes 2',3'-cGAMP (a STING ligand that promotes innate immunity), and is associated with cancer stemness in breast cancers and glioblastoma. Therefore, ENPP1 is considered a candidate therapeutic target and/or biomarker for early diagnosis of malignant tumors. In this study, we designed and synthesized a sensitive ENPP1 fluorescence probe, Tokyo Green (TG) mAMP. We used it to screen a chemical library for non-phosphate ENPP1 inhibitors. Structural optimization of a selected hit afforded a potent and specific ENPP1 inhibitor. We further found that ENPP1 mRNA expression in tissue samples from patients with triple-negative breast cancer was significantly inversely related to recurrence-free survival (RFS) and overall survival (OS), and TG-mAMP assay revealed a significant difference in ENPP1 activity between ENPP1 high-expressing and ENPP1 low-expressing samples. Our results suggest that TG-mAMP assay might be a rapid and inexpensive tool for predicting the prognosis of patients with malignant breast cancers.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Breast Neoplasms / diagnosis*
  • Breast Neoplasms / metabolism
  • Breast Neoplasms / pathology
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Drug Screening Assays, Antitumor
  • Enzyme Inhibitors / chemistry*
  • Enzyme Inhibitors / metabolism
  • Enzyme Inhibitors / pharmacology
  • Female
  • Fluorescent Dyes / chemistry*
  • Fluorescent Dyes / metabolism
  • Fluorescent Dyes / pharmacology
  • Gene Expression / drug effects
  • Humans
  • Microscopy, Fluorescence
  • Phosphoric Diester Hydrolases / genetics
  • Phosphoric Diester Hydrolases / metabolism
  • Prognosis
  • Protein Binding
  • Pyrophosphatases / antagonists & inhibitors*
  • Pyrophosphatases / genetics
  • Pyrophosphatases / metabolism
  • Rats
  • Triple Negative Breast Neoplasms / metabolism
  • Triple Negative Breast Neoplasms / pathology


  • Enzyme Inhibitors
  • Fluorescent Dyes
  • Phosphoric Diester Hydrolases
  • ectonucleotide pyrophosphatase phosphodiesterase 1
  • Pyrophosphatases