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

Abstract

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.

Development of a High-Throughput Assay to Identify Inhibitors of ENPP1

“…19 ENPP1 Inhibitor C (6-[(3-aminophenyl)methyl]-N,N,5-trimethyl- [1,2,4]triazolo [1,5-a]pyrimidin-7-amine) was identified as a selective inhibitor with an IC 50 of 10 µM. 17 POM-1 and PSB-069 are members of the polyoxometalate family of compounds, inorganic cluster metal complexes, that have been shown to inhibit ENPP1 18,19 as well as NTPDase 1 (CD39). The four compounds were tested first using subsaturating substrate concentrations (10 µM cGAMP or ATP).…”

“…Dose-response curves for the ENPP1 inhibitors were conducted with both substrates to determine IC 50 values and correlate the experimental values with those reported in the literature. 13,16,17…”

“…In addition, interference from optically active screening compounds can be problematic for colorimetric assays and fluorescent assays at wavelengths less than far red. [15][16][17] The AMP Glo method can be employed, but its limited sensitivity and the use of multiple coupling enzymes make it susceptible to interference. 7 Here, we describe a direct, high-throughput, sensitive, and robust ENPP1 assay based on the selective detection of AMP and GMP with fluorescent polarization readout 18,19 using the Transcreener AMP 2 /GMP 2 Assay.…”

Construction of a Prognostic Signature in Ewing’s Sarcoma: Based On Metabolism-Related Genes

“…Therefore, ENPP1 can also be used as a biomarker for the diagnosis of malignant tumors and used to predict the prognosis of patients. [24] .…”

Fluorescence Differentiation of ATP-Related Multiple Enzymatic Activities in Synovial Fluid as a Marker of Calcium Pyrophosphate Deposition Disease Using Kyoto Green

“…Enzymatic conversion of ATP by the combination of ENPP1 and apyrase was monitored in real time by Kyoto Green. Theoretically, active ENPP1 should enhance fluorescence emission at 521 nm due to production of PPi, whereas dephosphorylation of ATP into AMP and Pi by apyrase was [25]. The conventional ENPP1 assay employs p-nitrophenyl 5'-thymidine monophosphate (p-Nph-5'-TMP) as a substrate [26,27].…”