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

• Published in: SLAS discovery Vol. 26; no. 5; pp. 740 - 746
• Main Authors: Kumar, Meera, Lowery, Robert G.
• Format: Journal Article
• Language: English
• Published: Los Angeles, CA Elsevier Inc 01.06.2021; SAGE Publications
• Subjects: ATP hydrolysis; cancer; cGAMP; Drug Discovery - methods; ENPP1; Fluorescence Polarization Immunoassay - methods; High-Throughput Screening Assays - methods; Humans; Phosphodiesterase Inhibitors - chemistry; Phosphodiesterase Inhibitors - pharmacology; Phosphoric Diester Hydrolases - chemistry; Pyrophosphatases - antagonists & inhibitors; Pyrophosphatases - chemistry; Transcreener; Transcreener; ENPP1; cancer; cGAMP; ATP hydrolysis
• ISSN: 2472-5552; 2472-5560
• DOI: 10.1177/2472555220982321

The innate immune response to cancer is initiated by cytosolic DNA, where it binds to cGAS and triggers type I interferon (IFN) expression via the STING receptor, leading to activation of tumor-specific T cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as the primary enzyme responsible for degrading cGAMP, and therefore it is under intense investigation as a therapeutic target for cancer immunotherapy. ENPP1 hydrolyzes cGAMP to produce AMP and GMP, and hydrolyzes ATP and other nucleotides to monophosphates and pyrophosphate. We developed a robust, high-throughput screening (HTS)-compatible enzymatic assay method for ENPP1 using the Transcreener AMP2/GMP2 Assay, a competitive fluorescence polarization (FP) immunoassay that enables direct detection of AMP and GMP in a homogenous format. The monoclonal antibody used in the Transcreener AMP2/GMP2 Assay showed more than 104-fold selectivity for AMP and GMP versus cGAMP, and 3000-fold selectivity for AMP over ATP, indicating that the assay can be used for detection at initial velocity with either substrate. A working concentration of 100 pM ENPP1 was determined as optimal with a 60 min reaction period, enabling screening with very low quantities of enzyme. A Z′ value of 0.72 was determined using ATP as substrate, indicating a high-quality assay. Consistent with previous studies, we found that ENPP1 preferred ATP as a substrate when compared with other nucleotides like GTP, ADP, and GDP. ENPP1 showed a 20-fold selectivity for 2′3′cGAMP compared with 2′3′c-diGMP and showed no activity with 3′3′c-diAMP. The Transcreener AMP2/GMP2 Assay should prove to be a valuable tool for the discovery of ENPP1 lead molecules.