Related Experiment Video
Updated: Nov 22, 2025
A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1
Published on: April 16, 2021
Development of a High-Throughput Assay to Identify Inhibitors of ENPP1
Meera Kumar1, Robert G Lowery1
1BellBrook Labs, Madison, WI, USA.
Abstract:
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.
Insights
A new assay accurately measures ENPP1 activity, a key enzyme in cancer immunity. This high-throughput method aids in developing novel cancer immunotherapies by identifying ENPP1 inhibitors.
Area of Science:
- Immunology
- Biochemistry
- Drug Discovery
Background:
- The innate immune response to cancer involves cytosolic DNA activating cGAS, leading to type I interferon (IFN) production via STING, which activates tumor-specific T cells.
- Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) degrades cGAMP, a crucial molecule in this pathway, making it a significant therapeutic target for cancer immunotherapy.
- Developing effective ENPP1 inhibitors requires robust and high-throughput screening methods to identify lead compounds.
Purpose of the Study:
- To develop and validate a high-throughput screening (HTS)-compatible enzymatic assay for Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1).
- To enable direct detection of AMP and GMP, the hydrolysis products of ENPP1, in a homogenous format.
- To assess the assay's suitability for identifying ENPP1 inhibitors for cancer immunotherapy.
Main Methods:
- Utilized the Transcreener AMP2/GMP2 Assay, a competitive fluorescence polarization (FP) immunoassay.
- Optimized assay conditions, determining optimal ENPP1 concentration (100 pM) and reaction time (60 min).
- Evaluated assay performance using Z' factor and assessed ENPP1 substrate selectivity.
Main Results:
- The developed assay demonstrated high selectivity (>104-fold for AMP/GMP over cGAMP; 3000-fold for AMP over ATP).
- A Z' value of 0.72 indicated a high-quality assay suitable for HTS.
- ENPP1 showed substrate preference for ATP over other nucleotides and specific selectivity for 2'3'cGAMP over 2'3'c-diGMP.
Conclusions:
- The Transcreener AMP2/GMP2 Assay provides a robust, homogenous, and HTS-compatible method for measuring ENPP1 enzymatic activity.
- The assay's sensitivity and selectivity allow for screening with low enzyme quantities and accurate detection of hydrolysis products.
- This assay is a valuable tool for discovering novel ENPP1 inhibitors to advance cancer immunotherapy research.
