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

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.