BellBrook scientist Meera Kumar and Robert Lowery were recently published in SLAS Discovery for their work entitled Development of a High-Throughput Assay to Identify Inhibitors of ENPP1. Ectonucleotide pyrophosphatase/phosphodiesterase 1 or ENNP1 has recently become a target for cancer immunotherapy. ENNP1 hydrolyzes nucleotides such as cGAMP and ATP. Cytosolic DNA is a trigger for the innate immune response to cancer. cGAMP is a critical messenger of this immune response, and as such, regulation of ENNP1 could lead to breakthrough treatments.
In the paper, Kumar and Lowery describe the development of an HTS compatible ENPP1 activity assay to discover lead molecules for the enzyme. The technique uses the Transcreener AMP2/GMP2 Assay to measure cGAMP breakdown to AMP and GMP with a 104-fold selectivity. The assay reported a Z’ value of 0.72, demonstrating a robust HTS amiable method.
Also discussed in the article were ENPP1’s preferred substrates. ATP was selected to be hydrolyzed with greater efficiency when compared to other nucleotides such as ADP, GDP, and ATP. ENNP1 was much more selective for 2’3’ cGAMP with 20-fold selectivity over 2’3’ c-di-GMP and virtually no activity with 3’3’ c-diAMP.
Work done by the scientists at BellBrook Labs will enable further characterization on ENPP1. It also provides an avenue for other researchers to use the assay technique for HTS and lead discovery. By providing a simple single-step mix-and-read method, researchers can interrogate the target with greater efficiency to more quickly discover novel inhibitors and accelerate new treatments to the clinic.
Kumar, M., & Lowery, R. G. Development of a High-Throughput Assay to Identify Inhibitors of ENPP1. SLAS DISCOVERY: Advancing the Science of Drug Discovery, 2472555220982321. https://journals.sagepub.com/doi/abs/10.1177/2472555220982321