Structure-Aided Development of Small-Molecule Inhibitors of ENPP1, the Extracellular Phosphodiesterase of the Immunotransmitter cGAMP

Cancer cells initiate an innate immune response by synthesizing and exporting the small-molecule immunotransmitter cGAMP, which activates the anti-cancer Stimulator of Interferon Genes (STING) pathway in the host. An extracellular enzyme, ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), hydrolyzes cGAMP and negatively regulates this anti-cancer immune response. Small-molecule ENPP1 inhibitors are much needed as tools to study the basic biology of extracellular cGAMP and as investigational cancer immunotherapy drugs. Here, we surveyed structure-activity relationships around a series of cell-impermeable and thus extracellular-targeting phosphonate inhibitors of ENPP1. In addition, we solved the crystal structure of an exemplary phosphonate inhibitor to elucidate the interactions that drive potency. This study yielded several best-in-class inhibitors with Ki < 2 nM and excellent physicochemical and pharmacokinetic properties. Finally, we demonstrate that an ENPP1 inhibitor delays tumor growth in a breast cancer mouse model. Together, we have developed ENPP1 inhibitors that are excellent tool compounds and potential therapeutics. [Display omitted] •ENPP1 inhibitors are designed as extracellular cell-impermeable inhibitors•SAR studies lead to highly potent ENPP1 inhibitors under physiological conditions•Crystal structure of ENPP1: inhibitor complex reveals key interactions•Systemic dosing of ENPP1 inhibitor delays tumor growth in mice As the dominant hydrolase of the immunotransmitter cGAMP, the extracellular enzyme ENPP1 dampens the anti-cancer immune response. Carozza et al. have solved the crystal structure of ENPP1 in complex with a lead inhibitor, followed by structure-activity relationship studies. The resulting potent and specific extracellular ENPP1 inhibitor delays tumor growth in mice.