No abstract

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 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. Additionally, we solved the crystal structure of an exemplary phosphonate inhibitor to elucidate the interactions that drive potency. This study yielded several best-in-class compounds 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.

Significance The immune system strikes a careful balance between launching a robust response to threats and avoiding overactivation. The molecule cGAMP is an immunotransmitter that activates innate immunity and signals extracellularly, where it is subject to degradation by the enzyme ENPP1. Here, we engineer ENPP1 to lose activity toward cGAMP but not other substrates, thus creating a biochemically precise tool to understand how ENPP1 regulates extracellular cGAMP and thus innate immunity. We uncover that ENPP1's degradation of extracellular cGAMP has a long evolutionary history, and that this mechanism is critical for controlling diverse immune threats, including viral infection and inflammation.

2′,5′/3′,5′-cGMP-AMP (cGAMP) is a second messenger produced in response to cytosolic dsDNA that activates the stimulator of interferon genes (STING) pathway. We recently discovered that cGAMP is exported by cancer cells and that this extracellular signal is an immunotransmitter key to tumor detection and elimination by the innate immune system. The enhancement of extracellular cGAMP levels therefore holds great promise for managing cancer. However, there is still much more to understand about the basic biology of cGAMP before its full therapeutic potential can be realized. To answer these questions, we must be able to detect and quantitate cGAMP with an assay that is high-throughput, sensitive, and precise. Existing assays fall short of these needs. Here, we describe the development of cGAMP-Luc, a coupled enzyme assay that relies on the degradation of cGAMP to AMP by ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) and an optimized assay for the detection of AMP by luciferase. We also developed STING-CAP, a STING-mediated method to concentrate and purify cGAMP from any type of biological sample. We conclude that cGAMP-Luc is an economical high-throughput assay that matches the accuracy of and surpasses the detection limit of MS, the current gold standard of cGAMP quantitation. We propose that cGAMP-Luc is a powerful tool that may enable discoveries that advance insights into extracellular cGAMP levels in healthy and diseased tissues, such as cancer.

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 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. Additionally, we solved the crystal structure of an exemplary phosphonate inhibitor to elucidate the interactions that drive potency. This study yielded several best-in-class compounds 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. J.A.C., M.S., and L.L. designed the study. J.A.C., Y.A.S., and R.E.M. performed enzyme assays and analyzed the data. V.B. performed mouse experiments. J.A.B. and D.F. determined the crystal structure. J.A.C., M.S., and L.L. wrote the paper. All authors discussed the findings and commented on the manuscript. Competing interestsM.S. and L.L. are scientific cofounders of Angarus Therapeutics, which has exclusive licensing rights to patents PCT/US2018/50018 and PCT/US2020/015968. J.A.C., V.B., M.S., and L.L. are inventors on patents PCT/US2018/50018 and PCT/US2020/015968.