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.

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.

The biology of the immune second messenger cGAMP depends on its cellular localization. cGAMP, which is synthesized in response to cytosolic double-stranded DNA, also exists in the extracellular space as a paracrine immunotransmitter that enhances the anticancer immune response. However, the role of extracellular cGAMP is unexplored outside of cancer due to a lack of tools to systemically manipulate it. The extracellular enzyme ENPP1, the only known hydrolase of cGAMP, is a promising target. However, because ENPP1 also degrades extracellular ATP, using genetic knockouts of ENPP1 to study extracellular cGAMP leads to confounding effects. Here we report the H362A point mutation in ENPP1, the dominant cGAMP hydrolase, which selectively abolishes ENPP1’s ability to degrade cGAMP, while retaining activity toward other substrates. H362 is not necessary for binding cGAMP or the catalytically-essential zinc atoms but instead supports the in-line reaction geometry. H362 is evolutionarily conserved down to bacteria, suggesting an ancient origin for extracellular cGAMP biology. Enpp1H362A mice do not display the systemic calcification seen in Enpp1-/- mice, highlighting the substrate-specific phenotype of ENPP1. Remarkably, Enpp1H362A mice were resistant to HSV-1 infection, demonstrating the antiviral role of endogenous extracellular cGAMP. The ENPP1H362A mutation is the first genetic tool to enable exploration of extracellular cGAMP biology in a wide range of tissues and diseases.

Graft Versus Host Disease (GVHD) is an immune-mediated disease occurs as a complication of allogeneic Hematopoietic Stem Cell Transplantation (HSCT). Differences between donor and recipient Major Histocompatibility Complex (MHC) antigens initiate the disease. The immunocompetent cells of the donor recognize the cells of the immunocompromised host as non-self, thus commencing an immune response against them.The human Cytomegalovirus (hCMV) is a member of the herpesvirus family that has developed strategies to escape the immune surveillance and defense system via encoding a series of glycoproteins that down regulate host MHC antigens. The Unique Short (US) hCMV glycoproteins US2, US3, US6, US10 and US11 have shown variable capabilities to downregulate MHC class I and II. Theoretically, these capabilities could be utilized to downregulate the expression of host MHC antigens, thus inhibiting the allograft recognition and the subsequent immune response, which would prevent GVHD. In this systematic review, 620 literatures have been identified through a PubMed, Epistemonikos, and Google Scholar search. An inclusion criterion has been applied to these studies, of which 27 have been selected. This review found that the hCMV glycoproteins act as partner to downregulate MHC class I and class II, CMV glycoproteins regulate destruction of class I MHC molecules, and degrade MHC class II.The Preferred Reporting Items for Systematic Reviews (PRISMA) Statement has been used to increase the quality of the review, and thus a Population Intervention Comparison Outcome Study design (PICOS) model has been formulated.The findings of this research could be further studied and validated to offer an alternative approach to the current pharmacological preventive measures of GVHD, possibly without compromising patients' immunity.