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 K_i < 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.

癌细胞通过合成和输出小分子免疫递质 cGAMP 来启动先天免疫反应，从而激活宿主体内的抗癌干扰素刺激基因 (STING) 通路。胞外酶，外核苷酸焦磷酸酶磷酸二酯酶 1 (ENPP1)，可水解 cGAMP 并负向调节这种抗癌免疫反应。小分子 ENPP1 抑制剂非常需要作为研究细胞外 cGAMP 基础生物学的工具和研究性癌症免疫治疗药物。在这里，我们调查了一系列不可渗透细胞的 ENPP1 磷酸盐抑制剂的结构-活性关系。此外，我们还解析了示例性膦酸盐抑制剂的晶体结构，以阐明驱动效力的相互作用。这项研究产生了几种同类最佳的抑制剂，其K _i < 2 nM 且具有优异的理化和药代动力学特性。最后，我们证明 ENPP1 抑制剂可以延迟乳腺癌小鼠模型中的肿瘤生长。我们共同开发了 ENPP1 抑制剂，它们是优秀的工具化合物和潜在的治疗方法。