Agonists of mouse STING (TMEM173) shrink and even cure solid tumor by activating innate immunity; human STING agonists are needed to test this therapeutic hypothesis in man. The endogenous STING agonist is 2′3′-cGAMP, a 2nd messenger that signals the presence of cytosolic dsDNA. We report activity-guided partial purification and identification of ENPP1 as the dominant 2′3′-cGAMP hydrolyzing activity in cultured cells. The hydrolysis activity of ENPP1 was confirmed using recombinant protein and was depleted in tissue extracts and plasma from Enpp1-/- mice. We synthesized a hydrolysis-resistant bis-phosphothioate analog of 2′3′-cGAMP (2′3′-cGsAsMP) with similar affinity for human STING in vitro and 10 times more potent at inducing IFN-β secretion from human THP1 monocytes. Studies in mouse Enpp1-/- lung fibroblasts indicate that resistance to hydrolysis contributes significantly to its higher potency. 2′3′-cGsAsMP is therefore improved over natural 2′3′-cGAMP as a model agonist, and has potential as a vaccine adjuvant and cancer therapeutic.
