title: Hydrolysis of 2'3'-cGAMP by ENPP1 and design of nonhydrolyzable analogs.
abstract: Agonists of mouse STING (TMEM173) shrink and even cure solid tumors by activating innate immunity; human STING (hSTING) agonists are needed to test this therapeutic hypothesis in humans. The endogenous STING agonist is 2'3'-cGAMP, a second messenger that signals the presence of cytosolic double-stranded DNA. We report activity-guided partial purification and identification of ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP1) to be 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 bisphosphothioate analog of 2'3'-cGAMP (2'3'-cG(s)A(s)MP) that has similar affinity for hSTING in vitro and is ten times more potent at inducing IFN-β secretion from human THP1 monocytes. Studies in mouse Enpp1(-/-) lung fibroblasts indicate that resistance to hydrolysis contributes substantially to its higher potency. 2'3'-cG(s)A(s)MP is therefore improved over natural 2'3'-cGAMP as a model agonist and has potential as a vaccine adjuvant and cancer therapeutic.
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Lingyin
authors.initials: L
authors.lastname: Li
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.
authors.forename: Qian
authors.initials: Q
authors.lastname: Yin
authors.affiliation: Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, California, USA.
authors.forename: Pia
authors.initials: P
authors.lastname: Kuss
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Maliga
authors.affiliation: Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, California, USA.
authors.forename: José L
authors.initials: JL
authors.lastname: Millán
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Wu
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Mitchison
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000494:pharmacology*; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D006868:Hydrolysis; D016899:Interferon-beta; D008565:Membrane Proteins / Q000819:agonists* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D009712:Nucleotides, Cyclic / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D009946:Organothiophosphorus Compounds / Q000138:chemical synthesis / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015290:Second Messenger Systems / Q000235:genetics; D015398:Signal Transduction; D000099197:STING Protein
chemical_list: D000970:Antineoplastic Agents; D016899:Interferon-beta; D008565:Membrane Proteins; D009712:Nucleotides, Cyclic; D009946:Organothiophosphorus Compounds; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D011994:Recombinant Proteins; C532857:STING1 protein, human; D000099197:STING Protein; C530855:Sting1 protein, mouse; C584311:cyclic guanosine monophosphate-adenosine monophosphate; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1