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Hydrolysis of 2′3′-cGAMP by ENPP1 and design of nonhydrolyzable analogs

Nature Chemical Biology volume 10, pages 1043–1048 (2014)Cite this article

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An Erratum to this article was published on 18 August 2015

A Corrigendum to this article was published on 17 February 2015

This article has been updated

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′-cGsAsMP) 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′-cGsAsMP is therefore improved over natural 2′3′-cGAMP as a model agonist and has potential as a vaccine adjuvant and cancer therapeutic.

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Figure 1: There is 2′-5′ phosphodiesterase activity in the plasma membrane or heavy organelles and it is not due to PDE12.
Figure 2: ENPP1 is an efficient hydrolase for 2′3′-cGAMP.
Figure 3: ENPP1 is the dominant hydrolase activity for 2′3′-cGAMP.
Figure 4: Development of hydrolysis-resistant hSTING agonists.
Figure 5: ENPP1 dampens 2′3′-cGAMP signaling.

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Change history

  • 29 January 2015

    In the version of this article initially published, the chemical structures of the two cyclic dinucleotides in Figure 4a were incorrect at the triphosphate group, with each of the phosphates shown as containing two extra hydrogen atoms. The error has been corrected in the HTML and PDF versions of the article.

  • 15 July 2015

    In the version of this article published on 29 January 2015, in Figure 4 the hydroxyl group was depicted in the 3′ position on the deoxyribose ring of the cyclic dinucleotide precursor to 3′3′-cGAMP rather than in the 2′ position and the R group was in the 2′ position rather than in the 3′ position. This error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank P. Koch, P. Choi, K. Krukenberg, A. Groen, M. Loose, S. Kim and S. Gruver for helpful discussions. We thank R. Jiang for help with data analysis. We thank K. Chu for providing mouse livers. We thank S. Walker for sharing the MicroBeta plate reader and C. Fan for technical assistance. We thank J.J. Mekalanos for providing the DncV expression plasmid and T. Bernhard (both from Harvard Medical School) for providing the pTB146 plasmid. We thank G. Heffron and C. Sheahan for assistance with the NMR data collection and analysis. We thank R. Ward for help with manuscript preparation. This research was supported by the National Cancer Institute (CA139980, AR53102, AI050872 and 1K99AI108793-01). L. Li thanks the Jane Coffin Childs Fund for her postdoctoral fellowship.

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Authors and Affiliations

  1. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

    Lingyin Li, Zoltan Maliga & Timothy J Mitchison

  2. Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA

    Qian Yin & Hao Wu

  3. Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, California, USA

    Pia Kuss & José L Millán

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Contributions

L.L. and T.J.M. developed the hypothesis and designed the study. L.L., Q.Y., P.K. and Z.M. conducted the experiments. All authors interpreted and discussed the results. J.L.M. advised P.K., and H.W. advised Q.Y. Both J.L.M. and H.W. funded part of the research. L.L. and T.J.M. wrote the manuscript.

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Correspondence to Lingyin Li.

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Supplementary information

Supplementary Text and Figures

Supplementary Results and Supplementary Figures 1–15. (PDF 29368 kb)

Supplementary Data Set 1

Mass spectrometry analysis of fraction 26. (XLSX 77 kb)

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Li, L., Yin, Q., Kuss, P. et al. Hydrolysis of 2′3′-cGAMP by ENPP1 and design of nonhydrolyzable analogs. Nat Chem Biol 10, 1043–1048 (2014). https://doi.org/10.1038/nchembio.1661

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