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A kinase-cGAS cascade to synthesize a therapeutic STING activator


The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1,2,3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 ( study NCT04220866). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.

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Fig. 1: Discovery of MK-1454.
Fig. 2: Diastereoselective CDN synthesis catalysed by cGAS.
Fig. 3: Assembly of thiotriphosphate biocatalytic cascade.
Fig. 4: Cascade process to MK-1454 from nucleotide monothiophosphates without isolated intermediates.

Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information. Coordinates for the STING structure have been deposited in the Protein Data Bank (PDB ID 7MHC). DNA sequences of wild-type and evolved enzymes used in this study are available in the Supplementary Data files and have been deposited in Genbank (accession codes OL362244OL362267). Gene sequences are available in the Supplementary Data files. The enzymes are commercially available from Codexis, Inc., subject to existing license obligations and restrictions.


  1. Schrittwieser, J. H., Velikogne, S., Hall, M. & Kroutil, W. Artificial biocatalytic linear cascades for preparation of organic molecules. Chem. Rev. 118, 270–348 (2018).

    Article  CAS  PubMed  Google Scholar 

  2. Huffman, M. A. et al. Design of an in vitro biocatalytic cascade for the manufacture of islatravir. Science 366, 1255–1259 (2019).

    Article  CAS  PubMed  ADS  Google Scholar 

  3. Luo, X. et al. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast. Nature 567, 123–126 (2019).

    Article  CAS  PubMed  ADS  Google Scholar 

  4. Arnold, F. H. Directed evolution: bringing new chemistry to life. Angew Chem. Int. Ed. Engl. 57, 4143–4148 (2018).

    Article  CAS  PubMed  Google Scholar 

  5. Bowie, J. U. et al. Synthetic biochemistry: the bio-inspired cell-free approach to commodity chemical production. Trends Biotechnol. 38, 766–778 (2020).

    Article  CAS  PubMed  Google Scholar 

  6. Altman, M. D. et al. Cyclic di-nucleotide compounds as STING agonists. Patent WO2017027646A1 (2016).

  7. Walsh, C. T., Tu, B. P. & Tang, Y. Eight kinetically stable but thermodynamically activated molecules that power cell metabolism. Chem. Rev. 118, 1460–1494 (2018).

    Article  CAS  PubMed  Google Scholar 

  8. Cross, R. STING fever is sweeping through the cancer immunotherapy world. Chem. Eng. News 96, 24–26 (2018).

    Google Scholar 

  9. Burdette, D. L. et al. STING is a direct innate immune sensor of cyclic di-GMP. Nature 478, 515–518 (2011).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  10. Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z. J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786–791 (2013).

    Article  CAS  ADS  Google Scholar 

  11. Knouse, K. W. et al. Unlocking P(V): reagents for chiral phosphorothioate synthesis. Science 361, 1234–1238 (2018).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  12. Lioux, T. et al. Design, synthesis, and biological evaluation of novel cyclic adenosine-inosine monophosphate (cAIMP) analogs that activate stimulator of interferon genes (STING). J. Med. Chem. 59, 10253–10267 (2016).

    Article  CAS  PubMed  Google Scholar 

  13. Featherston, A. L. et al. Catalytic asymmetric and stereodivergent oligonucleoside synthesis. Science 371, 702–707 (2021).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  14. Yan, H., Wang, X., KuoLee, R. & Chen, W. Synthesis and immunostimulatory properties of the phosphorothioate analogues of cdiGMP. Bioorg. Med. Chem. Lett. 18, 5631–5634 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. Gaffney, B. L., Veliath, E., Zhao, J. & Jones, R. A. One-flask syntheses of c-di-GMP and the [Rp,Rp] and [Rp,Sp] thiophosphate analogues. Org. Lett. 12, 3269–3271 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zhang, X. et al. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol. Cell 51, 226–235 (2013).

    Article  CAS  PubMed  Google Scholar 

  17. Gao, P. et al. Structure-function analysis of STING activation by c[G(2',5')pA(3',5')p] and targeting by antiviral DMXAA. Cell 154, 748–762 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Li, L. et al. Hydrolysis of 2'3'-cGAMP by ENPP1 and design of nonhydrolyzable analogs. Nat. Chem. Biol. 10, 1043–1048 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Ablasser, A. et al. cGAS produces a 2'-5'-linked cyclic dinucleotide second messenger that activates STING. Nature 498, 380–384 (2013).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  20. Diner, E. J. et al. The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING. Cell. Rep. 3, 1355–1361 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gao, P. et al. Cyclic [G(2',5')pA(3',5')p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase. Cell 153, 1094–1107 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Eckstein, F. Nucleoside phosphorothioates. Annu. Rev. Biochem. 54, 367–402 (1985).

    Article  CAS  PubMed  Google Scholar 

  23. Du, M. & Chen, Z. J. DNA-induced liquid phase condensation of cGAS activates innate immune signaling. Science 361, 704–709 (2018).

    Article  CAS  ADS  Google Scholar 

  24. Thillier, V., Sallamand, C. C. B., Vasseur, J. J. & Debart, F. Solid‐phase synthesis of oligonucleotide 5′‐(α‐P‐Thio)triphosphates and 5′‐(α‐P‐Thio)(β,γ‐methylene)triphosphates. Eur. J. Org. Chem. 2015, 302–308 (2015).

    Article  CAS  Google Scholar 

  25. Ludwig, J. & Eckstein, F. Rapid and efficient synthesis of nucleoside 5'-O-(1-thiotriphosphates), 5'-triphosphates, and 2',3'-cyclophosphorothioates using 2-chloro-4H-1,3,2,-benzodioxaphosphorin-4-one. J. Org. Chem. 54, 631–635 (1989).

    Article  CAS  Google Scholar 

  26. Moran, J. R. & Whitesides, G. M. A practical enzymatic synthesis of (Sp)-adenosine 5'-O-(1-thiotriphosphate) ((Sp)-ATP-α-S)). J. Org. Chem. 49, 1984 (1984).

    Article  Google Scholar 

  27. Jaffe, E. K. & Cohn, M. 31P nuclear magnetic resonance spectra of the thiophosphate analogues of adenine nucleotides; effects of pH and Mg2+ binding. Biochemistry 17, 652–657 (1978).

    Article  CAS  PubMed  Google Scholar 

  28. Rex Sheu, K. F. & Frey, P. A. Enzymatic and 32P nuclear magnetic resonance study of adenylate kinase-catalyzed stereospecific phosphorylation of adenosine 5'-phosphorothioate. J. Biol. Chem. 252, 4445–4448 (1977).

    Article  CAS  PubMed  Google Scholar 

  29. Sandoval, B. A. & Hyster, T. K. Emerging strategies for expanding the toolbox of enzymes in biocatalysis. Curr. Opin. Chem. Biol. 55, 45–51 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ren, X. & Fasan, R. Engineered and artificial metalloenzymes for selective C–H functionalization. Curr. Opin. Green Sustain. Chem. 31, 100494 (2021).

    Article  PubMed  Google Scholar 

  31. Brandenberg, O. F., Fasan, R. & Arnold, F. H. Exploiting and engineering hemoproteins for abiological carbene and nitrene transfer reactions. Curr. Opin. Biotechnol. 47, 102–111 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Qu, G., Li, A., Acevedo-Rocha, C. G., Sun, Z. & Reetz, M. T. The crucial role of methodology development in directed evolution of selective enzymes. Angew Chem. Int. Ed. Engl. 59, 13204–13231 (2020).

    Article  CAS  PubMed  Google Scholar 

  33. Lim, J. & Kim, H. Y. Novel applications of biocatalysis to stereochemistry determination of 2′3′-cGAMP bisphosphorothioate (2′3′-cGSASMP). ACS Omega 5, 14173–14179 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Crans, D. C. & Whitesides, G. M. A convenient synthesis of disodium acetyl phosphate for use in in situ ATP cofactor regeneration. J. Org. Chem. 48, 3130–3132 (1983).

  35. Gottlieb, H. E., Kotlyar, V. & Nudelman, A. J. NMR chemical shifts of common laboratory solvents as trace impurities. J. Org. Chem. 62, 7512–7515 (1997).

    Article  CAS  PubMed  Google Scholar 

  36. Fulmer, G. R. et al. NMR chemical shifts of trace impurities: common laboratory solvents, organics, and gases in deuterated solvents relevant to the organometallic chemist. Organometallics 29, 2176–2179 (2010).

    Article  CAS  Google Scholar 

  37. Pan, B. S. et al. An orally available non-nucleotide STING agonist with antitumor activity. Science 369, eaba6098 (2020).

    Article  CAS  PubMed  Google Scholar 

  38. Studier, F. W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).

    Article  CAS  PubMed  Google Scholar 

  39. Abele, U. & Schulz, G. E. High-resolution structures of adenylate kinase from yeast ligated with inhibitor Ap5A, showing the pathway of phosphoryl transfer. Protein Sci. 4, 1262–1271 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Sekulic, N., Shuvalova, L., Spangenberg, O., Konrad, M. & Lavie, A. Structural characterization of the closed conformation of mouse guanylate kinase. J. Biol. Chem. 277, 30236–30243 (2002).

    Article  CAS  PubMed  Google Scholar 

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This research used resources at the Industrial Macromolecular Crystallography Association Collaborative Access Team beamline 17-ID, supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman–Woodward Medical Research Institute. This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. We acknowledge the help and support of the following people: C. Prier and A. Fryszkowska for comments on earlier versions of this manuscript; W. Pinto and F. Tenkorang for analytical assistance; K. Sirk for reaction optimization support; J. Corry and E. Fisher for helpful discussions; S. (G.) Xu, E. Frank and A. Struck for support with biochemical and cell-based assays used in this work; M. Childers, M. Lu, R. Otte, A. Haidle, T. Henderson, J. Jewell, L. Nogle and A. Beard for contributions to synthesis, purification and characterization of the molecules described in the article; L. Miller, A. Petkova, J. Riggins and A. Sowell-Kantz for support of enzyme evolution.

Author information

Authors and Affiliations



J.A.M., J.C.M., N.M.M., M.B.-G., M.A.H., C.A., D.D., B.F.M., K.H., G.S.M., J.N.K., A.M., W.P., I.F., O.A., A.C., D.V., M.B., K.A.C. and M.D.T. contributed to enzyme discovery and engineering. Z.L., N.S.M., J.V.O., P.S.F., F.P., J.A.M., M.S.W., C.A., F.-R.T., J.H.F., X.B., R.S.B., J.H.F., K.S., E.G., E.H., E.L.R., S.C., N.R., J.P.S., F.W., S.A. and Z.E.X.D. contributed to reaction optimization, characterization and/or process development. B.M.A., W.C., J.L., M.D.A., C.A.L., D.S., B.W.T., J.N.C., A.N., D.A. and M.L.M. contributed to CDN analogue synthesis and characterization. M.A.H., S.P.M., P.N.D., D.T., P.G.B., B.D.S., R.T.R., L.C.C., D.J.B., G.R.H., K.R.C. and M.L.M. provided intellectual contributions. J.A.M., Z.L., B.M.A., M.B.-G., M.A.H., M.L.M., R.T.R. and L.C.C. prepared the manuscript.

Corresponding authors

Correspondence to John A. McIntosh, Zhijian Liu or Brian M. Andresen.

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Competing interests

The authors are current or former employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA or Codexis, Inc., which are assignees for patents governing chemical matter, processes and enzyme sequences reported in the article.

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Nature thanks Elaine O’Reilly and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Abbreviations, synthetic methods, Supplementary Figs. 1–23, Tables 1 and 2, NMR spectra and references.

Supplementary Data 1

Wild-type cGAS variant sequences.

Supplementary Data 2

Wild-type adenylate and guanylate kinase sequences.

Supplementary Data 3

Acetate kinase sequences.

Supplementary Data 4

Sequences of evolved cGAS, GK, AK and AcK variants.

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McIntosh, J.A., Liu, Z., Andresen, B.M. et al. A kinase-cGAS cascade to synthesize a therapeutic STING activator. Nature 603, 439–444 (2022).

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