Abstract
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 (ClinicalTrials.gov 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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
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 OL362244–OL362267). Gene sequences are available in the Supplementary Data files. The enzymes are commercially available from Codexis, Inc., subject to existing license obligations and restrictions.
References
Schrittwieser, J. H., Velikogne, S., Hall, M. & Kroutil, W. Artificial biocatalytic linear cascades for preparation of organic molecules. Chem. Rev. 118, 270–348 (2018).
Huffman, M. A. et al. Design of an in vitro biocatalytic cascade for the manufacture of islatravir. Science 366, 1255–1259 (2019).
Luo, X. et al. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast. Nature 567, 123–126 (2019).
Arnold, F. H. Directed evolution: bringing new chemistry to life. Angew Chem. Int. Ed. Engl. 57, 4143–4148 (2018).
Bowie, J. U. et al. Synthetic biochemistry: the bio-inspired cell-free approach to commodity chemical production. Trends Biotechnol. 38, 766–778 (2020).
Altman, M. D. et al. Cyclic di-nucleotide compounds as STING agonists. Patent WO2017027646A1 (2016).
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).
Cross, R. STING fever is sweeping through the cancer immunotherapy world. Chem. Eng. News 96, 24–26 (2018).
Burdette, D. L. et al. STING is a direct innate immune sensor of cyclic di-GMP. Nature 478, 515–518 (2011).
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).
Knouse, K. W. et al. Unlocking P(V): reagents for chiral phosphorothioate synthesis. Science 361, 1234–1238 (2018).
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).
Featherston, A. L. et al. Catalytic asymmetric and stereodivergent oligonucleoside synthesis. Science 371, 702–707 (2021).
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).
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).
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).
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).
Li, L. et al. Hydrolysis of 2'3'-cGAMP by ENPP1 and design of nonhydrolyzable analogs. Nat. Chem. Biol. 10, 1043–1048 (2014).
Ablasser, A. et al. cGAS produces a 2'-5'-linked cyclic dinucleotide second messenger that activates STING. Nature 498, 380–384 (2013).
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).
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).
Eckstein, F. Nucleoside phosphorothioates. Annu. Rev. Biochem. 54, 367–402 (1985).
Du, M. & Chen, Z. J. DNA-induced liquid phase condensation of cGAS activates innate immune signaling. Science 361, 704–709 (2018).
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).
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).
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).
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).
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).
Sandoval, B. A. & Hyster, T. K. Emerging strategies for expanding the toolbox of enzymes in biocatalysis. Curr. Opin. Chem. Biol. 55, 45–51 (2020).
Ren, X. & Fasan, R. Engineered and artificial metalloenzymes for selective C–H functionalization. Curr. Opin. Green Sustain. Chem. 31, 100494 (2021).
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).
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).
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).
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).
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).
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).
Pan, B. S. et al. An orally available non-nucleotide STING agonist with antitumor activity. Science 369, eaba6098 (2020).
Studier, F. W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).
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).
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).
Acknowledgements
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
Contributions
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
Ethics declarations
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.
Peer review
Peer review information
Nature thanks Elaine O’Reilly and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
Rights and permissions
About this article
Cite this article
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). https://doi.org/10.1038/s41586-022-04422-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41586-022-04422-9
This article is cited by
-
Stereocontrolled access to thioisosteres of nucleoside di- and triphosphates
Nature Chemistry (2024)
-
Activating STING/TBK1 suppresses tumor growth via degrading HPV16/18 E7 oncoproteins in cervical cancer
Cell Death & Differentiation (2024)
-
cGAMP-activated cGAS–STING signaling: its bacterial origins and evolutionary adaptation by metazoans
Nature Structural & Molecular Biology (2023)
-
Chemically programmed STING-activating nano-liposomal vesicles improve anticancer immunity
Nature Communications (2023)
-
Beyond DNA sensing: expanding the role of cGAS/STING in immunity and diseases
Archives of Pharmacal Research (2023)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
