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.
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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.
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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.
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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.
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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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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). https://doi.org/10.1038/s41586-022-04422-9
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DOI: https://doi.org/10.1038/s41586-022-04422-9
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