Abstract

Stimulator of interferon genes (STING) is a receptor in the endoplasmic reticulum that propagates innate immune sensing of cytosolic pathogen-derived and self DNA1. The development of compounds that modulate STING has recently been the focus of intense research for the treatment of cancer and infectious diseases and as vaccine adjuvants2. To our knowledge, current efforts are focused on the development of modified cyclic dinucleotides that mimic the endogenous STING ligand cGAMP; these have progressed into clinical trials in patients with solid accessible tumours amenable to intratumoral delivery3. Here we report the discovery of a small molecule STING agonist that is not a cyclic dinucleotide and is systemically efficacious for treating tumours in mice. We developed a linking strategy to synergize the effect of two symmetry-related amidobenzimidazole (ABZI)-based compounds to create linked ABZIs (diABZIs) with enhanced binding to STING and cellular function. Intravenous administration of a diABZI STING agonist to immunocompetent mice with established syngeneic colon tumours elicited strong anti-tumour activity, with complete and lasting regression of tumours. Our findings represent a milestone in the rapidly growing field of immune-modifying cancer therapies.

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Data availability

All data generated or analysed during this study are included in this published article (and its Supplementary Information files). Structure datasets generated during the current study are available in the PDB repository under accession numbers 6DXG and 6DXL. Additional data are available from the corresponding author on reasonable request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

We thank B. Geddes for helpful suggestions and S. Romeril for discussions and comments.

Reviewer information

Nature thanks B. Stockwell and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Joshi M. Ramanjulu, G. Scott Pesiridis, Jingsong Yang

  2. These authors jointly supervised this work: James Smothers, Axel Hoos, John Bertin

Affiliations

  1. Pattern Recognition Receptor DPU, GlaxoSmithKline, Collegeville, PA, USA

    • Joshi M. Ramanjulu
    • , G. Scott Pesiridis
    • , Robert Singhaus
    • , Jean-Luc Tran
    • , Patrick Moore
    • , John Mehlmann
    • , Joseph Romano
    • , Angel Morales
    • , James Kang
    • , Lara Leister
    • , Todd L. Graybill
    • , Adam K. Charnley
    • , Kamelia Behnia
    • , Amaya I. Wolf
    • , Viera Kasparcova
    • , Michael A. Reilly
    • , Kevin P. Foley
    • , Peter J. Gough
    • , Robert W. Marquis
    •  & John Bertin
  2. Immuno-Oncology & Combinations DPU, GlaxoSmithKline, Collegeville, PA, USA

    • Jingsong Yang
    • , Shu-Yun Zhang
    • , Michael Adam
    • , Christopher B. Hopson
    • , Yiqian Lian
    • , Kevin J. Duffy
    • , Jerry Adams
    • , James Smothers
    •  & Axel Hoos
  3. Cellzome, GlaxoSmithKline R&D, Heidelberg, Germany

    • Stephanie Lehmann
    • , H. Christian Eberl
    • , Marcel Muelbaier
    • , Marcus Bantscheff
    •  & Giovanna Bergamini
  4. Platform Technology & Science, GlaxoSmithKline, Collegeville, PA, USA

    • Nestor Concha
    • , Jessica L. Schneck
    • , Jim Clemens
    • , Guosen Ye
    • , Neysa Nevins
    • , Kelvin Nurse
    • , Liping Wang
    • , Yue Li
    • , Michael Klein
    •  & Jeffrey Guss

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Contributions

J.M.R. conceived the dimer concept and designed compound 2, and conceived the concept for compound 3 and synthetic chemistry for compound 4. G.S.P. identified compound 1. J.M.R., G.S.P. and J.Y. were co-leaders and oversaw the research program. J.M.R., G.S.P and J.Y. wrote the manuscript with assistance from all other authors. N.C. performed HDX studies and determined X-ray structures with assistance from L.W. R.S. synthesized compounds 2 and 4. S.-Y.Z., M.A., and C.B.H. conducted the in vivo efficacy study in CT-26 tumour-bearing mice. J.-L.T. conducted in vivo pharmacodynamics studies in wild-type and Sting−/− mice. P.M. performed in vitro functional experiments in PBMCs. S.L., H.C.E., M.M., M.B., and G.B. designed, performed and analysed chemoproteomics experiments. M.K. and J.L.S. developed and assisted with the high-throughput screening assay. J.C. conducted PBMC assays from different haplotype donors. J.M., J.R., A.M., L.L., T.L.G., A.K.C., G.Y., and Y. Li contributed to design, optimization of synthetic route and preparation of compounds. N.N. carried out structure-based design analysis. A.I.W., V.K., and P.M. characterized agonist activity. K.N. purified STING protein. J.G. conducted thermal shift experiments. K.B. and M.A.R. designed and supervised pharmacokinetic studies. K.P.F. was co-leader during program initiation. P.J.G. supervised biology and provided advice. Y. Lian, K.J.D., and J.A. contributed to compound selection. R.W.M. contributed to chemistry strategy and provided advice. J.K. contributed to optimization of synthetic route and preparation of compounds. J.S., A.H. and J.B jointly supervised the program.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Joshi M. Ramanjulu.

Extended data figures and tables

  1. Extended Data Fig. 1 Co-crystal structures and superimposition of compounds 1 and 2.

    a, Superposition of compound 1 (PDB code: 6DXG) and the diABZI compound 2 (PDB code: 6DXL) bound to human STING (aa 149–379). b, Intermolecular contacts in the complex of compounds 1 and 2 bound to human STING (aa 149–379). Magenta, compound 1; green, compound 2. Corresponding subunits of STING shown in same colour for compounds 1 and 2. c, Electron density (1.0σ) of compound 1. d, Electron density of (0.5σ) of compound 2.

  2. Extended Data Fig. 2 Selectivity of compound 2 determined by affinity enrichment chemoproteomics.

    To identify any potential off-target liabilities early on, an affinity enrichment-based chemoproteomics strategy was applied to compound 2. Compound 5, an active analogue containing a primary amine functionality, was covalently immobilized on sepharose beads and was used to affinity-capture potential target proteins from a THP1 cell lysate. Pull-down experiments were performed in the absence of free compound 2 to delineate target proteins from background or in the presence of compound 2 over a range of concentrations. All proteins captured by the beads under the different conditions were eluted and subsequently quantified by isotope tagging of tryptic peptides followed by LC–MS/MS analysis to establish a competition-binding curve and determine a half-maximal inhibition (IC50) value. The IC50 values obtained in these experiments represent a measure of target affinity, but are also affected by the affinity of the target for the bead-immobilized ligand. The latter effect can be deduced by determining the depletion of the target proteins by the beads, such that apparent dissociation constants (\({K}_{{\rm{d}}}^{{\rm{app}}}\)) can be determined, which are largely independent from the bead ligand (see Supplementary Methods for details). Notably, only two proteins were captured and competed in a dose-dependent manner within a 1,000-fold window, namely STING and orosomucoid1 (ORM1, alpha-1-acid glycoprotein 1 precursor). The mean \({K}_{{\rm{d}}}^{{\rm{app}}}\) value for STING was determined as 1.6 nM, demonstrating high potency of compound 2 on the target protein not only in an artificial biochemical assay system using truncated protein but also against the full-length endogenous human protein. The mean \({K}_{{\rm{d}}}^{{\rm{app}}}\)value of the only identified off-target protein, ORM1, was determined as 79 nM giving a comfortable selectivity window of approximately 40-fold. ORM1 is an acute phase reactant, an abundant plasma protein with known drug binding properties, and is known to be expressed in monocytes.

  3. Extended Data Fig. 3 Superimposition of co-crystal structures of cGAMP and compound 2.

    a, Superimposition of bound conformations of cGAMP (yellow) and diABZI compound 2 (green) bound to human STING (aa 149–379). b, Superimposition of bound structures of cGAMP and diABZI compound 2. Source data

  4. Extended Data Fig. 4 Bound conformations of cGAMP and compound 2.

    a, Conformations of cGAMP bound to human STING (aa 149–379). b, diABZI compound 2 bound to human STING (aa 149–379).

  5. Extended Data Fig. 5 Anti-CD8 depletion antibody validation by flow cytometry.

    a, Schematic of CD8T cell depletion scheme with timings consistent with efficacy studies. b, c, Flow cytometry quantification of CD4 and CD8 T cells from vehicle-treated (b) or anti-CD8 antibody (BioXcell: clone 2.43)-treated (c) BALB/c mice. Blood taken before dosing and after the third dose and spleen samples validate effective depletion of CD8+ T cells. Similar results observed 72 h after dose 1 and dose2. d, Flow cytometry gating strategy. Flow cytometry staining and gating blood samples were collected via tail snip for pre-dose bleeds and via cardiac puncture under isoflurane following the third dose. An equal volume of blood was added to flow staining buffer (PBS + 0.5% BSA), and samples were incubated in mouse Fc blocker. Spleen samples were processed to cell suspension, resuspended in flow staining buffer, and incubated with mouse Fc blocker. Samples were stained with live/dead aqua dye, CD45–PE, CD3–V421, and CD8–APC. Gating strategy reports the percentage positive population of live cells → CD45+ → CD3+. All samples were run on BD Canto II and analysed with FACSDiva software. Source data

  6. Extended Data Table 1 Screening statistics and results for compound 1
  7. Extended Data Table 2 X-ray diffraction data collection and refinement statistics
  8. Extended Data Table 3 Kinome inhibition

Supplementary information

  1. Supplementary Information

    This file contains the Supplementary Methods section and the Supplementary information as a combined single document

  2. Reporting Summary

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DOI

https://doi.org/10.1038/s41586-018-0705-y

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