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Dual-display of small molecules enables the discovery of ligand pairs and facilitates affinity maturation

Abstract

In contrast to standard fragment-based drug discovery approaches, dual-display DNA-encoded chemical libraries have the potential to identify fragment pairs that bind simultaneously and benefit from the chelate effect. However, the technology has been limited by the difficulty in unambiguously decoding the ligand pairs from large combinatorial libraries. Here we report a strategy that overcomes this limitation and enables the efficient identification of ligand pairs that bind to a target protein. Small organic molecules were conjugated to the 5′ and 3′ ends of complementary DNA strands that contain a unique identifying code. DNA hybridization followed by an inter-strand code-transfer created a stable dual-display DNA-encoded chemical library of 111,100 members. Using this approach we report the discovery of a low micromolar binder to alpha-1-acid glycoprotein and the affinity maturation of a ligand to carbonic anhydrase IX, an established marker of renal cell carcinoma. The newly discovered subnanomolar carbonic anhydrase IX binder dramatically improved tumour targeting performance in vivo.

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Figure 1: Design, synthesis and encoding of the dual-display DNA-encoded chemical library.
Figure 2: Affinity-based selection procedure for the isolation of simultaneously binding fragment pairs from a dual-display chemical library.
Figure 3: Selection results from the 111,100-member dual-display library.
Figure 4: Hit validation of selected pharmacophore pair A-117/B-113 against AGP.
Figure 5: Hit validation of selected pharmacophore pair A-493/B-202 binding to CAIX.
Figure 6: Long-lasting residence of selected library compound–dye conjugate at the tumour site.

References

  1. Carter, P. J. Potent antibody therapeutics by design. Nature Rev. Immunol. 6, 343–357 (2006).

    CAS  Google Scholar 

  2. Sliwkowski, M. X. & Mellman, I. Antibody therapeutics in cancer. Science 341, 1192–1198 (2013).

    CAS  PubMed  Google Scholar 

  3. McCafferty, J., Griffiths, A. D., Winter, G. & Chiswell, D. J. Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552–554 (1990).

    CAS  PubMed  Google Scholar 

  4. Kang, A. S., Barbas, C. F., Janda, K. D., Benkovic, S. J. & Lerner, R. A. Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces. Proc. Natl Acad. Sci. USA 88, 4363–4366 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Boder, E. T. & Wittrup, K. D. Yeast surface display for screening combinatorial polypeptide libraries. Nature Biotechnol. 15, 553–557 (1997).

    CAS  Google Scholar 

  6. Wilson, D. S., Keefe, A. D. & Szostak, J. W. The use of mRNA display to select high-affinity protein-binding peptides. Proc. Natl Acad. Sci. USA 98, 3750–3755 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Hanes, J. & Pluckthun, A. In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl Acad. Sci. USA 94, 4937–4942 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Ellington, A. D. & Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).

    CAS  PubMed  Google Scholar 

  9. Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510 (1990).

    CAS  PubMed  Google Scholar 

  10. Alley, S. C., Okeley, N. M. & Senter, P. D. Antibody–drug conjugates: targeted drug delivery for cancer. Curr. Opin. Chem. Biol. 14, 529–537 (2010).

    CAS  PubMed  Google Scholar 

  11. Chari, R. V. Targeted cancer therapy: conferring specificity to cytotoxic drugs. Acc. Chem. Res. 41, 98–107 (2008).

    CAS  PubMed  Google Scholar 

  12. Neri, D. & Bicknell, R. Tumour vascular targeting. Nature Rev. Cancer 5, 436–446 (2005).

    CAS  Google Scholar 

  13. Krall, N., Scheuermann, J. & Neri, D. Small targeted cytotoxics: current state and promises from DNA-encoded chemical libraries. Angew. Chem. Int. Ed. 52, 1384–1402 (2013).

    CAS  Google Scholar 

  14. Thurber, G. M. et al. Single-cell and subcellular pharmacokinetic imaging allows insight into drug action in vivo. Nature Commun. 4, 1504 (2013).

    Google Scholar 

  15. Franzini, R. M., Neri, D. & Scheuermann, J. DNA-encoded chemical libraries advancing beyond conventional small-molecule libraries. Acc. Chem. Res. 47, 1247–1255 (2014).

    CAS  PubMed  Google Scholar 

  16. Kleiner, R. E., Dumelin, C. E. & Liu, D. R. Small-molecule discovery from DNA-encoded chemical libraries. Chem. Soc. Rev. 40, 5707–5717 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Mannocci, L., Leimbacher, M., Wichert, M., Scheuermann, J. & Neri, D. 20 years of DNA-encoded chemical libraries. Chem. Commun. 47, 12747–12753 (2011).

    CAS  Google Scholar 

  18. Mannocci, L. et al. High-throughput sequencing allows the identification of binding molecules isolated from DNA-encoded chemical libraries. Proc. Natl Acad. Sci. USA 105, 17670–17675 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Brenner, S. & Lerner, R. A. Encoded combinatorial chemistry. Proc. Natl Acad. Sci. USA 89, 5381–5383 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Dower, W. J., Barrett, R. W., Gallop, M. A. & Needels, M. C. Method of synthesizing diverse collections of oligomers. WO patent 1993006121 (1993).

  21. Clark, M. A. et al. Design, synthesis and selection of DNA-encoded small-molecule libraries. Nature Chem. Biol. 5, 647–654 (2009).

    CAS  Google Scholar 

  22. Gartner, Z. J. et al. DNA-templated organic synthesis and selection of a library of macrocycles. Science 305, 1601–1605 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Hansen, M. H. et al. A yoctoliter-scale DNA reactor for small-molecule evolution. J. Am. Chem. Soc. 131, 1322–1327 (2009).

    CAS  PubMed  Google Scholar 

  24. Li, Y., Zhao, P., Zhang, M., Zhao, X. & Li, X. Multistep DNA-templated synthesis using a universal template. J. Am. Chem. Soc. 135, 17727–17730 (2013).

    CAS  PubMed  Google Scholar 

  25. Wrenn, S. J., Weisinger, R. M., Halpin, D. R. & Harbury, P. B. Synthetic ligands discovered by in vitro selection. J. Am. Chem. Soc. 129, 13137–13143 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Melkko, S., Dumelin, C. E., Scheuermann, J. & Neri, D. On the magnitude of the chelate effect for the recognition of proteins by pharmacophores scaffolded by self-assembling oligonucleotides. Chem. Biol. 13, 225–231 (2006).

    CAS  PubMed  Google Scholar 

  27. Krishnamurthy, V. M., Estroff, L. A. & Whitesides, G. M. in Fragment-based Approaches in Drug Discovery (eds Jahnke, W. & Erlanson, D. A.) Ch. 2, 11–53 (Methods and Principles in Medicinal Chemistry series, Wiley-VCH, 2006).

    Google Scholar 

  28. Melkko, S., Scheuermann, J., Dumelin, C. E. & Neri, D. Encoded self-assembling chemical libraries. Nature Biotechnol. 22, 568–574 (2004).

    CAS  Google Scholar 

  29. Scheibe, C., Bujotzek, A., Dernedde, J., Weber, M. & Seitz, O. DNA-programmed spatial screening of carbohydrate–lectin interactions. Chem. Sci. 2, 770–775 (2011).

    CAS  Google Scholar 

  30. Ciobanu, M. et al. Selection of a synthetic glycan oligomer from a library of DNA-templated fragments against DC-SIGN and inhibition of HIV gp120 binding to dendritic cells. Chem. Commun. 47, 9321–9323 (2011).

    CAS  Google Scholar 

  31. Shuker, S. B., Hajduk, P. J., Meadows, R. P. & Fesik, S. W. Discovering high-affinity ligands for proteins: SAR by NMR. Science 274, 1531–1534 (1996).

    CAS  PubMed  Google Scholar 

  32. Rees, D. C., Congreve, M., Murray, C. W. & Carr, R. Fragment-based lead discovery. Nature Rev. Drug Discov. 3, 660–672 (2004).

    CAS  Google Scholar 

  33. Hajduk, P. J. & Greer, J. A decade of fragment-based drug design strategic advances and lessons learned. Nature Rev. Drug Discov. 6, 211–219 (2007).

    CAS  Google Scholar 

  34. Pellecchia, M. Fragment-based drug discovery takes a virtual turn. Nature Chem. Biol. 5, 274–275 (2009).

    CAS  Google Scholar 

  35. Dumelin, C. E., Scheuermann, J., Melkko, S. & Neri, D. Selection of streptavidin binders from a DNA-encoded chemical library. Bioconj. Chem. 17, 366–370 (2006).

    CAS  Google Scholar 

  36. Weber, P. C., Ohlendorf, D. H., Wendoloski, J. J. & Salemme, F. R. Structural origins of high-affinity biotin binding to streptavidin. Science 243, 85–88 (1989).

    CAS  PubMed  Google Scholar 

  37. Gilabert, M. A. et al. Differential substrate behaviour of phenol and aniline derivatives during oxidation by horseradish peroxidase: kinetic evidence for a two-step mechanism. Biochim. Biophys. Acta 1699, 235–243 (2004).

    CAS  PubMed  Google Scholar 

  38. Fournier, T., Medjoubi, N. N. & Porquet, D. Alpha-1-acid glycoprotein. Biochim. Biophys. Acta 1482, 157–171 (2000).

    CAS  PubMed  Google Scholar 

  39. McDonald, P. C., Winum, J. Y., Supuran, C. T. & Dedhar, S. Recent developments in targeting carbonic anhydrase IX for cancer therapeutics. Oncotarget 3, 84–97 (2012).

    PubMed  PubMed Central  Google Scholar 

  40. Muselaers, S., Mulders, P., Oosterwijk, E., Oyen, W. & Boerman, O. Molecular imaging and carbonic anhydrase IX-targeted radioimmunotherapy in clear cell renal cell carcinoma. Immunotherapy 5, 489–495 (2013).

    CAS  PubMed  Google Scholar 

  41. Supuran, C. T. Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nature Rev. Drug Discov. 7, 168–181 (2008).

    CAS  Google Scholar 

  42. Ahlskog, J. K. et al. Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours. Br. J. Cancer 101, 645–657 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Gram, H. et al. In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library. Proc. Natl Acad. Sci. USA 89, 3576–3580 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Krall, N. et al. A small-molecule drug conjugate for the treatment of carbonic anhydrase IX expressing tumors. Angew. Chem. Int. Ed. 53, 4231–4235 (2014).

    CAS  Google Scholar 

  45. Perrino, E. et al. Curative properties of non-internalizing antibody–drug conjugates based on maytansinoids. Cancer Res. 74, 2569–2578 (2014).

    CAS  PubMed  Google Scholar 

  46. Rini, B. I., Campbell, S. C. & Escudier, B. Renal cell carcinoma. Lancet 373, 1119–1132 (2009).

    CAS  PubMed  Google Scholar 

  47. Krall, N., Pretto, F. & Neri, D. A bivalent small molecule–drug conjugate directed against carbonic anhydrase IX can elicit complete tumour regression in mice. Chem. Sci. 5, 3640–3644 (2014).

    CAS  Google Scholar 

  48. Ginj, M. et al. Radiolabeled somatostatin receptor antagonists are preferable to agonists for in vivo peptide receptor targeting of tumors. Proc. Natl Acad. Sci. USA 103, 16436–16441 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Low, P. S., Henne, W. A. & Doorneweerd, D. D. Discovery and development of folic-acid-based receptor targeting for imaging and therapy of cancer and inflammatory diseases. Acc. Chem. Res. 41, 120–129 (2008).

    CAS  PubMed  Google Scholar 

  50. Kularatne, S. A., Wang, K., Santhapuram, H. K. & Low, P. S. Prostate-specific membrane antigen targeted imaging and therapy of prostate cancer using a PSMA inhibitor as a homing ligand. Mol. Pharmacol. 6, 780–789 (2009).

    CAS  Google Scholar 

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Acknowledgements

This work was supported by ETH Zürich, the Swiss National Science Foundation (SNSF), Philochem AG and Krebsliga Schweiz/Krebsforschung Schweiz (KFS-2839-08-2011). R.M.F. acknowledges a VPFW-ETH postdoctoral fellowship endowed by ETH Zurich and Marie-Curie actions. The authors thank M. Jaggi, I. Mafli and A. Nauer for help with library and ligand synthesis, C. Aquino and L. Opitz (Functional Genomics Center Zurich) for help with high-throughput DNA sequencing, Y. Zhang, F. Buller, H. Röst, M. Stravs, G. Jackson and A. Rabenseifner for help with sequencing data analysis and software implementation, and L. Urner for help with NMR spectra analysis. The authors also thank C. Hess, G. Hausammann, T. Hemmerle, M. Weber, E. Perrino, A. Baumann, M. Bühler and A. Zemann for help with experimental work. The authors are grateful to I. Jelezarov for critically reviewing the ITC data and to F. Samain for discussions. The authors thank J. Kunze and D. Reker for technical support with protein graphics implementation. Instant JChem (ChemAxon) was used for structure and data management (http://www.chemaxon.com).

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M.W., D.N. and J.S. designed the project. M.W. and J.S. constructed the dual-display library. W.D. and R.F. provided target proteins. W.D. designed and performed the selections. M.W., W.D. and J.S. analysed high-throughput DNA screening data. M.W. and P.S. performed ITC experiments. M.W. performed hit validation experiments. M.W., N.K. and F.P. performed in vivo experiments. M.W., D.N. and J.S. wrote the manuscript.

Corresponding authors

Correspondence to Dario Neri or Jörg Scheuermann.

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Competing interests

D.N. is a co-founder and shareholder of Philochem AG (Otelfingen, Switzerland) and J.S. is a board member of Philochem AG.

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Wichert, M., Krall, N., Decurtins, W. et al. Dual-display of small molecules enables the discovery of ligand pairs and facilitates affinity maturation. Nature Chem 7, 241–249 (2015). https://doi.org/10.1038/nchem.2158

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