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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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. 1

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

  2. 2

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

  3. 3

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

  4. 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).

  5. 5

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

  6. 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).

  7. 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).

  8. 8

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

  9. 9

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

  10. 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).

  11. 11

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

  12. 12

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

  13. 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).

  14. 14

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

  15. 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).

  16. 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).

  17. 17

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

  18. 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).

  19. 19

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

  20. 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. 21

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

  22. 22

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

  23. 23

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

  24. 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).

  25. 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).

  26. 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).

  27. 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).

  28. 28

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

  29. 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).

  30. 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).

  31. 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).

  32. 32

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

  33. 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).

  34. 34

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

  35. 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).

  36. 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).

  37. 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).

  38. 38

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

  39. 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).

  40. 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).

  41. 41

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

  42. 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).

  43. 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).

  44. 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).

  45. 45

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

  46. 46

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

  47. 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).

  48. 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).

  49. 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).

  50. 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).

Download references

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).

Author information

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.

Correspondence to Dario Neri or Jörg Scheuermann.

Ethics declarations

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.

Supplementary information

Supplementary information

Supplementary information (PDF 5762 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading