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Stereo- and regiodefined DNA-encoded chemical libraries enable efficient tumour-targeting applications

Abstract

The encoding of chemical compounds with amplifiable DNA tags facilitates the discovery of small-molecule ligands for proteins. To investigate the impact of stereo- and regiochemistry on ligand discovery, we synthesized a DNA-encoded library of 670,752 derivatives based on 2-azido-3-iodophenylpropionic acids. The library was selected against multiple proteins and yielded specific ligands. The selection fingerprints obtained for a set of protein targets of pharmaceutical relevance clearly showed the preferential enrichment of ortho-, meta- or para-regioisomers, which was experimentally verified by affinity measurements in the absence of DNA. The discovered ligands included novel selective enzyme inhibitors and binders to tumour-associated antigens, which enabled conditional chimeric antigen receptor T-cell activation and tumour targeting.

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Fig. 1: Library design, synthesis and encoding.
Fig. 2: Selection against CAIX, hit validation and conversion of ligands to CAR T-cell activators.
Fig. 3: Selection against CREBBP bromodomain, hit validation and selectivity determination.
Fig. 4: Selections against PI3KCA variants.
Fig. 5: Selections against tumour-associated antigens and human CtIP.

Data availability

The main data supporting the findings of this study are available within this article and its Supplementary Information. Software for the evaluation of high-throughput DNA sequencing was reported previously87.

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Acknowledgements

We acknowledge financial support from the ETH Zürich, the Swiss National Science Foundation (grant no. 310030_182003/1), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement 670603), the Federal Commission for Technology and Innovation (KTI, grant no. 12803.1 VOUCH-LS), the Clinical Research Priority Program ‘ImmunoCure’ of the University of Zurich to M.G.M. and D.N., a University Research Priority Project Translational Cancer Research grant to R.M., a Swiss Cancer Research grant to M.G.M. and D.N. (KFS-3846-02-2016) and the National Cancer Institute (R35 CA197582 to P.K.V.) (TSRI ms. no. 29970). Work in the Sartori laboratory is supported by the Swiss National Science Foundation (grant no. 31003A_176161) and the Swiss Cancer Research Foundation (grant no. KFS-4702-02-2019). M.M. thanks the EPSRC Centre for Doctoral Training in Synthesis for Biology and Medicine (EP/L015838/1) for studentship support, supported by AstraZeneca, Diamond Light Source, Defence Science and Technology Laboratory, Evotec, GlaxoSmithKline, Janssen, Novartis, Pfizer, Syngenta, Takeda, UCB and Vertex. S.J.C. thanks St Hugh’s College for research funding. We further thank D. Bianchi and C. Harvey for the synthesis of some of the molecules described in this article and M. Catalano for providing the anti-FITC antibody used in the ELISA procedures. We thank A. Martinelli for his help with data evaluation and we are most grateful to B. Pfeiffer for help with the NMR measurements. We are also grateful to the Functional Genomics Center Zurich for help with high-throughput DNA sequencing. Instant JChem (ChemAxon) was used for the structure and data management (http://www.chemaxon.com).

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Contributions

N.F., J.S. and D.N. designed the project. N.F. constructed the library. N.F. and G.B. performed the affinity selections and analysed the HT-sequencing results. J.S. helped with the evaluation of HT-sequencing. C.P. performed the FACS and UniCAR-T killing assay experiments. J.M. and S.C. performed ex vivo experiments on SK-RC-52 positive mice. R.D.L. produced the hTNC and mTNC. S.Y. and P.K.V. constructed the expression vectors for the PI3K proteins and carried out the large-scale production in insect cells and the purification of these proteins. A.T., N.L.M. and A.A.S. produced CtIP proteins. R.M. and M.G.M. were involved in the cloning of the UniCAR plasmid. M.M. and S.J.C. produced CREBBP and BRD4(1) proteins. N.F. performed the resynthesis and the validation of all hit compounds. The manuscript was written with contributions from all the authors. The manuscript was written by N.F., R.A.L., J.S., D.N. and corrected by all the authors.

Corresponding authors

Correspondence to Jörg Scheuermann or Dario Neri.

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Competing interests

D.N. is co-founder and shareholder of Philochem AG (http://www.philochem.com), a company active in the field of DELs.

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Peer review information Nature Chemistry thanks Andreas Brunschweige, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–35, Tables 1–8, abbreviations, additional materials and general methods, scaffold and library synthesis details, affinity selection results, hit re-synthesis and validation, lists of the two sets of building blocks used for DEL construction, 1H- and 13C NMR data, LC-MS data and references.

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Favalli, N., Bassi, G., Pellegrino, C. et al. Stereo- and regiodefined DNA-encoded chemical libraries enable efficient tumour-targeting applications. Nat. Chem. (2021). https://doi.org/10.1038/s41557-021-00660-y

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