Abstract
DNA-encoded chemical libraries (DELs) consist of large chemical compound collections individually linked to DNA barcodes, facilitating pooled construction and screening. However, screening campaigns often fail if the molecular arrangement of the building blocks is not conducive to an efficient interaction with a protein target. Here we postulated that the use of rigid, compact and stereo-defined central scaffolds for DEL synthesis may facilitate the discovery of very specific ligands capable of discriminating between closely related protein targets. We synthesized a DEL comprising 3,735,936 members, featuring the four stereoisomers of 4-aminopyrrolidine-2-carboxylic acid as central scaffolds. The library was screened in comparative selections against pharmaceutically relevant targets and their closely related protein isoforms. Hit validation results revealed a strong impact of stereochemistry, with large affinity differences between stereoisomers. We identified potent isozyme-selective ligands against multiple protein targets. Some of these hits, specific to tumour-associated antigens, demonstrated tumour-selective targeting in vitro and in vivo. Collectively, constructing DELs with stereo-defined elements contributed to high library productivity and ligand selectivity.
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Data availability
All data supporting the findings of this study are available within the article and Supplementary Information. Next-generation sequencing data of all selection duplicates are provided as separate text files. Source Data Figs. 1–6 and Supplementary Figs. 14, 19, 20, 43–46 and 53–63 are provided with the paper. The Fastq file, containing raw high throughput Illumina sequence counts, is not provided in this article and Supplementary Information, but can be made available to readers upon justified request addressed to the corresponding authors. Source data are provided with this paper.
Code availability
Software for the evaluation of HTDS has previously been reported5.
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Acknowledgements
The authors thank A. Galbiati and A. Zana for the support with the in vivo experiments. Furthermore, the authors thank C. Pellegrino for the help with flow cytometry measurements. The authors received no specific funding for this work.
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All authors have contributed to the manuscript preparation (S. O., L. L., F. M., A. E., L. P., S. P., M. M., K. S., J. S., D. Y., M. J., N. B., D. B., R. K., S. C., D. N., N. F. and G. B.). S.O., G.B., N.F., S.C. and D.N. designed the experiments. S.O. synthesized the library with support from G.B., N.F. and F.M. S.O. and G.B. performed the selections. S.O., G.B., N.F. and J.S. analysed the HTS data. Hits were synthesized and validated by S.O. and G.B. with support from L.P., L.L., S.P., N.F. and A.E. A.E., L.L., S.O., M.M., L.P., G.B. and N.F. expressed and biotinylated the proteins with exception of Nsp14, which was provided by N.B., M.J., D.Y. and K.S. In vitro and in vivo experiments were performed by S.O., G.B. and S.C.
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D.N. is co-founder, CEO, CSO and President of the Scientific Advisory Board of Philogen. S.O., L.L., A.E., F.M., S.P., M.M., L.P., S.C., N.F. and G.B. are employed by Philochem AG, the research and development unit of the Philogen. R.K. is member of the Board of Directors Chair and Scientific Advisory Board of NeoTX Therapeutics LTD. D.B. is CSO of the Discovery Division at NeoTX Therapeutics LTD. All other authors do not declare any conflict of interest.
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Supplementary Information
Supplementary Figs. 1–279, Supplementary Tables 1–7, results, experimental data, procedural details, synthesis and characterization data, NMR spectra, mass spectrometry spectra and source data files.
Supplementary Table 1
Source data file for supplementary figures.
Supplementary Data 1
Next-generation sequencing raw data of all selection duplicates are provided as separate text files.
Source data
Source Data Fig. 2
Numerical source data of FP measurements, SPR sensograms, affinity constant values and quantitative biodistributions.
Source Data Fig. 3
Numerical source data of FP measurements.
Source Data Fig. 4
Numerical source data of FP measurements and of the co-elution experiment.
Source Data Fig. 4
Unprocessed microscope image of immunofluorescence staining of human submandibular salivary glands with PSMA-617*.
Source Data Fig. 4
Unprocessed microscope image of immunofluorescence staining of human submandibular salivary glands with compound 45.
Source Data Fig. 5
Numerical source data of FP measurements.
Source Data Fig. 6
Numerical source data of FP measurements and affinity constant values.
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Oehler, S., Lucaroni, L., Migliorini, F. et al. A DNA-encoded chemical library based on chiral 4-amino-proline enables stereospecific isozyme-selective protein recognition. Nat. Chem. 15, 1431–1443 (2023). https://doi.org/10.1038/s41557-023-01257-3
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DOI: https://doi.org/10.1038/s41557-023-01257-3