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DNA-encoded library-enabled discovery of proximity-inducing small molecules

Nature Chemical Biology volume 20, pages 170–179 (2024)Cite this article

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Abstract

Small molecules that induce protein–protein associations represent powerful tools to modulate cell circuitry. We sought to develop a platform for the direct discovery of compounds able to induce association of any two preselected proteins, using the E3 ligase von Hippel–Lindau (VHL) and bromodomains as test systems. Leveraging the screening power of DNA-encoded libraries (DELs), we synthesized ~1 million DNA-encoded compounds that possess a VHL-targeting ligand, a variety of connectors and a diversity element generated by split-and-pool combinatorial chemistry. By screening our DEL against bromodomains in the presence and absence of VHL, we could identify VHL-bound molecules that simultaneously bind bromodomains. For highly barcode-enriched library members, ternary complex formation leading to bromodomain degradation was confirmed in cells. Furthermore, a ternary complex crystal structure was obtained for our most enriched library member with BRD4BD1 and a VHL complex. Our work provides a foundation for adapting DEL screening to the discovery of proximity-inducing small molecules.

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Fig. 1: Library design and synthesis.
Fig. 2: CIP–DEL screening analysis and evaluation of compounds synthesized off-DNA.
Fig. 3: Correlations between CIP–DEL screening results and off-DNA validation data.
Fig. 4: Ternary complex crystal structure of CIP-1 with BRD4 and VCB.
Fig. 5: CIP–DEL screening against BRD2BD1 and BRDTBD1 with off-DNA profiling of two shared hits.

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Data availability

All data supporting the findings of this study are available within the article and Supplementary Information. The X-ray crystal structure for the VCB–CIP-1–BRD4 complex is available on the PDB (code: 8EWV). Source data for Figs. 2h and 5h are provided with the paper. The FASTQ files, containing raw Illumina sequencing data, are available at https://doi.org/10.5281/zenodo.8253891. Source data are provided with this paper.

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Acknowledgements

J. Capece, J. Poirier, P. Michaels, C. Rakiec, A. Lindeman, T. Dice and R. Tichkule are gratefully acknowledged for excellent technical and analytical support. B. Hua, C. Gerry, W. Wang, A. Reidenbach, K. Lim, S. Gill, B. Tong, L. Chung, C. Gampe and N. Smith are gratefully acknowledged for their support, guidance and valuable feedback during the preparation of this manuscript. We also thank D. Dovala and M. Romanowski (Novartis Institutes for Biomedical Research) for supplying the protein reagents used in SPR. The research was supported in part by the National Institute of General Medical Sciences (award R35GM127045 to S.L.S.) and by the Novartis Institutes for Biomedical Research (NIBR) Scholar’s Program.

Author information

Authors and Affiliations

  1. Chemical Biology and Therapeutics Science, Broad Institute, Cambridge, MA, USA

    Jeremy W. Mason, Yuen Ting Chow, Liam Hudson, Matthias V. Westphal, Zher Yin Tan, Connor W. Coley, Paul A. Clemons, Shuang Liu & Stuart L. Schreiber

  2. Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Cambridge, MA, USA

    Jeremy W. Mason, Liam Hudson, Matthias V. Westphal, Simone Bonazzi, Karin Briner & Frédéric J. Zécri

  3. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Yuen Ting Chow, Zher Yin Tan, Shuang Liu & Stuart L. Schreiber

  4. Chemical Biology and Therapeutics, Novartis Institutes for BioMedical Research, Cambridge, MA, USA

    Antonin Tutter & Gregory Michaud

  5. Structural and Biophysical Chemistry, Novartis Institutes for BioMedical Research, Emeryville, CA, USA

    Wei Shu & Xiaolei Ma

  6. Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Novartis Campus, Basel, Switzerland

    Frédéric Berst

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Contributions

J.W.M. conceived the project, designed and synthesized the CIP–DEL library, performed the BRD4 screening, analyzed the data and produced screening hits off-DNA. Y.T.C. performed the BRD2/BRDT screening, synthesized hits off-DNA and profiled the hits from these screens. L.H. and M.V.W. contributed to the design, screening, data analysis and off-DNA hit synthesis. A.T. developed and conducted all NanoBiT, HiBiT, CTG and TR-FRET experiments from the BRD4 screening and oversaw these experiments for BRD2/BRDT compound profiling. G.M. generated and analyzed all SPR data. W.S. and X.M. ran crystallization screens and solved the ternary complex structure. Z.Y.T. contributed to off-DNA compound synthesis. C.W.C. and P.A.C. developed the CIP–DEL data analysis pipeline. S.L. supervised and supported all BRD2/BRDT screening and profiling. S.B., F.B., K.B., F.J.Z. and S.L.S. provided context for the framing of the original goals, supervision, guidance, operational support, assisted in the interpretation of experimental outcomes and made recommendations for a subset of the reported experiments. J.W.M. and S.L.S. wrote the manuscript, and all authors read and edited the paper.

Corresponding authors

Correspondence to Shuang Liu, Frédéric J. Zécri or Stuart L. Schreiber.

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Competing interests

X.M. is a shareholder of Terremoto Biosciences. C.W.C. is an advisor to Anagenex. P.A.C. is an advisor to nference, Pfizer and Belharra Therapeutics. S.L.S. is a shareholder and serves on the Board of Directors of Jnana Therapeutics and Kojin Therapeutics; is a shareholder and advises Kisbee Therapeutics, Belharra Therapeutics, Magnet Biomedicine, Exo Therapeutics and Eikonizo Therapeutics; advises Vividian Therapeutics, Eisai Co, Ono Pharma Foundation, F-Prime Capital Partners and the Genomics Institute of the Novartis Research Foundation and is a Novartis Faculty Scholar. The remaining authors declare no competing interests.

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Nature Chemical Biology thanks Andrea Testa and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Distribution of barcode counts for each CIP-DEL library member across each screening condition.

(a) Barcode counts for the beads-only screen. (b) Barcode counts for the BRD4 (-) VHL screen. (c) Barcode counts for the BRD4 (+) VHL screen.

Extended Data Fig. 2 Individual building block barcode counts for each screening condition.

Raw sequencing counts for each building block (x-axis) at each cycle (BB1, BB2, and BB3) are shown for each of the selection conditions (beads only = top row; BRD4 (-) VHL = middle row; BRD4 (+) VHL = bottom row).

Extended Data Fig. 3 CIP-1 ternary complex structural analysis.

(a) Six copies of the ternary complex are present in each asymmetric unit. An overlay of the six ternary complexes shows the structures are highly similar with RMS deviations of 1.06–1.37 Å. (b) The electron density for CIP-1 is well defined in 4 of the 6 complexes of the asymmetric unit. 2Fo – Fc maps contoured at 2.5σ and 1σ are shown for CIP-1 in a ternary complex with well-defined density. (c) BRD4BD1 protein surface with residues colored based on interactions with CIP-1 (blue), VHL (red), or both (purple). CIP-1 is shown in sticks. (d) VHL protein surface with residues colored based on interactions with CIP-1 (blue), BRD4BD1 (red), or both (purple). (e) 2D diagram of CIP-1 interactions with BRD4BD1. (f) 2D diagram of CIP-1 interactions with VHL.

Extended Data Fig. 4 Comparison of the small molecule components of VHL PROTAC ternary complex structures.

(a) Overlaying the VHL binding moieties of ternary complex assemblies, the diverse projection vectors of the connectors and target protein ligands are shown. The initial point of divergence for the bifunctional molecules is the carbon atom following the terminal amide of the VH032 ligand. (b) Comparison of the buried surface area for four VHL-targeting CIPs.

Extended Data Fig. 5 Comparison of the ternary complex assemblies for four VHL-targeting bifunctional degraders.

The first three structures show bromodomains targets (BRD4BD1, BRD4BD2, and SMARCA4BD) and the fourth complex is a non-bromodomain targeting degrader (Bcl-XL). VHL proteins are aligned at the bottom of each structure and the target protein orientation is shown at the top. Key details for each complex are provided in the table. Inset: the three bromodomain containing complexes are overlaid with VHL aligned at the bottom of the structures, highlighting the diverse target protein orientations.

Supplementary information

Supplementary Information

Supplementary Tables 1–5, Supplementary Figs. 1–21, Supplementary Note, Supplementary References and Spectral Data.

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Source data

Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 5

Unprocessed western blots.

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Mason, J.W., Chow, Y.T., Hudson, L. et al. DNA-encoded library-enabled discovery of proximity-inducing small molecules. Nat Chem Biol 20, 170–179 (2024). https://doi.org/10.1038/s41589-023-01458-4

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