Molecules that covalently bind macromolecular targets have found widespread applications as activity-based probes and as irreversibly binding drugs. However, the general reactivity of the electrophiles needed for covalent bond formation makes control of selectivity difficult. There is currently no rapid, unbiased screening method to identify new classes of covalent inhibitors from highly diverse pools of candidate molecules. Here we describe a phage display method to directly screen for ligands that bind to protein targets through covalent bond formation. This approach makes use of a reactive linker to form cyclic peptides on the phage surface while simultaneously introducing an electrophilic ‘warhead’ to covalently react with a nucleophile on the target. Using this approach, we identified cyclic peptides that irreversibly inhibited a cysteine protease and a serine hydrolase with nanomolar potency and exceptional specificity. This approach should enable rapid, unbiased screening to identify new classes of highly selective covalent inhibitors for diverse molecular targets.
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All data presented in this manuscript are available from the corresponding author upon reasonable request. The TEV protease-expressing plasmid sequence is available at GenBank with accession number MN480436. Characterization data for cyclization linker and probes are available in the Supplementary Information. Source data are provided with this paper.
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We thank the Vincent Coates Foundation Mass Spectrometry Laboratory at Stanford University for providing technical assistance with mass spectrometry. We also thank D. Waugh (National Cancer Institute) for providing the TEV expression construct, pDZ2087, and C. Heinis (EPFL) for providing the phage library. This work was supported by Swiss National Science Foundation Postdoc Mobility fellowship P2ELP3_155323 P300PB_164725 (to S.C.) and by funding from National Institutes of Health grants R01 EB026285 and R01 EB026285 02S1 (to M.B.).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Strategy for the synthesis of DCA-VS, derivatizing a vinyl sulfone cysteine reactive warhead with 1,3-Dichloroacetone.
The cysteine protease reactive vinyl sulfone warhead was efficiently conjugated to the 1,2-dichloroacetone linker through oxime-ketone reaction.
Extended Data Fig. 2 Strategy for the synthesis of DCA-DPP, derivatizing a diphenylphosphonate serine reactive warhead with 1,3-Dichloroacetone.
a, Acetic Anhydride, paraformaldehyde, triphenyl phosphite, acetic acid, 5 h, 120 °C, 46 % b, i. HBr in acetic acid ii. (Boc-aminooxy)acetic acid, DCC, DIPEA, DMF, RT, O/N, 67 % c, i. TFA in DCM, RT, 1 h ii. Dichloroacetone, DMF, RT, O/N, 72 %.
The carboxyl side chain of P1 glutamine was deprotected and reacted with chlorotrityl resin. The derived resin can be used for directly synthesizing linear ABPs bearing a glutamine-VS motif at the C-terminus.
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Chen, S., Lovell, S., Lee, S. et al. Identification of highly selective covalent inhibitors by phage display. Nat Biotechnol 39, 490–498 (2021). https://doi.org/10.1038/s41587-020-0733-7
Nature Methods (2021)