2026-10-11T01:37:39Z https://api.figshare.com/v2/oai

oai:figshare.com:article/34018695 2026-09-29T00:04:27Z category_1 category_4 category_12 category_14 category_19 category_21 category_734 category_931 category_64 category_69 portal_63 item_type_6 month_year_09_2026

De Novo Discovery of Cysteine-Targeted Reversible Covalent Cyclic Peptides with Prolonged Residence Time via Phage Display Yuqian Zhang (615460) Mengzhu Qi (17756760) Xiankai Liu (188445) Yukun Zhou (8257716) Yanmei Wang (392327) Xing Xiao (134047) Yapei Wu (4168285) Wei Gao (2085) Yang Liu (4829) Yiwu Zheng (1466191) Biophysics Biochemistry Cell Biology Molecular Biology Pharmacology Biotechnology Biological Sciences not elsewhere classified Information Systems not elsewhere classified Cancer Inorganic Chemistry prolonged residence time mechanistic analysis reveals maintaining prolonged residence de novo identification de novo discovery complex cellular environment 5 – 100 transient binding dynamics binding potency relative unsaturated nitrile electrophiles reversible covalent warhead cysteine residues offers 32 </ b retains binding selected warhead designing electrophiles k </ </ sub work establishes rich conditions react rapidly powerful strategy potent ligand nanomolar affinities mediated targeting keep pace keap1 enabled keap1 cys434 generating potent general strategy fold improvement displayed peptides capture keap1 balanced reactivity >< sub 7 nm Reversible covalent targeting of cysteine residues offers a powerful strategy for generating potent and tunable ligands, but designing electrophiles with balanced reactivity, stability, and reversible binding remains challenging. Here, we systematically optimized reversible α,β-unsaturated nitrile electrophiles and incorporated the selected warhead with prolonged residence time into phage-displayed peptides to construct a library of reversible covalent cyclic peptides. Screening this library against Keap1 enabled the de novo identification of ligands with nanomolar affinities, demonstrating a 5–100-fold improvement in binding potency relative to the corresponding noncovalent cyclic peptides. The most potent ligand, cyclic peptide 32, exhibits a Ki of 3.7 nM, retains binding under thiol-rich conditions, and can capture Keap1 from a complex cellular environment. Mechanistic analysis reveals that peptide-mediated targeting in cyclic peptide 32 positions the reversible covalent warhead to react rapidly with Keap1 Cys434 while maintaining prolonged residence, allowing covalent bond formation to keep pace with the transient binding dynamics of the peptide–protein interaction. Overall, this work establishes a general strategy for integrating tunable reversible covalent chemistry into genetically encoded peptide libraries, enabling de novo discovery of high-affinity ligands. 2026-09-28T00:00:00Z Text Journal contribution 10.1021/jacs.6c17599.s001 https://figshare.com/articles/journal_contribution/De_Novo_Discovery_of_Cysteine-Targeted_Reversible_Covalent_Cyclic_Peptides_with_Prolonged_Residence_Time_via_Phage_Display/34018695 CC BY-NC 4.0