A simple method for developing lysine targeted covalent protein reagents

被引:20
作者
Gabizon, Ronen [1 ]
Tivon, Barr [1 ]
Reddi, Rambabu N. [1 ]
van den Oetelaar, Maxime C. M. [2 ]
Amartely, Hadar [3 ]
Cossar, Peter J. [2 ]
Ottmann, Christian [2 ]
London, Nir [1 ]
机构
[1] Weizmann Inst Sci, Dept Chem & Struct Biol, IL-7610001 Rehovot, Israel
[2] Eindhoven Univ Technol, Inst Complex Mol Syst, Dept Biomed Engn, Lab Chem Biol, POB 513, NL-5600MB Eindhoven, Netherlands
[3] Hebrew Univ Jerusalem, Alexander Silberman Inst Life Sci, Wolfson Ctr Appl Struct Biol, IL-9190401 Jerusalem, Israel
基金
以色列科学基金会;
关键词
PEPTIDE; DESIGN; INHIBITORS; DISCOVERY; PROXIMITY; CELLS; IDENTIFICATION; STRATEGY; PLATFORM; ENABLES;
D O I
10.1038/s41467-023-42632-5
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Peptide-based covalent probes can target shallow protein surfaces not typically addressable using small molecules, yet there is a need for versatile approaches to convert native peptide sequences into covalent binders that can target a broad range of residues. Here we report protein-based thio-methacrylate esters-electrophiles that can be installed easily on unprotected peptides and proteins via cysteine side chains, and react efficiently and selectively with cysteine and lysine side chains on the target. Methacrylate phosphopeptides derived from 14-3-3-binding proteins irreversibly label 14-3-3 sigma via either lysine or cysteine residues, depending on the position of the electrophile. Methacrylate peptides targeting a conserved lysine residue exhibit pan-isoform binding of 14-3-3 proteins both in lysates and in extracellular media. Finally, we apply this approach to develop protein-based covalent binders. A methacrylate-modified variant of the colicin E9 immunity protein irreversibly binds to the E9 DNAse, resulting in significantly higher thermal stability relative to the non-covalent complex. Our approach offers a simple and versatile route to convert peptides and proteins into potent covalent binders. The combination of a covalent electrophile with a peptide or protein-based scaffold enables the targeting of shallow protein surfaces, but the approaches to convert native peptide sequences into covalent binders are missing. Here, the authors report the design of protein-based thiomethacrylate ester electrophiles that can be installed on unprotected peptides and proteins via cysteine side chains and react efficiently and selectively with cysteine and lysine side chains on the target.
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页数:16
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