Home Journals Articles Updates About
CN
image
收藏切换
Exploring Protein Bioconjugation: A Redox-Based Strategy for Tryptophan Targeting
收藏切换
PDF
Qian-Qian Yang, Shuai-Jiang Liu, Wei Huang, Cheng Peng, Bo Han*
Research. Vol 7 Article ID 0410
Less
收藏切换
Research. Vol 7 Article ID 0410
• Perspective •
Exploring Protein Bioconjugation: A Redox-Based Strategy for Tryptophan Targeting
Full
Qian-Qian Yang, Shuai-Jiang Liu, Wei Huang, Cheng Peng, Bo Han*
Affiliations
  • State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, P. R. China.
Published: 2024-07-04 doi: 10.34133/research.0410
Outline
收藏切换

Amino acid bioconjugation technology has emerged as a pivotal tool for linking small-molecule fragments with proteins, antibodies, and even cells. The study in Nature by Chang and Toste introduces a redox-based strategy for tryptophan bioconjugation, employing N-sulfonyloxaziridines as oxidative cyclization reagents, demonstrating high efficiency comparable to traditional click reactions. Meanwhile, this tool provides feasible methods for investigating the mechanisms underlying functional tryptophan-related biochemical processes, paving the way for protein function exploration, activity-based proteomics for functional amino acid identification and characterization, and even the design of covalent inhibitors.

Qian-Qian Yang, Shuai-Jiang Liu, Wei Huang, Cheng Peng, Bo Han. Exploring Protein Bioconjugation: A Redox-Based Strategy for Tryptophan Targeting[J]. Research, 2024 , 7 (7) : 0410 . DOI: 10.34133/research.0410
Full Text
Less
In recent years, covalent inhibitors [1,2] and molecular chemical probes [3] designed based on nucleophilic amino acids have attracted widespread attention in the field of drug discovery. Compared to traditional non-covalent inhibitors, covalent inhibitors provide higher affinity by forming stable covalent bonds with target proteins. Additionally, covalent inhibitors help to circumvent some potential resistance mechanisms, especially when facing undruggable proteins [4–6] or kinases [7] (such as SHP2 and KRAS) that lack traditional binding pockets due to mutations. Covalent inhibitors can expand their therapeutic potential, offering possibilities for developing new therapeutic strategies, leading to an increasing number of small-molecule covalent inhibitors successfully entering the market [8]. The development of reactive “warheads” that rapidly and effectively covalently bind to specific amino acid residues is pivotal in the creation of covalent inhibitors. With the advancement of amino acid bioconjugation chemistry [9], numerous novel warheads have emerged in recent years, targeting a variety of amino acid residues including cysteine [10], methionine [11], tyrosine [12], etc. [13]. Unlike traditional approaches to targeting amino acid residues bearing highly nucleophilic groups, tryptophan residue, which contains an indole segment, shows weak nucleophilicity under physiological pH conditions, making it difficult to achieve covalent targeting using conventional acid–base catalysis methods. Currently, while a few methods offer strategies for covalently targeting tryptophan, achieving this under physiological conditions remains challenging [14,15], leading to the underdevelopment of bioconjugation reagents specific to tryptophan.
In a recent publication in Nature, Chang and Toste introduced a novel redox-based strategy for tryptophan bioconjugation [16], further advancing the selective coupling methods previously established for methionine [11]. This innovative approach draws inspiration from the indole oxidative cyclization biosynthesis, akin to the Communesin core [17]. Their approach utilized N-sulfonyloxaziridines (Davis reagents or Davis oxaziridines) [18], which are stable, aprotic, and neutral oxidizing agents. These reagents are capable of oxidizing a wide variety of nucleophiles with high regioselectivity and stereoselectivity [19], thereby presenting a novel strategy for tryptophan bioconjugation. This method, termed tryptophan chemical ligation by cyclization (Trp-CLiC), achieves selective and rapid functionalization of tryptophan at the levels of peptides, proteins, and proteomes, with a reaction rate comparable to traditional click chemistry reactions [20]. The authors demonstrated the broad application of this method in revealing functional tryptophan-related biochemical processes, including systematically identifying tryptophan residues involved in cation–π interactions, and deciphering these cation–π motifs could functionally regulate protein-mediated phase-separation processes, with disease-related mutations or post-translational modifications that disrupt these interactions leading to alterations in protein localization (Fig. 1).
Using biomimetic and computational chemistry screening, the authors found that N-sulfonyl oxaziridine showed high reaction efficiency, and after further optimization of substituents, they obtained the Ox-W18 with about 90% selectivity. Additionally, the Trp-CLiC method can be successfully applied to various known protein labeling, including site-specific antibody functionalization and monitoring protein unfolding in solution and cells. Furthermore, an activity-based protein proteomics (ABPP) approach based on Trp-CLiC has also been established for identifying new functional tryptophan sites in the cell proteome. Using this method, 906 tryptophan residue binding sites were identified on protein surfaces, mainly associated with undruggable proteins and highly enriched in various subcellular regions, especially in phase-separated subcellular compartments. Using the NPM1 protein as a model, further investigation into the relationship between functional tryptophan residues on the protein surface and cellular phase separation revealed that the 2 labeled tryptophan residues could engage in lysine–tryptophan cation–π interactions. Moreover, acylation modifications resulted in alterations in protein localization and phase separation, demonstrating the regulatory role of cation–π interactions in protein phase separation and localization.
Despite the relatively small proportion of tryptophan in the human body [10], tryptophan plays a key role in various proteins. The widespread application of the novel oxidative covalent coupling method in tryptophan-specific bioconjugation not only overcomes the challenges of targeting tryptophan for covalent coupling but also enriches the types of developed warheads, providing new avenues for the development of bioconjugation techniques targeting other amino acids. Firstly, the Trp-CLiC redox method serves as an effective tool for developing covalent small-molecule inhibitors and activators, while also aiding in the exploration of protein functionality, activity-based proteomics for functional amino acid identification and characterization, and the synthesis of antibody–drug conjugates. Secondly, by leveraging the structural characteristics of amino acid residues to develop various types of potential reaction pathways and integrating interdisciplinary knowledge from biochemistry, medicinal chemistry and computational chemistry, new insights are brought to the development of covalent inhibitors based on protein conjugation technology.
Moreover, compared to in vitro bioconjugation, the complexity of the biological environment often results in a lack of specificity in chemically based protein bioconjugation. Thus, covalent modifications of endogenous proteins in their native biological milieu are largely underdeveloped. The development of highly specific protein bioconjugation strategies, coupled with quantitative analysis using instruments of high sensitivity and accuracy, will be essential for future exploration and modification of endogenous proteins. The utilization of gene editing tools like the CRISPR/Cas9 system [21], which enables the expression of genetically engineered proteins as endogenous proteins, in conjunction with the aforementioned chemical methods, offers abundant opportunities for the chemical modification of endogenous proteins in live cells. Furthermore, with the burgeoning development of various deep learning models, systematic analysis of accumulated data may enable prediction of novel bioconjugation methods and covalent binding sites on proteins in the future, significantly expediting research progress and accelerating exploration in this field [22].
References
Less
1.
Eaton JK, Furst L, Ruberto RA, Moosmayer D, Hilpmann A, Ryan MJ, Zimmermann K, Cai LL, Niehues M, Badock V, et al. Selective covalent targeting of GPX4 using masked nitrile-oxide electrophiles. Nat Chem Biol. 2020;16(5):497–506.
2.
Janes MR, Zhang J, Li L-S, Hansen R, Peters U, Guo X, Chen Y, Babbar A, Firdaus SJ, Darjania L, et al. Targeting KRAS mutant cancers with a covalent G12C-specific inhibitor. Cell. 2018;172(3):578–589.
3.
Chen Y, Craven GB, Kamber RA, Cuesta A, Zhersh S, Moroz YS, Bassik MC, Taunton J. Direct mapping of ligandable tyrosines and lysines in cells with chiral sulfonyl fluoride probes. Nat Chem. 2023;15(11):1616–1625.
4.
Ostrem JML, Shokat KM. Direct small-molecule inhibitors of KRAS: From structural insights to mechanism-based design. Nat Rev Drug Discov. 2016;15(11):77–785.
5.
Weiss A, Lorthiois E, Barys L, Beyer KS, Bomio-Confaglia C, Burks H, Chen X, Cui X, de Kanter R, Dharmarajan L, et al. Discovery, preclinical characterization, and early clinical activity of JDQ443, a structurally novel, potent, and selective covalent oral inhibitor of KRASG12C. Cancer Discov. 2022;12(6):1500–1517.
6.
Xie X, Yu T, Li X, Zhang N, Foster LJ, Peng C, Huang W, He G. Recent advances in targeting the “undruggable” proteins: From drug discovery to clinical trials. Signal Transduct Target Ther. 2023;8(1):335.
7.
Abdeldayem A, Raouf YS, Constantinescu SN, Moriggl R, Gunning PT. Advances in covalent kinase inhibitors. Chem Soc Rev. 2020;49(9):2617–2687.
8.
Skoulidis F, Li BT, Dy GK, Price TJ, Falchook GS, Wolf J, Italiano A, Schuler M, Borghaei H, Barlesi F, et al. Sotorasib for lung cancers with KRAS p.G12C mutation. N Engl J Med. 2023;384(25):2371–2381.
9.
Chauhan P, Ragendu V, Kumar M, Molla R, Dev Mishra S, Basaa S, Rai V. Chemical technology principles for selective bioconjugation of proteins and antibodies. Chem Soc Rev. 2024;53(1):380–449.
10.
Hartmann P, Bohdan K, Hartmann P, Bohdan K, Hommrich M, Juliá F, Vogelsang L, Eirich J, Zangl R, Farès C, et al. Chemoselective umpolung of thiols to episulfoniums for cysteine bioconjugation. Nat Chem. 2024;16(3):380–388.
11.
Lin S, Yang XY, Jia S, Weeks AM, Hornsby M, Lee PS, Nichiporuk RV, Iavarone AT, Wells JA, Toste FD, et al. Redox-based reagents for chemoselective methionine bioconjugation. Science. 2017;355(6325):597–602.
12.
Li BX, Kim DK, Bloom S, Huang RY-C, Qiao JX, Ewing WR, Oblinsky DG, Scholes GD, MacMillan DWC. Redox-based reagents for chemoselective methionine bioconjugation. Nat Chem. 2021;13(9):902–908.
13.
Vantourout JC, Adusumalli SR, Knouse KW, Flood DT, Ramirez A, Padial NM, Istrate A, Maziarz K, deGruyter JN, Merchant RR, et al. Serine-selective bioconjugation. J Am Chem Soc. 2020;142(41):17236–17242.
14.
Hansen MB, Hubálek F, Skrydstrup T, Hoeg-Jensen T. Transition metal-free tryptophan-selective bioconjugation of proteins. J Am Chem Soc. 2016;138(34):10798–10801.
15.
Hoopes CR, Garcia FJ, Sarkar AM, Kuehl NJ, Barkan DT, Collins NL, Meister GE, Bramhall TR, Hsu CH, Jones MD, et al. Donor–acceptor pyridinium salts for photo-induced electron-transfer-driven modification of tryptophan in peptides, proteins, and proteomes using visible light. J Am Chem Soc. 2022;144(14):6227–6236.
16.
Xie X, Moon PJ, Crossley SWM, Bischoff AJ, He D, Li G, Dao N, Gonzalez-Valero A, Reeves AG, McKenna JM, et al. Oxidative cyclization reagents reveal tryptophan cation–π interactions. Nature. 2024;627(8004):680–687.
17.
Tang M-C, Zou Y, Watanabe K, Walsh CT, Tang Y. Oxidative cyclization in natural product biosynthesis. Chem Rev. 2017;117(8):5226–5333.
18.
Davis FA. Recent applications of N-sulfonyloxaziridines (Davis oxaziridines) in organic synthesis. Tetrahedron. 2018;74(26):3198–3214.
19.
Michaelis DJ, Yoon TP. Advances in the chemistry of oxaziridines. Chem Rev. 2014;114(16):8016–8036.
20.
Hu J-J, He P-Y, Li Y-M. Chemical modifications of tryptophan residues in peptides and proteins. J Pept Sci. 2021;27(1):e3286.
21.
Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157(6):1262–1278.
22.
Du H, Jiang D, Gao J, Zhang X, Jiang L, Zeng Y, Wu Z, Shen C, Xu L, Cao D, et al. Proteome-wide profiling of the covalent-druggable cysteines with a structure-based deep graph learning network. Research. 2022;2022:DOI:10.34133/2022/9873564.
Appendix
Less
Year 2024 volume 7 Issue 7
PDF
114
56
Cite this Article
BibTeX
Article Info
doi: 10.34133/research.0410
  • Receive Date：2024-04-28
  • Online Date：2025-08-07
  • Published：2024-07-04
Article Data
Affiliations
History
  • Received：2024-04-28
  • Accepted：2024-05-22
Affiliations
    State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, P. R. China.

Corresponding:

* Address correspondence to:
References
Share
https://castjournals.cast.org.cn/joweb/research/EN/10.34133/research.0410
Share to
QR

Scan QR to access full text

Cite this article
BibTeX
Citations
表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
关闭全屏
  • BibTeX
  • EndNote
  • RefWorks
  • TxT
Links
Articles
Latest Articles
Most Read
Collections
Updates
Events
News
Multimedia
About
About Us
Contact
No. 86 Xueyuan South Road, Haidian District, Beijing
100081
010-62199257
qkjq@cast.org.cn

Copyright © 2025 China Association for Science and Technology. All rights reserved. For all open access content, the relevant licensing terms apply.
Sponsored by the Office of the Leading Group for Cybersecurity and Informatization of CAST, and supported by Science and Technology Review Publishing House