How to Utilize Unnatural Amino Acids to Develop Highly Selective Deubiquitinase Probes?

1. How to Develop Highly Selective Chemical Probes for Deubiquitinases (DUBs)?
 
Deubiquitinases (DUBs) are responsible for removing ubiquitin modifications from proteins and play critical roles in various pathological processes such as viral infection, neurodegenerative diseases, and cancer, making them promising therapeutic targets. The human genome encodes approximately 100 DUBs; however, the lack of chemical tools capable of specifically recognizing and characterizing their activities has limited the study of their functions under physiological and pathological conditions. Although commercially available broad-spectrum fluorescent substrates (e.g., C-terminal ubiquitin sequences or fluorescently labeled ubiquitin molecules) have been widely used in biochemical analyses of DUBs, there is an urgent need to develop highly selective probes targeting individual DUBs.
 
2. How to Screen DUB Substrate Preferences Using Unnatural Amino Acids?
 
A team led by Prof. Marcin Drag and Dr. Wioletta Rut from the Wrocław University of Science and Technology in Poland published a study in Chemical Science focused on constructing highly selective chemical probes for DUBs based on a ubiquitin (Ub) scaffold. The core strategy involves incorporating unnatural amino acids into the conserved C-terminal Ub sequence to systematically screen DUB substrate recognition preferences.
 
 
 
Using two cancer-associated DUBs—MERS PLpro and UCH-L3—as models, the researchers constructed two types of chemical substrate libraries: one for screening P2 site preferences (Ac-Leu-Arg-P2-Gly-ACC) and another mixed substrate library for evaluating P3 and P4 site specificities. Screening results revealed that MERS PLpro prefers glycine at the P2 site, while UCH-L3 favors aliphatic amino acids. At the P3 site, UCH-L3 strongly prefers arginine, whereas MERS PLpro is more tolerant of hydrophobic residues. Based on these specificity profiles, the authors synthesized corresponding substrates and validated their high selectivity through kinetic experiments.
 
3. Can Selective Ubiquitin-Based Irreversible Probes Be Constructed?
 
Building on the clarified substrate specificities, the authors further designed Ub-based activity probes. These probes feature an N-terminal detection tag (e.g., biotin) and a C-terminal嫁接 irreversible electrophilic group—vinyl methyl ester (VME)—which can covalently bind to the DUB active site.
Kinetic analyses demonstrated that the constructed probes, B-Ub-UCHL3-2-VME and B-Ub-MERS27-VME, exhibited efficient and specific labeling capabilities for UCH-L3 and MERS PLpro, respectively, significantly outperforming wild-type Ub-ACC substrates. Subsequent experiments in cell lysates further confirmed the high selectivity and cellular activity of these probes.
 
4. Does This Strategy Provide a Universal Platform for DUB Research Tool Development?
 
By embedding unnatural amino acids into the ubiquitin molecular framework, this study successfully developed a strategy for constructing DUB chemical probes with both activity and selectivity. This method is not only applicable to UCH-L3 and MERS PLpro but also holds potential for extension to other DUBs and ubiquitin-like specific proteases, providing powerful chemical tools for exploring the roles of DUBs in disease mechanisms and developing targeted therapies.

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