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  • SBI-0206965 Advances in the total chemical synthesis

    2020-06-29

    Advances in the total chemical synthesis of ubiquitin have enabled the efficient synthesis of new and improved ubiquitin-based reagents. Using an optimized linear synthesis, ubiquitin can now be easily obtained in high yield and purity [35]. Using this synthetic methodology, ubiquitin can be functionalized with any reactive group, dye or label that is compatible with standard peptide synthesis procedures at any specific position.
    Active-site directed probes and their applications The discovery and study of DUBs and Ubl deconjugating enzymes has been greatly accelerated by the development of activity-based probes (ABPs). These ubiquitin-based probes are suicide substrates that specifically react with the active site cysteine nucleophile of DUBs in an activity-based manner. Their covalent nature can be used to visualize and purify an entire family of active proteins simultaneously. ABPs contain three essential elements (Figure 2A). The targeting element confers specificity for the desired enzyme targets. In the case of DUB probes, the targeting element is ubiquitin. Secondly, the reactive group or warhead, which reacts with the active site of the target enzyme after the targeting element has bound. Finally, a recognition or retrieval element is incorporated to allow for the selective retrieval or visualization of the enzyme-ABP complex [34]. The first of these ubiquitin ABPs was ubiquitin-nitrile, which allowed the labeling of a proteasome-bound cysteine dependent DUB [36]. Many improvements to probe design and synthesis have been reported since [32, 33, 34, 37••]. For instance, these developments led to the discovery that OTU class enzymes act as deubiquitylases [38]. Last year, our lab reported the discovery of a novel warhead for use in activity-based profiling and purification of active deubiquitylating enzymes []. The functionality of this warhead was nicely confirmed and complemented in work reported by Sommer et al. []. The introduction of a chemically inert terminal alkyne moiety at the C-terminus of ubiquitin (Ub-Prg) renders it highly reactive toward deubiquitylating enzymes. It was shown that this novel Ub-Prg probe reacts with all SBI-0206965 of cysteine DUBs. An advantage of this alkyne-based probe is that it is very reactive toward DUBs but does not react with unrelated Cys-proteases under the same conditions. Further reviews on general ABP chemistry can be found elsewhere [40].
    DUB specificity profiling Ubiquitin-based ABPs can be used to identify known and putative deubiquitylating enzymes from cell lysates by mass spectrometry. In addition, active DUBs in cell lysates can be directly visualized after SDS-PAGE separation using western blotting or fluorescence scanning (Figure 2B). This direct visualization can be used to assay novel DUB inhibitors for putative enzyme specificity (Figure 2C), recently described by Altun et al. []. Using the same principle, De Jong et al. [42] demonstrated that the DUB inhibitor b-AP15 has little selectivity among DUBs (Figure 2C). While inhibitor selectivity profiling is feasible, these experiments are not trivial to perform due to the covalent nature of the ABPs and the high affinity for targets. Careful timing and elevated inhibitor concentrations in these experiments are crucial.
    Native chemical ligation to synthesize ubiquitin chain specific probes A number of non-enzymatic ways to obtain ubiquitin conjugates have been reported in recent years [43]. An important technology enabling the synthesis of natively isopeptide-linked assay reagents is the use of native chemical ligation (NCL) as depicted in Figure 3A. In an NCL a native peptide bond is formed between two peptides, one containing a C-terminal thioester and another containing an N-terminal cysteine residue [44]. To generate diubiquitin modules, which are normally linked through an isopeptide bond between the C-terminal glycine residue of ubiquitin and the ɛ-amine of lysine, this technology needed to be adapted. By introducing SBI-0206965 a thiolysine residue (Figure 3B) that harbors a thiol moiety positioned either at the δ- or γ-position of a lysine residue (Figure 3C) in the sequence of ubiquitin, an acceptor ubiquitin is created onto which a ubiquitin molecule can be ligated [35, 45, 46, 47]. With this technology, all isopeptide-linked ubiquitin chain topologies can be synthesized chemically.