Covalent inhibition of SUMO and ubiquitin-specific cysteine proteases by an in situ thiol–alkyne addition

Abstract

Posttranslational modification of proteins with ubiquitin and ubiquitin-like modifiers such as SUMO can be reverted by specific proteases, also referred to as deubiquitinases and isopeptidases, most of which are cysteine-dependent. We have found that the replacement of the conserved C-terminal glycine with propargylamine converts SUMO and ubiquitin to highly efficient covalent inhibitors of their cognate cysteine proteases. Attack of the catalytic cysteine onto the terminal alkyne results in the formation of a vinyl sulfide linkage. Although this reaction is reminiscent of the inhibitory mechanism of the isosteric nitrile inhibitors it was unexpected due to the low electrophilicity of the alkyne group. We show that a precise location of the functional group in the active site of the protease is crucial for the reaction, which was not inhibited by the presence of a radical scavenger. Furthermore, a mutational study of key catalytic residues in the SUMO-protease Senp1, that is H533A and D550A of the catalytic triad and Q597A as part of the oxyanion hole, revealed that these residues are not required for the observed covalent adduct formation. We therefore propose that the reaction is an in situ thiol-alkyne addition. Due to the high chemical inertness of the alkyne moiety the respective protease inhibitors should be well-suited for cellular and therapeutic applications. In keeping with this idea, selective labeling with propargylated SUMO and Ub probes was observed in lysates of cell lines expressing the cognate proteases after transient transfection.

Covalent protein modification: the current landscape of residue-specific electrophiles

“…The bioorthogonality of terminal alkynes has recently been questioned due to the observed covalent modification of cysteines by propargyl amides [47,48,49 ]. Ubiquitin propargylated at the C-terminus (Ub-Prg) inhibits a number of deubiquitinating enzymes (DUBs) through covalent modification of the active-site cysteine nucleophile [47] (Figure 2h).…”

Isomorphous Crystals from Diynes and Bromodiynes Involved in Hydrogen and Halogen Bonds

“…Better knowledge of the behavior of these interactions could bring additional tools in crystal engineering and in material design, as well as for efficient and fruitful drug design. Indeed, terminal alkynes [43,44] and haloalkynes [45][46][47] are biologically important classes of molecules. …”

Total Chemical Synthesis of SUMO and SUMO‐Based Probes for Profiling the Activity of SUMO‐Specific Proteases

“…SUMO conjugates have been prepared before by semi‐synthesis using intein chemistry15 or by synthesis and ligation of peptide fragments 16, 17, 18, 19. In this report, we present a fully synthetic linear solid‐phase peptide synthesis (SPPS) approach towards full‐length SUMO conjugates, which enables facile parallel synthesis procedures.…”