Exploring the Versatility of the Covalent Thiol-Alkyne Reaction with Substituted Propargyl Warheads: A Deciding Role for the Cysteine Protease

Terminal unactivated alkynes are nowadays considered the golden standard for cysteine-reactive warheads in activity-based probes (ABPs) targeting cysteine deubiquitinating enzymes (DUBs). In this work, we study the versatility of the thiol-alkyne addition reaction in more depth. Contrary to previous findings with UCHL3, we now show that covalent adduct formation can progress with substituents on the terminal or internal alkyne position. Strikingly, acceptance of alkyne substituents is strictly DUB-specific as this is not conserved among members of the same subfamily. Covalent adduct formation with the catalytic cysteine residue was validated by gel analysis and mass spectrometry of intact ABP-treated USP16CD(WT) and catalytically inactive mutant USP16CD(C205A). Bottom-up mass spectrometric analysis of the covalent adduct with a deuterated propargyl ABP provides mechanistic understanding of the in situ thiol-alkyne reaction, identifying the alkyne rather than an allenic intermediate as the reactive species. Furthermore, kinetic analysis revealed that introduction of (bulky/electron-donating) methyl substituents on the propargyl moiety decreases the rate of covalent adduct formation, thus providing a rational explanation for the commonly lower level of observed covalent adduct compared to unmodified alkynes. Altogether, our work extends the scope of possible propargyl derivatives in cysteine targeting ABPs from unmodified terminal alkynes to internal and substituted alkynes, which we anticipate will have great value in the development of ABPs with improved selectivity profiles.

Automated summary

This study delves into the versatility of the thiol-alkyne addition reaction, showing that covalent adduct formation can occur with substituents on different alkynes positions, depending on the specific DUB. Mass spectrometric analysis confirmed the covalent adduct formation with the catalytic cysteine residue, while kinetic analysis revealed the impact of methyl substituents on the rate of formation. These findings extend the potential of propargyl derivatives in cysteine targeting ABPs, offering insights into developing ABPs with enhanced selectivity.