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

Abstract

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.

A Comprehensive Guide for Assessing Covalent Inhibition in Enzymatic Assays Illustrated with Kinetic Simulations

“…It is paramount to select the appropriate algebraic model for data analysis, as the inhibitor binding mode changes the obtainable parameters as well as the compatibility with experimental methods. Covalent EI* adduct formation should be validated by direct detection with MS, X‐ray crystallography or NMR (Harris et al., 2018; Liclican et al., 2020; Mons et al., 2019; Mons et al., 2021). Reversibility of covalent adduct formation is commonly assessed in rapid/jump dilution or washout assays with detection of regained enzymatic activity after dilution/washout (Copeland, Basavapathruni, Moyer, & Scott, 2011), MS detection of unbound inhibitor upon denaturation or digestion‐mediated dissociation (Bradshaw et al., 2015), or competitive binding of a (selective) irreversible (activity‐based) probe (Liclican et al., 2020; Smith et al., 2017).…”

Inhibiting UCH-L5: Rational Design of a Cyclic Ubiquitin-Based Peptide Inhibitor

“…Furthermore, we also tested the competitive labeling of other DUBs in the UCH family, namely UCH-L1, UCH-L3, and BAP1. Our results indicate a stronger reactivity of Cy5-UbVME to UCH-L5, L1, and L3 than BAP1, which was successfully labeled with Rho-UbPA (Ekkebus et al, 2013;Mons et al, 2021). Therefore, we used both Cy5-UbVME and Rho-UbPA probes for all the enzymes in our competition assay.…”

Facile Preparation of UFMylation Activity-Based Probes by Chemoselective Installation of Electrophiles at the C-Terminus of Recombinant UFM1

“…Epoxides are known as cysteine-reactive groups that undergo ring-opening upon reaction with nucleophilic thiols. 27,28 Alkynes have been shown to react with deconjugating enzymes, 29,30 although for UFM1, only the propargyl group (UFM1-PA) has previously been tested for UFSP1 and UFSP2 de-UFMylases. 16 UFM1-PA was shown to be inactive with human UFSP1 and required extended incubation to engage with human UFSP2.…”