Ubiquitin-like proteins (Ubls) such as SUMO, NEDD8, ISG15, URM1, UFM1, FAT10, ATG8/ATG12, and FUBI are essential regulators of cellular homeostasis, controlling processes from protein stability and trafficking to immune signaling and autophagy. Their conjugation-deconjugation cycles are mediated by cascades of E1, E2, and E3 enzymes and reversed by Ubl-specific proteases (ULPs), many of which are cysteine-dependent. Deciphering these dynamic and reversible pathways requires tools that directly capture the active forms of these enzymes. Activity-based probes (ABPs) have become indispensable for this task, providing covalent, mechanism-based snapshots of enzymatic activity in complex systems. This review highlights chemistry-centric strategies for the design and synthesis of Ubl-targeting ABPs. We summarize synthetic and semisynthetic approaches that install electrophilic warheads onto Ubl backbones, methods for C-terminal ligation (native chemical ligation, activated cysteine ligation, hydrazide chemistry), and strategies for incorporating reporter tags or bioorthogonal handles. Probe development is organized by target class, including Ubl isopeptidases, E1/E2 conjugating enzymes, and E3 ligases. Representative examples illustrate how chemical design choices are tailored for specific applications-ranging from live-cell activity profiling to proteomic mapping and inhibitor discovery. Together, these methodologies establish a versatile chemical toolkit for dissecting Ubl biology, enabling the discovery of novel enzymes, the mapping of substrate networks, and the development of potential therapeutic modulators.
Chanda et al. (Mon,) studied this question.