From Covalent Traps to Fluorescent Beacons: The Expanding Arsenal of Chemical Probes for Studying Ubiquitin and Ubiquitin‐Like Proteins

Ubiquitin (Ub) and ubiquitin-like proteins (Ubls) orchestrate diverse cellular processes through reversible post-translational modification of target proteins. Their conjugation is governed by a cascade of E1 activating, E2 conjugating, and E3 ligating enzymes, while deconjugation is mediated by deubiquitinases (DUBs) and Ubl-specific proteases. Profiling the catalytic activity of these enzymes is essential for understanding the dynamics and specificity of Ub/Ubl signaling. Activity-based probes (ABPs) have emerged as powerful tools to covalently label active enzymes through electrophilic warheads that target catalytic residues. Unlike conventional affinity-based approaches, ABPs capture functional states of enzymes in complex biological systems. This review provides a comprehensive analysis of ABPs designed for the Ub/Ubl signal pathways, encompassing probes for Ub, SUMO, NEDD8, ISG15, FAT10, UFM1, URM1, Atg8-family modifiers, and FUBI (MNSFβ). We discuss key elements of probe design, including recognition domains, electrophilic warheads (e.g., vinyl sulfones, vinyl methyl esters, propargylamine, azapeptide esters), and detection tags. Particular emphasis is placed on emerging azapeptide ester-based probes, which structurally mimic native enzyme-substrate intermediates and offer high selectivity and reactivity. ABPs targeting E1, E2, and HECT/RBR E3 ligases are also highlighted, expanding their utility beyond classical DUB profiling. We further compare warhead chemistries, enzyme selectivity, and labeling strategies, and examine structural insights derived from probe-enzyme complexes. Collectively, these tools have transformed our ability to interrogate Ub/Ubl-regulating enzymes in vitro and in cells. The review concludes with perspectives on next-generation probe development, including cell-permeable designs, spatiotemporal control, and applications in systems biology and drug discovery.