E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates^1,2. However, rather than functioning individually, many neddylated cullin–RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family—which together account for nearly half of all ubiquitin ligases in humans—form E3–E3 super-assemblies^3–7. Here, by studying CRLs in the SKP1–CUL1–F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3–E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3–E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3–E3 super-assembly may therefore underlie widespread ubiquitylation.

Covalent inhibition has become more accepted in the past two decades, as illustrated by the clinical approval of several irreversible inhibitors designed to covalently modify their target. Elucidation of the structure‐activity relationship and potency of such inhibitors requires a detailed kinetic evaluation. Here, we elucidate the relationship between the experimental read‐out and the underlying inhibitor binding kinetics. Interactive kinetic simulation scripts are employed to highlight the effects of in vitro enzyme activity assay conditions and inhibitor binding mode, thereby showcasing which assumptions and corrections are crucial. Four stepwise protocols to assess the biochemical potency of (ir)reversible covalent enzyme inhibitors targeting a nucleophilic active site residue are included, with accompanying data analysis tailored to the covalent binding mode. Together, this will serve as a guide to make an educated decision regarding the most suitable method to assess covalent inhibition potency. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol I: Progress curve analysis of substrate association competitionBasic Data Analysis Protocol 1A: Two‐step irreversible covalent inhibitionBasic Data Analysis Protocol 1B: One‐step irreversible covalent inhibitionBasic Data Analysis Protocol 1C: Two‐step reversible covalent inhibitionBasic Data Analysis Protocol 1D: Two‐step irreversible covalent inhibition with substrate depletionBasic Protocol II: Incubation time–dependent potency IC₅₀(t)Basic Data Analysis Protocol 2: Two‐step irreversible covalent inhibitionBasic Protocol III: Preincubation time–dependent inhibition without dilutionBasic Data Analysis Protocol 3: Preincubation time–dependent inhibition without dilutionBasic Data Analysis Protocol 3Ai: Two‐step irreversible covalent inhibitionAlternative Data Analysis Protocol 3Aii: Two‐step irreversible covalent inhibitionBasic Data Analysis Protocol 3Bi: One‐step irreversible covalent inhibitionAlternative Data Analysis Protocol 3Bii: One‐step irreversible covalent inhibitionBasic Data Analysis Protocol 3C: Two‐step reversible covalent inhibitionBasic Protocol IV: Preincubation time–dependent inhibition with dilution/competitionBasic Data Analysis Protocol 4: Preincubation time–dependent inhibition with dilutionBasic Data Analysis Protocol 4Ai: Two‐step irreversible covalent inhibitionAlternative Data Analysis Protocol 4Aii: Two‐step irreversible covalent inhibitionBasic Data Analysis Protocol 4Bi: One‐step irreversible covalent inhibitionAlternative Data Analysis Protocol 4Bii: One‐step irreversible covalent inhibition

Within druggable target space, new small-molecule modalities, particularly covalent inhibitors and targeted degraders, have expanded the repertoire of medicinal chemists. Molecules with such modes of action have a large potential not only as drugs but also as chemical probes. Criteria have previously been established to describe the potency, selectivity, and properties of small-molecule probes that are qualified to enable the interrogation and validation of drug targets. These definitions have been tailored to reversibly acting modulators but fall short in their applicability to other modalities. While initial guidelines have been proposed, we delineate here a full set of criteria for the characterization of covalent, irreversible inhibitors as well as heterobifunctional degraders ("proteolysis-targeting chimeras", or PROTACs) and molecular glue degraders. We propose modified potency and selectivity criteria compared to those for reversible inhibitors. We discuss their relevance and highlight examples of suitable probe and pathfinder compounds.

We present the development of a native chemical ligation handle that also functions as a masked electrophile that can be liberated during synthesis when required. This handle can thus be used for the synthesis of complex activity‐based probes. We describe the use of this handle in the generation of linkage‐specific activity‐based deubiquitylating enzyme probes that contain substrate context and closely mimic the native ubiquitin isopeptide linkage. We have generated activity‐based probes based on all seven isopeptide‐linked diubiquitin topoisomers and demonstrated their structural integrity and ability to label DUBs in a linkage‐specific manner.