Converting lead compounds into drug candidates is a crucial step in drug development, requiring early assessment of potency, selectivity, and off-target effects. We have utilized activity-based chemical proteomics to determine the potency and selectivity of deubiquitylating enzyme (DUB) inhibitors in cell culture models. Importantly, we characterized the small molecule PR-619 as a broad-range DUB inhibitor, and P22077 as a USP7 inhibitor with potential for further development as a chemotherapeutic agent in cancer therapy. A striking accumulation of polyubiquitylated proteins was observed after both selective and general inhibition of cellular DUB activity without direct impairment of proteasomal proteolysis. The repertoire of ubiquitylated substrates was analyzed by tandem mass spectrometry, identifying distinct subsets for general or specific inhibition of DUBs. This enabled identification of previously unknown functional links between USP7 and enzymes involved in DNA repair.

O-GlcNAc hydrolase, OGA, removes O-linked N-acetylglucosamine (O-GlcNAc) from myriad nucleocytoplasmic proteins. Through co-expression and assembly of OGA fragments we determined the 3-D structure of human OGA, revealing an unusual helix exchanged dimer that lays a structural foundation for an improved understanding of substrate recognition and regulation of OGA. Structures of OGA in complex with a series of inhibitors define a precise blueprint for the design of inhibitors having clinical value.

Activity-based protein profiling (ABPP) has emerged as a powerful strategy to study the activity of enzymes in complex proteomes. The aim of ABPP is to selectively visualize only the active forms of particular enzymes using chemical probes termed activity-based probes (ABPs). These probes are directed to the active site of a particular target protein (or protein family) where they react in a mechanism-based manner with an active site residue. This results in the selective labeling of only the catalytically active form of the enzyme, usually in a covalent manner. Besides the monitoring of a specific enzymatic activity, ABPP strategies have also been used to identify and characterize (unknown) protein functions, to study up- and down-regulation of enzymatic activity in various disease states, to discover and evaluate putative new enzyme inhibitors, and to identify the protein targets of covalently binding natural products. In this Topical Review we will provide a brief overview of some of the recent developments in the field of ABPP.

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