As the scope of modern genomics technologies increases, so does the need for informative chemical tools to study functional biology. Activity-based probes (ABPs) provide a powerful suite of reagents to probe the biochemistry of living organisms. These probes, featuring a specificity motif, a reactive chemical group and a reporter tag, are opening-up large swathes of protein chemistry to investigation in vitro, as well as in cellular extracts, cells and living organisms in vivo. Glycoside hydrolases, by virtue of their prominent biological and applied roles, provide a broad canvas on which ABPs may illustrate their functions. Here we provide an overview of glycosidase ABP mechanisms, and review recent ABP work in the glycoside hydrolase field, encompassing their use in medical diagnosis, their application for generating chemical genetic disease models, their fine-tuning through conformational and reactivity insight, their use for high-throughput inhibitor discovery, and their deployment for enzyme discovery and dynamic characterization. Highlights  Glycosidases carry out many essential functions across all domains of life  Activity-based probes can interrogate complex biological samples for glycosidase activity  Glycosidase probes can characterize enzymes of biomedical/biotechnological interest  Analysis of conformational itineraries may inform the design of future probes.  Activity-based probes assist high-throughput discovery of inhibitors

Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and endo -β-1,4-xylanases in the secretomes of Aspergillus niger , by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme–substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.

Heparan sulfate proteoglycans (HSPGs) mediate essential interactions throughout the extracellular matrix (ECM), providing signals that regulate cellular growth and development. Altered HSPG composition during tumorigenesis strongly aids cancer progression. Heparanase (HPSE) is the principal enzyme responsible for extracellular heparan sulfate catabolism and is markedly up-regulated in aggressive cancers. HPSE overactivity degrades HSPGs within the ECM, facilitating metastatic dissemination and releasing mitogens that drive cellular proliferation. Reducing extracellular HPSE activity reduces cancer growth, but few effective inhibitors are known, and none are clinically approved. Inspired by the natural glycosidase inhibitor cyclophellitol, we developed nanomolar mechanism-based, irreversible HPSE inhibitors that are effective within physiological environments. Application of cyclophellitol-derived HPSE inhibitors reduces cancer aggression in cellulo and significantly ameliorates murine metastasis. Mechanism-based irreversible HPSE inhibition is an unexplored anticancer strategy. We demonstrate the feasibility of such compounds to control pathological HPSE-driven malignancies.

The design of structurally diverse enzymes is constrained by long-range interactions that are necessary for accurate folding. We introduce an atomistic and machine learning strategy for the combinatorial assembly and design of enzymes (CADENZ) to design fragments that combine with one another to generate diverse, low-energy structures with stable catalytic constellations. We applied CADENZ to endoxylanases and used activity-based protein profiling to recover thousands of structurally diverse enzymes. Functional designs exhibit high active-site preorganization and more stable and compact packing outside the active site. Implementing these lessons into CADENZ led to a 10-fold improved hit rate and more than 10,000 recovered enzymes. This design-test-learn loop can be applied, in principle, to any modular protein family, yielding huge diversity and general lessons on protein design principles.

Activity-based protein profiling has emerged as a powerful discovery tool in chemical biology and medicinal chemistry research. Success of activity-based protein profiling hinges on the presence of compounds that can covalently and irreversibly bind to enzymes, do so selectively in the context of complex biological samples, and subsequently report on the selected pool of proteins. Such tagged molecules featuring an electrophilic trap, termed activity-based probes, have been developed with most success for serine hydrolases and various protease families (serine proteases, cysteine proteases, proteasomes). This concept presents the current progress and future directions in the design of activity-based probes targeting retaining glycosidases, enzymes that employ a double displacement mechanism in the hydrolysis of glycosidic bonds with overall retention. In contrast to inverting glycosidases, retaining glycosidases form a covalent intermediate with their substrates during the catalytic process and are therefore amenable to activity-based protein profiling studies.