Mechanism-based heparanase inhibitors reduce cancer metastasis in vivo.

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.

Xylose-Configured Cyclophellitols as Selective Inhibitors for Glucocerebrosidase.

Glucocerebrosidase (GBA), a lysosomal retaining ß-d-glucosidase, has recently been shown to hydrolyze ß-d-xylosides and to transxylosylate cholesterol. Genetic defects in GBA cause the lysosomal storage disorder Gaucher disease (GD), and also constitute a risk factor for developing Parkinson's disease. GBA and other retaining glycosidases can be selectively visualized by activity-based protein profiling (ABPP) using fluorescent probes composed of a cyclophellitol scaffold having a configuration tailored to the targeted glycosidase family. GBA processes ß-d-xylosides in addition to ß-d-glucosides, this in contrast to the other two mammalian cellular retaining ß-d-glucosidases, GBA2 and GBA3. Here we show that the xylopyranose preference also holds up for covalent inhibitors: xylose-configured cyclophellitol and cyclophellitol aziridines selectively react with GBA over GBA2 and GBA3 in vitro and in vivo, and that the xylose-configured cyclophellitol is more potent and more selective for GBA than the classical GBA inhibitor, conduritol B-epoxide (CBE). Both xylose-configured cyclophellitol and cyclophellitol aziridine cause accumulation of glucosylsphingosine in zebrafish embryo, a characteristic hallmark of GD, and we conclude that these compounds are well suited for creating such chemically induced GD models.

Development of Non-Hydrolysable Oligosaccharide Activity-Based Inactivators for Endoglycanases: A Case Study on α-1,6 Mannanases.

There is a vast genomic resource for enzymes active on carbohydrates. Lagging far behind, however, are functional chemical tools for the rapid characterization of carbohydrate-active enzymes. Activity-based probes (ABPs) offer one chemical solution to these issues with ABPs based upon cyclophellitol epoxide and aziridine covalent and irreversible inhibitors representing a potent and widespread approach. Such inhibitors for enzymes active on polysaccharides are potentially limited by the requirement for several glycosidic bonds, themselves substrates for the enzyme targets. Here, it is shown that non-hydrolysable trisaccharide can be synthesized and applied even to enzymes with challenging subsite requirements. It was found that incorporation of carbasugar moieties, which was accomplished by cuprate-assisted regioselective trans-diaxial epoxide opening of carba-mannal synthesised for this purpose, yields inactivators that act as powerful activity-based inhibitors for α-1,6 endo-mannanases. 3-D structures at 1.35-1.47 Šresolutions confirm the design rationale and binding to the enzymatic nucleophile. Carbasugar oligosaccharide cyclophellitols offer a powerful new approach for the design of robust endoglycosidase inhibitors, while the synthesis procedures presented here should allow adaptation towards activity-based endoglycosidase probes as well as configurational isosteres targeting other endoglycosidase families.

Dynamics of Ligand Binding to a Rigid Glycosidase*.

The single-domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, which is one of the most abundant renewable biomaterials in nature. We demonstrate that BCX in solution undergoes minimal structural changes during turnover. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow conversion, which is attributed to an enzyme-induced substrate distortion. A model is proposed in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates the acyl formation step of the hydrolysis reaction.

Direct Stereoselective Aziridination of Cyclohexenols with 3-Amino-2-(trifluoromethyl)quinazolin-4(3H)-one in the Synthesis of Cyclitol Aziridine Glycosidase Inhibitors.

Cyclophellitol aziridine and its configurational and functional isomers are powerful covalent inhibitors of retaining glycosidases, and find application in fundamental studies on glycosidases, amongst others in relation to inherited lysosomal storage disorders caused by glycosidase malfunctioning. Few direct and stereoselective aziridination methodologies are known for the synthesis of cyclophellitol aziridines. Herein, we present our studies on the scope of direct 3-amino-2-(trifluoromethyl)quinazolin-4(3H)-one-mediated aziridination on a variety of configurational and functional cyclohexenol isosters. We demonstrate that the aziridination can be directed by an allylic or homoallylic hydroxyl through H-bonding and that steric hindrance plays a key role in the diastereoselectivity of the reaction.

Gluco-1 H-imidazole: A New Class of Azole-Type ß-Glucosidase Inhibitor.

Gluco-azoles competitively inhibit glucosidases by transition-state mimicry and their ability to interact with catalytic acid residues in glucosidase active sites. We noted that no azole-type inhibitors described, to date, possess a protic nitrogen characteristic for 1 H-imidazoles. Here, we present gluco-1 H-imidazole, a gluco-azole bearing a 1 H-imidazole fused to a glucopyranose-configured cyclitol core, and three close analogues as new glucosidase inhibitors. All compounds inhibit human retaining ß-glucosidase, GBA1, with the most potent ones inhibiting this enzyme (deficient in Gaucher disease) on a par with glucoimidazole. None inhibit glucosylceramide synthase, cytosolic ß-glucosidase GBA2 or α-glucosidase GAA. Structural, physical and computational studies provide first insights into the binding mode of this conceptually new class of retaining ß-glucosidase inhibitors.

Spiro-epoxyglycosides as Activity-Based Probes for Glycoside Hydrolase Family 99 Endomannosidase/Endomannanase.

N-Glycans direct protein function, stability, folding and targeting, and influence immunogenicity. While most glycosidases that process N-glycans cleave a single sugar residue at a time, enzymes from glycoside hydrolase family 99 are endo-acting enzymes that cleave within complex N-glycans. Eukaryotic Golgi endo-1,2-α-mannosidase cleaves glucose-substituted mannose within immature glucosylated high-mannose N-glycans in the secretory pathway. Certain bacteria within the human gut microbiota produce endo-1,2-α-mannanase, which cleaves related structures within fungal mannan, as part of nutrient acquisition. An unconventional mechanism of catalysis was proposed for enzymes of this family, hinted at by crystal structures of imino/azasugars complexed within the active site. Based on this mechanism, we developed the synthesis of two glycosides bearing a spiro-epoxide at C-2 as electrophilic trap, to covalently bind a mechanistically important, conserved GH99 catalytic residue. The spiro-epoxyglycosides are equipped with a fluorescent tag, and following incubation with recombinant enzyme, allow concentration, time and pH dependent visualization of the bound enzyme using gel electrophoresis.

From covalent glycosidase inhibitors to activity-based glycosidase probes.

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.

Selective labelling of GBA2 in cells with fluorescent ß-d-arabinofuranosyl cyclitol aziridines.

GBA2, the non-lysosomal ß-glucosylceramidase, is an enzyme involved in glucosylceramide metabolism. Pharmacological inhibition of GBA2 by N-alkyl iminosugars is well tolerated and benefits patients suffering from Sandhoff and Niemann-Pick type C diseases, and GBA2 inhibitors have been proposed as candidate-clinical drugs for the treatment of parkinsonism. With the ultimate goal to unravel the role of GBA2 in (patho)physiology, we sought to develop a GBA2-specific activity-based probe (ABP). A library of probes was tested for activity against GBA2 and the two other cellular retaining ß-glucosidases, lysosomal GBA1 and cytosolic GBA3. We show that ß-d-arabinofuranosyl cyclitol aziridine (ß-d-Araf aziridine) reacts with the GBA2 active site nucleophile to form a covalent and irreversible bond. Fluorescent ß-d-Araf aziridine probes potently and selectively label GBA2 both in vitro and in cellulo, allowing for visualization of the localization of overexpressed GBA2 using fluorescence microscopy. Co-staining with an antibody selective for the lysosomal ß-glucosylceramidase GBA1, shows distinct subcellular localization of the two enzymes. We proffer our ABP technology for further delineating the role and functioning of GBA2 in disease and propose the ß-d-Araf aziridine scaffold as a good starting point for the development of GBA2-specific inhibitors for clinical development.

Correction: Glycosylated cyclophellitol-derived activity-based probes and inhibitors for cellulases.

[This corrects the article DOI: 10.1039/D0CB00045K.].

Glycosylated cyclophellitol-derived activity-based probes and inhibitors for cellulases.

Cellulases and related ß-1,4-glucanases are essential components of lignocellulose-degrading enzyme mixtures. The detection of ß-1,4-glucanase activity typically relies on monitoring the breakdown of purified lignocellulose-derived substrates or synthetic chromogenic substrates, limiting the activities which can be detected and complicating the tracing of activity back to specific components within complex enzyme mixtures. As a tool for the rapid detection and identification of ß-1,4-glucanases, a series of glycosylated cyclophellitol inhibitors mimicking ß-1,4-glucan oligosaccharides have been synthesised. These compounds are highly efficient inhibitors of HiCel7B, a well-known GH7 endo-ß-1,4-glucanase. An elaborated activity-based probe facilitated the direct detection and identification of ß-1,4-glucanases within a complex fungal secretome without any detectable cross-reactivity with ß-d-glucosidases. These probes and inhibitors add valuable new capacity to the growing toolbox of cyclophellitol-derived probes for the activity-based profiling of biomass-degrading enzymes.

An overview of activity-based probes for glycosidases.

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.

Dynamic and Functional Profiling of Xylan-Degrading Enzymes in Aspergillus Secretomes Using Activity-Based Probes.

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 ß-xylosidases and endo-ß-1,4-xylanases in the secretomes of Aspergillus niger, by the use of chemical probes inspired by the ß-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.

Towards broad spectrum activity-based glycosidase probes: synthesis and evaluation of deoxygenated cyclophellitol aziridines.

Activity-based protein profiling has emerged as a powerful tool for visualizing glycosidases in complex biological samples. Several configurational cyclophellitol isomers have been shown to display high selectivity as probes for glycosidases processing substrates featuring the same configuration. Here, a set of deoxygenated cyclophellitols are presented which enable inter-class profiling of ß-glucosidases and ß-galactosidases.

Catalytic decarboxylative alkenylation of enolates.

A palladium-catalyzed decarboxylative alkenylation of stabilized enolates has been developed, which gives rise to alkenylated dicarbonyl products from enol carbonates regioselectively with concomitant installation of a quaternary all-carbon center. The broad scope of the reaction has been demonstrated by successfully utilizing a range of enolates and external phenol nucleophiles.