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

ABSTRACT: Cellulases and related β-1,4-glucanases are essential components of lignocellulose-degrading enzyme mixtures. The detection of β-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 β-1,4-glucanases, a series of glycosylated cyclophellitol inhibitors mimicking β-1,4-glucan oligosaccharides have been synthesised. These compounds are highly efficient inhibitors of HiCel7B, a well-known GH7 endo-β-1,4-glucanase. An elaborated activity-based probe facilitated the direct detection and identification of β-1,4-glucanases within a complex fungal secretome without any detectable cross-reactivity with β-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.

SUBMITTER: de Boer C

PROVIDER: S-EPMC8341922 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

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

| S-EPMC8637750 | biostudies-literature

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

Project description:Cellulases and related β-1,4-glucanases are essential components of lignocellulose-degrading enzyme mixtures. The detection of β-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 β-1,4-glucanases, a series of glycosylated cyclophellitol inhibitors mimicking β-1,4-glucan oligosaccharides have been synthesised. These compounds are highly efficient inhibitors of HiCel7B, a well-known GH7 endo-β-1,4-glucanase. An elaborated activity-based probe facilitated the direct detection of β-1,4-glucanases within a complex fungal secretome without any detectable cross-reactivity with β-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.

2021-09-09 | PXD019930 | Pride

New Irreversible α-l-Iduronidase Inhibitors and Activity-Based Probes.

Project description:Cyclophellitol aziridines are potent irreversible inhibitors of retaining glycosidases and versatile intermediates in the synthesis of activity-based glycosidase probes (ABPs). Direct 3-amino-2-(trifluoromethyl)quinazolin-4(3H)-one-mediated aziridination of l-ido-configured cyclohexene has enabled the synthesis of new covalent inhibitors and ABPs of α-l-iduronidase, deficiency of which underlies the lysosomal storage disorder mucopolysaccharidosis type I (MPS I). The iduronidase ABPs react covalently and irreversibly in an activity-based manner with human recombinant α-l-iduronidase (rIDUA, Aldurazyme® ). The structures of IDUA when complexed with the inhibitors in a non-covalent transition state mimicking form and a covalent enzyme-bound form provide insights into its conformational itinerary. Inhibitors 1-3 adopt a half-chair conformation in solution (4 H3 and 3 H4 ), as predicted by DFT calculations, which is different from the conformation of the Michaelis complex observed by crystallographic studies. Consequently, 1-3 may need to overcome an energy barrier in order to switch from the 4 H3 conformation to the transition state (2, 5 B) binding conformation before reacting and adopting a covalent 5 S1 conformation. rIDUA can be labeled with fluorescent Cy5 ABP 2, which allows monitoring of the delivery of therapeutic recombinant enzyme to lysosomes, as is intended in enzyme replacement therapy for the treatment of MPS I patients.

| S-EPMC6343074 | biostudies-literature

Novel Inhibitors and Activity-Based Probes Targeting Trypsin-Like Serine Proteases.

Project description:The trypsin-like proteases (TLPs) play widespread and diverse roles, in a host of physiological and pathological processes including clot dissolution, extracellular matrix remodelling, infection, angiogenesis, wound healing and tumour invasion/metastasis. Moreover, these enzymes are involved in the disruption of normal lung function in a range of respiratory diseases including allergic asthma where several allergenic proteases have been identified. Here, we report the synthesis of a series of peptide derivatives containing an N-alkyl glycine analogue of arginine, bearing differing electrophilic leaving groups (carbamate and triazole urea), and demonstrate their function as potent, irreversible inhibitors of trypsin and TLPs, to include activities from cockroach extract. As such, these inhibitors are suitable for use as activity probes (APs) in activity-based profiling (ABP) applications.

| S-EPMC9068901 | biostudies-literature

A sensitive gel-based method combining distinct cyclophellitol-based probes for the identification of acid/base residues in human retaining β-glucosidases.

Project description:Retaining β-exoglucosidases operate by a mechanism in which the key amino acids driving the glycosidic bond hydrolysis act as catalytic acid/base and nucleophile. Recently we designed two distinct classes of fluorescent cyclophellitol-type activity-based probes (ABPs) that exploit this mechanism to covalently modify the nucleophile of retaining β-glucosidases. Whereas β-epoxide ABPs require a protonated acid/base for irreversible inhibition of retaining β-glucosidases, β-aziridine ABPs do not. Here we describe a novel sensitive method to identify both catalytic residues of retaining β-glucosidases by the combined use of cyclophellitol β-epoxide- and β-aziridine ABPs. In this approach putative catalytic residues are first substituted to noncarboxylic amino acids such as glycine or glutamine through site-directed mutagenesis. Next, the acid/base and nucleophile can be identified via classical sodium azide-mediated rescue of mutants thereof. Selective labeling with fluorescent β-aziridine but not β-epoxide ABPs identifies the acid/base residue in mutagenized enzyme, as only the β-aziridine ABP can bind in its absence. The Absence of the nucleophile abolishes any ABP labeling. We validated the method by using the retaining β-glucosidase GBA (CAZy glycosylhydrolase family GH30) and then applied it to non-homologous (putative) retaining β-glucosidases categorized in GH1 and GH116: GBA2, GBA3, and LPH. The described method is highly sensitive, requiring only femtomoles (nanograms) of ABP-labeled enzymes.

| S-EPMC4271221 | biostudies-literature

Design and Synthesis of Activity-Based Probes and Inhibitors for Bleomycin Hydrolase.

Project description:Bleomycin hydrolase (BLMH) is a neutral cysteine aminopeptidase that has been ascribed roles in many physiological and pathological processes, yet its primary biological function remains enigmatic. In this work, we describe the results of screening of a library of fluorogenic substrates to identify non-natural amino acids that are optimally recognized by BLMH. This screen identified several substrates with kcat/KM values that are substantially improved over the previously reported fluorogenic substrates for this enzyme. The substrate sequences were used to design activity-based probes that showed potent labeling of recombinant BLMH as well as endogenously expressed BLMH in cell extracts, and in intact cells. Importantly, we identify potent BLMH inhibitors that are able to fully inhibit endogenous BLMH activity in intact cells. These probes and inhibitors will be valuable new reagents to study BLMH function in cellular and animal models of human diseases where BLMH is likely to be involved.

| S-EPMC4546515 | biostudies-literature

Scaffold diversity for enhanced activity of glycosylated inhibitors of fungal adhesion.

Project description:Candida albicans is one of the most prevalent fungal pathogens involved in hospital acquired infections. It binds to glycans at the surface of epithelial cells and initiates infection. This process can be blocked by synthetic carbohydrates that mimic the structure of cell surface glycans. Herein we report the evaluation of a series of divalent glycosides featuring aromatic (benzene, squaramide) and bicyclic aliphatic (norbornene) scaffolds, with the latter being the first examples of their kind as small molecule anti-adhesion glycoconjugates. Galactosides 1 and 6, built on an aromatic core, were most efficient inhibitors of adhesion of C. albicans to buccal epithelial cells, displacing up to 36% and 48%, respectively, of yeast already attached to epithelial cells at 138 μM. Remarkably, cis-endo-norbornene 21 performed comparably to benzene-core derivatives. Conformational analysis reveals a preference for compounds 1 and 21 to adopt folded conformations. These results highlight the potential of norbornenes as a new class of aliphatic scaffolds for the synthesis of anti-adhesion compounds.

| S-EPMC8126883 | biostudies-literature

Confirming target engagement for reversible inhibitors in vivo by kinetically tuned activity-based probes.

Project description:The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPLA1 and LYPLA2, two enzymes for which selective, in vivo active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo.

| S-EPMC3392194 | biostudies-literature

Rational design of inhibitors and activity-based probes targeting Clostridium difficile virulence factor TcdB.

Project description:Clostridium difficile is a leading cause of nosocomial infections. The major virulence factors of this pathogen are the multi-domain toxins TcdA and TcdB. These toxins contain a cysteine protease domain (CPD) that autoproteolytically releases a cytotoxic effector domain upon binding intracellular inositol hexakisphosphate. Currently, there are no known inhibitors of this protease. Here, we describe the rational design of covalent small molecule inhibitors of TcdB CPD. We identified compounds that inactivate TcdB holotoxin function in cells and solved the structure of inhibitor-bound protease to 2.0 Å. This structure reveals the molecular basis of CPD substrate recognition and informed the synthesis of activity-based probes for this enzyme. The inhibitors presented will guide the development of therapeutics targeting C. difficile, and the probes will serve as tools for studying the unique activation mechanism of bacterial toxin CPDs.

| S-EPMC3005307 | biostudies-literature

Affinity-based probes based on type II kinase inhibitors.

Project description:Protein kinases are key components of most mammalian signal transduction networks and are therapeutically relevant drug targets. Efforts to study protein kinase function would benefit from new technologies that are able to profile kinases in complex proteomes. Here, we describe active site-directed probes for profiling kinases in whole cell extracts and live cells. These probes contain general ligands that stabilize a specific inactive conformation of the ATP-binding sites of protein kinases, as well as trifluoromethylphenyl diazirine and alkyne moieties that allow covalent modification and enrichment of kinases, respectively. A diverse group of serine/threonine and tyrosine kinases were identified as specific targets of these probes in whole cell extracts. In addition, a number of kinase targets were selectively labeled in live cells. Our chemical proteomics approach should be valuable for interrogating protein kinase active sites in physiologically relevant environments.

| S-EPMC3523701 | biostudies-literature

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