Carbohydrate Bis-acetal-Based Substrates as Tunable Fluorescence-Quenched Probes for Monitoring exo-Glycosidase Activity

Tunable Förster resonance energy transfer (FRET)-quenched substrates are useful for monitoring the activity of various enzymes within their relevant physiological environments. Development of FRET-quenched substrates for exo-glycosidases, however, has been hindered by their constrained pocket-shaped active sites. Here we report the design of a new class of substrate that overcomes this problem. These Bis-Acetal-Based Substrates (BABS) bear a hemiacetal aglycon leaving group that tethers fluorochromes in close proximity, also positioning them distant from the active site pocket. Following cleavage of the glycosidic bond, the liberated hemiacetal spontaneously breaks down, leading to separation of the fluorophore and quencher. We detail the synthesis and characterization of GlcNAc-BABS, revealing a striking 99.9% quenching efficiency. These substrates are efficiently turned over by the human exo-glycosidase O-GlcNAcase (OGA). We find the hemiacetal leaving group rapidly breaks down, enabling quantitative monitoring of OGA activity. We expect this strategy to be broadly useful for the development of substrate probes for monitoring exo-glycosidases, as well as a range of other enzymes having constrained pocket-shaped active sites

Enzymatic Characterization and Inhibition of the Nuclear Variant of Human O-GlcNAcase

Increasing cellular O-GlcNAc levels through pharmacological inhibition of O-GlcNAcase, the enzyme responsible for removal of the O-GlcNAc post-translational modification, is being increasingly used to aid in discerning the roles played by this form of intracellular glycosylation. Interestingly, two forms of O-GlcNAcase have been studied; a full-length isoform that is better characterized, and a shorter nuclear-localized variant, arising from failure to splice out one intron, which has not been as well characterized. Given the increasing use of O-GlcNAcase inhibitors as research tools, we felt that a clear understanding of how these inhibitors affect both isoforms of O-GlcNAcase is important for proper interpretation of studies making use of these inhibitors in cell culture and in vivo. Here we describe an enzymatic characterization of the nuclear variant of human O-GlcNAcase. We find that this short nuclear variant of O-GlcNAcase, which has the identical catalytic domain as the full-length enzyme, has similar trends in a pH-rate profile and Taft linear free energy analysis as the full-length enzyme. These findings strongly suggest that both enzymes use broadly similar transition states. Consistent with this interpretation, the short isoform is potently inhibited by several previously described inhibitors of full-length O-GlcNAcase including PUGNAc, NAG-thiazoline, and the selective O-GlcNAcase inhibitor NButGT. These findings contrast with earlier studies and suggest that studies using O-GlcNAcase inhibitors in cultured cells or in vivo can be interpreted with the knowledge that both these forms of O-GlcNAcase are inhibited when present

O-GlcNAcase Catalyzes Cleavage of Thioglycosides without General Acid Catalysis

O-GlcNAcase catalyzes the removal of N-acetylglucosamine residues from serine and threonine residues of post-translationally modified proteins using a catalytic mechanism involving substrate-assisted catalysis and general acid/base catalysis. Since thioglycosides are widely perceived as resistant to hydrolysis by glycosidases, it was surprising to find that O-GlcNAcase also catalyzes the efficient hydrolysis of S-glycosides. Brønsted analyses and pH-activity studies of the O-GlcNAcase-catalyzed hydrolysis of a series of aryl S- and O-glycosides reveal that O-GlcNAcase effects hydrolysis of thioglycosides without the assistance of general acid catalysis. α-Deuterium kinetic isotope effects for O- and S-glycosides, as well as Taft-like analyses using N-fluoroacetyl-β-glycosides, suggest that O-GlcNAcase accomplishes hydrolysis of thioglycosides by stabilizing late transition states. For S-glycosides this transition state shows greater nucleophilic participation from the 2-acetamido group than for O-glycosides. The rate constants governing the O-GlcNAcase-catalyzed hydrolysis of O- and S-glycosides as compared to those previously determined for the spontaneous hydrolysis of structurally similar O,O- and O,S-acetals show a similar ratio. O-GlcNAcase therefore demonstrates similar catalytic proficiency toward both O- and S-glycosides. We conclude that O-GlcNAcase is a bifunctional catalyst capable of efficiently cleaving thioglycosides without general acid catalysis, an observation that may have biological implications

A highly concise preparation of O-deacetylated arylthioglycosides of N-acetyl-D-glucosamine from 2-acetamido-3,4,6-tri-O-acetyl2-deoxy-a-D-glucopyranosyl chloride and aryl thiols or disulfides

An expedient and mild route to a range of aryl 2-acetamido-2-deoxy-1-thio-beta-D-glucopyranosides has been devised from 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranosyl chloride and arylthiols or aryl disulfides using phase transfer catalysis conditions. This simple procedure compresses up to three synthetic steps into a one-pot reaction, obviating the need for tedious workups and chromatography and directly furnishes crystalline materials in good yields. The procedure is compatible with a range of thiols and disulfides and may be amenable for preparing a wide range of thioglycosides with various glycons and aglycons

A 1-acetamido derivative of 6-epi-valienamine: an inhibitor of a diverse group of β-N-acetylglucosaminidases

The synthesis of an analogue of 6-epi-valienamine bearing an acetamido group and its characterisation as an inhibitor of β-N-acetylglucosaminidases are described. The compound is a good inhibitor of both human O-GlcNAcase and human β-hexosaminidase, as well as two bacterial β-N-acetylglucosaminidases. A 3-D structure of the complex of Bacteroides thetaiotaomicron BtGH84 with the inhibitor shows the unsaturated ring is surprisingly distorted away from its favoured solution phase conformation and reveals potential for improved inhibitor potency

Structural snapshots of the reaction coordinate for O-GlcNAc transferase

Visualization of the reaction coordinate undertaken by glycosyltransferases has remained elusive, but is critical for understanding this important class of enzyme. Using substrates and substrate mimics, we describe structural snapshots of all species along the kinetic pathway for human O-GlcNAc transferase, an intracellular enzyme that catalyzes installation of a dynamic post-translational modification. The structures reveal key features of the mechanism and show that substrate participation is important during catalysis

A Divergent Synthesis to Generate Targeted Libraries of Inhibitors for Endo-N-Acetylglucosaminidases

Cell active inhibitors of glycoside processing enzymes are valuable research tools that help us understand the physiological roles of this diverse class of enzymes. endo-N-Acetylglucosaminidases have gained increased attention for their important roles in both mammals and human pathogens; however, metabolically stable cell active inhibitors of these enzymes are lacking. Here, we describe a divergent synthetic strategy involving elaboration of a thiazoline core scaffold. We illustrate the potential of this approach by using the copper catalysed azide-alkyne click (CuAAC) reaction, in combination with a suitable catalyst to avoid poisoning by the thiazoline moiety, to generate a targeted panel of candidate inhibitors of endo-N-acetylglucosaminidases and chitinase

Structures of Lactate Dehydrogenase A (LDHA) In Apo, Ternary an Inhibitor-Bound Forms

Lactate dehydrogenase (LDH) is an essential metabolic enzyme that catalyzes the interconversion of pyruvate and lactate using NADH/NAD + as a co-substrate. Many cancer cells exhibit a glycolytic phenotype known as the Warburg effect, in which elevated LDH levels enhance the conversion of glucose to lactate, making LDH an attractive therapeutic target for oncology. Two known inhibitors of the human muscle LDH isoform, LDHA, designated 1 and 2 , were selected, and their IC 50 values were determined to be 14.4 ± 3.77 and 2.20 ± 0.15 µ M , respectively. The X-ray crystal structures of LDHA in complex with each inhibitor were determined; both inhibitors bind to a site overlapping with the NADH-binding site. Further, an apo LDHA crystal structure solved in a new space group is reported, as well as a complex with both NADH and the substrate analogue oxalate bound in seven of the eight molecules and an oxalate only bound in the eighth molecule in the asymmetric unit. In this latter structure, a kanamycin molecule is located in the inhibitor-binding site, thereby blocking NADH binding. These structures provide insights into LDHA enzyme mechanism and inhibition and a framework for structure-assisted drug design that may contribute to new cancer therapie

Selective Fluorogenic β-Glucocerebrosidase Substrates for Convenient Analysis of Enzyme Activity in Cell and Tissue Homogenates

Within mammals, there are often several functionally related glycoside hydrolases, which makes monitoring their activities problematic. This problem is particularly acute for the enzyme β-glucocerebrosidase (GCase), the malfunction of which is a key driver of Gaucher's disease (GD) and a major risk factor for Parkinson's disease (PD). Humans harbor two other functionally related β-glucosidases known as GBA2 and GBA3, and the currently used fluorogenic substrates are not selective, which has driven the use of complicated subtractive assays involving the use of detergents and inhibitors. Here we describe the preparation of fluorogenic substrates based on the widely used nonselective substrate resorufin β-d-glucopyranoside. Using recombinant enzymes, we show that these substrates are highly selective for GCase. We also demonstrate their value through the analysis of GCase activity in brain tissue homogenates from transgenic mice expressing mutant human GCase and patient fibroblasts expressing mutant GCase. This approach simplifies the analysis of cell and tissue homogenates and should facilitate the analysis of clinical and laboratory tissues and samples.

O-GlcNAc Modification of tau Directly Inhibits Its Aggregation without Perturbing the Conformational Properties of tau Monomers

Abstract The aggregation of the microtubule-associated protein tau into paired helical filaments to form neurofibrillary tangles constitutes one of the pathological hallmarks of Alzheimer's disease. Tau is post-translationally modified by the addition of N-acetyl-D-glucosamine O-linked to several serine and threonine residues (O-GlcNAc). Previously, increased O-GlcNAcylation of tau has been shown to block the accumulation of tau aggregates within a tauopathy mouse model. Here we show that O-GlcNAc modification of full-length human tau impairs the rate and extent of its heparin-induced aggregation without perturbing its activity toward microtubule polymerization. O-GlcNAcylation, however, does not impact the "global-fold" of tau as measured by a Förster resonance energy transfer assay. Similarly, nuclear magnetic resonance studies demonstrated that O-GlcNAcylation only minimally perturbs the local structural and dynamic features of a tau fragment (residues 353-408) spanning the last microtubule binding repeat to the major GlcNAc-acceptor Ser400. These data indicate that the inhibitory effects of O-GlcNAc on tau aggregation may result from enhanced monomer solubility or the destabilization of fibrils or soluble aggregates, rather than by altering the conformational properties of the monomeric protein. This work further underscores the potential of targeting the O-GlcNAc pathway for potential Alzheimer's disease therapeutics