Identification of Orthosteric and Allosteric Pharmacological Chaperones for Mucopolysaccharidosis Type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal inherited disease caused by mutations in gene encoding the lysosomal enzyme N-acetyl-alpha-glucosaminidase (NAGLU). These mutations result in reduced NAGLU activity, preventing it from catalyzing the hydrolysis of the glycosaminoglycan heparan sulfate (HS). There are currently no approved treatments for MPS IIIB. A novel approach in the treatment of lysosomal storage diseases is the use of pharmacological chaperones (PC). In this study, we used a drug repurposing approach to identify and characterize novel potential PCs for NAGLU enzyme. We modeled the interaction of natural and artificial substrates within the active cavity of NAGLU (orthosteric site) and predicted potential allosteric sites. We performed a virtual screening for both the orthosteric and the predicted allosteric site against a curated database of human tested molecules. Considering the binding affinity and predicted blood-brain barrier permeability and gastrointestinal absorption, we selected atovaquone and piperaquine as orthosteric and allosteric PCs. The PCs were evaluated by their capacity to bind NAGLU and the ability to restore the enzymatic activity in human MPS IIIB fibroblasts These results represent novel PCs described for MPS IIIB and demonstrate the potential to develop novel therapeutic alternatives for this and other protein deficiency diseases.

Structural variation of the 3-acetamido-4,5,6-trihydroxyazepane iminosugar through epimerization and C-alkylation leads to low micromolar HexAB and NagZ inhibitors.

We report the synthesis of seven-membered iminosugars derived from a 3S-acetamido-4R,5R,6S-trihydroxyazepane scaffold and their evaluation as inhibitors of functionally related exo-N-acetylhexosaminidases including human O-GlcNAcase (OGA), human lysosomal ß-hexosaminidase (HexAB), and Escherichia coli NagZ. Capitalizing on the flexibility of azepanes and the active site tolerances of hexosaminidases, we explore the effects of epimerization of stereocenters at C-3, C-5 and C-6 and C-alkylation at the C-2 or C-7 positions. Accordingly, epimerization at C-6 (L-ido) and at C-5 (D-galacto) led to selective HexAB inhibitors whereas introduction of a propyl group at C-7 on the C-3 epimer furnished a potent NagZ inhibitor.

Emerging roles of protein O-GlcNAcylation in cardiovascular diseases: Insights and novel therapeutic targets.

Cardiovascular diseases (CVDs) are the leading cause of death globally. While the major focus of pharmacological and non-pharmacological interventions has been on targeting disease pathophysiology and limiting predisposing factors, our understanding of the cellular and molecular mechanisms underlying the pathogenesis of CVDs remains incomplete. One mechanism that has recently emerged is protein O-GlcNAcylation. This is a dynamic, site-specific reversible post-translational modification of serine and threonine residues on target proteins and is controlled by two enzymes: O-linked ß-N-acetylglucosamine transferase (OGT) and O-linked ß-N-acetylglucosaminidase (OGA). Protein O-GlcNAcylation alters the cellular functions of these target proteins which play vital roles in pathways that modulate vascular homeostasis and cardiac function. Through this review, we aim to give insights on the role of protein O-GlcNAcylation in cardiovascular diseases and identify potential therapeutic targets in this pathway for development of more effective medicines to improve patient outcomes.

Synthesis of novel homoazanucleosides and their peptidyl analogs.

Synthesis of novel homoazanucleosides and their peptidyl analogs as hybrid molecules comprised of amino acids, an iminosugar and natural nucleobases is reported for the first time. A pluripotent amino-substituted chiral polyhydroxypyrrolidine, possessing orthogonally different functional groups on either arm of the pyrrolidine ring, served as an ideal substrate for the synthesis of the proposed peptidyl homoazanucleosides. The acid sensitive primary benzyloxy group, on one arm of the pyrrolidine ring, after selective deprotection, was utilized for the introduction of nucleobases to obtain the homoazanucleosides. The amino group on the other side offered the opportunity to be coupled with amino acids to deliver the desired peptidyl homoazanucleosides. Glycosidase inhibition studies revealed that the acetamido derivatives of homoazanucleosides were found to be sub-millimolar inhibitors of ß-N-acetyl-glucosaminidase.

High-level expression of ß-N-Acetylglucosaminidase BsNagZ in Pichia pastoris to obtain GlcNAc.

ß-N-Acetylglucosaminidases (NAGase) can remove N-acetylglucosamine (GlcNAc) from the non-reducing end of chitin or chitosan. GlcNAc has many important physiological functions in organism, which can be used for the treatment of rheumatoid arthritis clinically and be used as food antioxidant, infant food additive and diabetic sweetener. Thus, it is very important to develop genetic-engineering strains with high-yield NAGase to hydrolyze chitin into GlcNAc. Here, the NAGase gene of Bacillus subtilis 168 (BsnagZ) was synthesized according to the codon bias of Pichia pastoris and expressed in P. pastoris. The expression level of BsNagZ in P. pastoris increased over the induced time and the highest activity reached 0.76 U/mL at the 7th day. The recombinant BsNagZ was purified for characterization. The optimal temperature and pH are 60 °C and 6.0, respectively. It can both keep over 80% activities after pre-incubation at 55 °C for one hour and at 4 °C for 12 h from pH 4.5 to 10.0. To further improve the expression level of BsNagZ, a recombinant strain with four copy BsnagZs was screened using a high concentration of zeocin. The highest BsNagZ activity reached 3.2 U/mL at the 12th day, which was fourfold higher than that of single-copy strain. Combined with commercial chitinase CtnSg, GlcNAc can be produced by recombinant BsNagZ when used colloidal chitin as the substrate. Our study highlights that the NAGase was first successfully expressed in P. pastoris and GlcNAc can be produced via NAGase hydrolyzing the colloidal chitin.

O-GlcNAcylation of the human epidermal growth factor receptor.

The reversible O-linked attachment of single ß-D-N-acetylglucosamine (GlcNAc) moieties to serine/threonine residues in target proteins is a frequently occurring post-translational modification affecting the functionality of many cellular systems. In this report we present experimental evidence suggesting that the epidermal growth factor receptor (EGFR) is subjected to O-GlcNAcylation in human carcinoma epidermoid A431 cells and human lung carcinoma A549 cells. However, no signal was detected in human cervix adenocarcinoma HeLa cells or in mouse EGFR-T17 fibroblasts ectopically expressing the human EGFR. We detected a positive O-GlcNAcylation signal in the immunoprecipitated EGFR by Western blotting using two distinct specific anti-O-GlcNAc antibodies even after N-deglycosylation of the receptor using peptide-N-glycosidase F (PNGase F). Conversely, the presence of EGFR was detected by Western blotting using an anti-EGFR antibody in the immunocomplex of O-GlcNAcylated proteins immunoprecipitated with an anti-O-GlcNAc antibody. These signals were enhanced when the O-linked ß-N-acetylglucosaminidase (OGA) inhibitor Thiamet G was added to prevent the deglycosylation of the GlcNAc moiety(ies). Moreover, we also detected a positive signal in the immunoprecipitated and N-deglycosylated EGFR using PNGase F, and tunicamycin when the cells were metabolically labeled with azido-GlcNAc (GlcNAz), biotinylated and probed with a streptavidin-labeled peroxidase. Finally, EGFR and O-linked ß-N-acetylglucosamine transferase (OGT) co-immunoprecipitate, and incubation of the immunoprecipitated EGFR with the immunoprecipitated OGT in the presence of uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) resulted in a significant enhancement of the EGFR O-GlcNAcylation signal as detected by Western blotting using an anti-O-GlcNAc antibody. We conclude that the human EGFR is subjected to O-GlcNAcylation in the A431 and A549 tumor cell lines.

GM130 gain-of-function induces cell pathology in a model of lysosomal storage disease.

Cell pathology in lysosomal storage diseases is characterized by the formation of distended vacuoles with characteristics of lysosomes. Our previous studies in mucopolysaccharidosis type IIIB (MPSIIIB), a disease in which a genetic defect induces the accumulation of undigested heparan sulfate (HS) fragments, led to the hypothesis that abnormal lysosome formation was related to events occurring at the Golgi level. We reproduced the enzyme defect of MPSIIIB in HeLa cells using tetracycline-inducible expression of shRNAs directed against α-N-acetylglucosaminidase (NAGLU) and addressed this hypothesis. HeLa cells deprived of NAGLU accumulated abnormal lysosomes. The Golgi matrix protein GM130 was over-expressed. The cis- and medial-Golgi compartments were distended, elongated and formed circularized ribbons. The Golgi microtubule network was enlarged with increased amounts of AKAP450, a partner of GM130 controlling this network. GM130 down-regulation prevented pathology in HeLa cells deprived of NAGLU, whereas GM130 over-expression in control HeLa cells mimicked the pathology of deprived cells. We concluded that abnormal lysosomes forming in cells accumulating HS fragments were the consequence of GM130 gain-of-function and subsequent alterations of the Golgi ribbon architecture. These results indicate that GM130 functions are modulated by HS glycosaminoglycans and therefore possibly controlled by extracellular cues.

Inactivation kinetics of formaldehyde on N-acetyl-ß-D-glucosaminidase from Nile tilapia (Oreochromis niloticus).

Formaldehyde is a widely used sanitizer in aquaculture in China, while the appropriate concentration is not available to be used effectively and without damage to tilapia much less to its reproductive function. N-acetyl-ß-D-glucosaminidase (EC 3.2.1.52, NAGase), hydrolyzing the oligomers of N-acetyl-ß-D-glucosamine into monomer, is proved to be correlated with reproduction of male animals. In this paper, NAGase from spermary of tilapia was chosen as the material to study the effects of formaldehyde on its activity in order to further investigate the effects of formaldehyde use on tilapia reproduction. The results showed the relationship between the residual enzyme activity and the concentration of formaldehyde was concentration dependent, and the IC50 value was estimated to be 3.2 ± 0.1 %. Appropriate concentration of formaldehyde leaded to competitive reversible inhibition on tilapia NAGase. Moreover, formaldehyde could reduce the thermal and pH stability of the enzyme. The inactivation kinetics of formaldehyde on the enzyme was studied using the kinetic method of substrate reaction. The inactivation model was setup, and the rate constants were determined. The results showed that the inactivation of formaldehyde on tilapia NAGase was a slow, reversible reaction with partially residual activity. The results will give some basis to determine the concentration of formaldehyde used in tilapia culture.

Evidence of the involvement of O-GlcNAc-modified human RNA polymerase II CTD in transcription in vitro and in vivo.

The RNA polymerase II C-terminal domain (CTD), which serves as a scaffold to recruit machinery involved in transcription, is modified post-translationally. Although the O-GlcNAc modification of RNA polymerase II CTD was documented in 1993, its functional significance remained obscure. We show that O-GlcNAc transferase (OGT) modified CTD serine residues 5 and 7. Drug inhibition of OGT and OGA (N-acetylglucosaminidase) blocked transcription during preinitiation complex assembly. Polymerase II and OGT co-immunoprecipitated, and OGT is a component of the preinitiation complex. OGT shRNA experiments showed that reduction of OGT causes a reduction in transcription and RNA polymerase II occupancy at several B-cell promoters. These data suggest that the cycling of O-GlcNAc on and off of polymerase II occurs during assembly of the preinitiation complex. Our results define unexpected roles for both the CTD and O-GlcNAc in the regulation of transcription initiation in higher eukaryotes.

Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells.

Glycosyltransferases are ubiquitous enzymes that catalyze the assembly of glycoconjugates throughout all kingdoms of nature. A long-standing problem is the rational design of probes that can be used to manipulate glycosyltransferase activity in cells and tissues. Here we describe the rational design and synthesis of a nucleotide sugar analog that inhibits, with high potency both in vitro and in cells, the human glycosyltransferase responsible for the reversible post-translational modification of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc). We show that the enzymes of the hexosamine biosynthetic pathway can transform, both in vitro and in cells, a synthetic carbohydrate precursor into the nucleotide sugar analog. Treatment of cells with the precursor lowers O-GlcNAc in a targeted manner with a single-digit micromolar EC(50). This approach to inhibition of glycosyltransferases should be applicable to other members of this superfamily of enzymes and enable their manipulation in a biological setting.

O-GlcNAcylation involvement in high glucose-induced cardiac hypertrophy via ERK1/2 and cyclin D2.

Continuous hyperglycemia is considered to be the most significant pathogenesis of diabetic cardiomyopathy, which manifests as cardiac hypertrophy and subsequent heart failure. O-GlcNAcylation has attracted attention as a post-translational protein modification in the past decade. The role of O-GlcNAcylation in high glucose-induced cardiomyocyte hypertrophy remains unclear. We studied the effect of O-GlcNAcylation on neonatal rat cardiomyocytes that were exposed to high glucose and myocardium in diabetic rats induced by streptozocin. High glucose (30 mM) incubation induced a greater than twofold increase in cell size and increased hypertrophy marker gene expression accompanied by elevated O-GlcNAcylation protein levels. High glucose increased ERK1/2 but not p38 MAPK or JNK activity, and cyclin D2 expression was also increased. PUGNAc, an inhibitor of ß-N-acetylglucosaminidase, enhanced O-GlcNAcylation and imitated the effects of high glucose. OGT siRNA and ERK1/2 inhibition with PD98059 treatment blunted the hypertrophic response and cyclin D2 upregulation. OGT inhibition also prevented ERK1/2 activation. We also observed concentric hypertrophy and similar changes of O-GlcNAcylation level, ERK1/2 activation and cyclin D2 expression in myocardium of diabetic rats induced by streptozocin. In conclusion, O-GlcNAcylation plays a role in high glucose-induced cardiac hypertrophy via ERK1/2 and cyclin D2.

RNAi suppression of ß-N-acetylglucosaminidase (BmFDL) for complex-type N-linked glycan synthesis in cultured silkworm cells.

Glycoproteins have various biological functions including enzymatic activity, protein stability and others. Due to the presence of paucimannosidic N-linked glycans, recombinant proteins from an insect cell expression system may not be suitable for therapeutic use. Because baculovirus expression systems (BESs) are used to produce recombinant proteins, it is of interest to modify the endogenous N-glycosylation pathway in insects to mimic that of mammals. Using a soaking RNAi sensitive cell line, BmN4-SID1, has enabled us to suppress Bombyx mori FDL (BmFDL), an N-linked glycan-specific ß-N-acetylglucosaminidase. Western blotting and MALDI-TOF MS demonstrated that the BmFDL depletion almost completely converted the paucimannosidic structures of the recombinant proteins produced by BES into a complex-type structure. This highly efficient, simple and low-cost method can be used for mass production of secretion proteins with complex-type N-linked glycans.

The susceptibility of soil enzymes to inhibition by leaf litter tannins is dependent on the tannin chemistry, enzyme class and vegetation history.

By inhibiting soil enzymes, tannins play an important role in soil carbon (C) and nitrogen (N) mineralization. The role of tannin chemistry in this inhibitory process, in conjunction with enzyme classes and isoforms, is less well understood. Here, we compared the inhibition efficiencies of mixed tannins (MTs, mostly limited to angiosperms) and condensed tannins (CTs, produced mostly by gymnosperms) against the potential activity of ß-glucosidase (BG), N-acetyl-glucosaminidase (NAG), and peroxidase in two soils that differed in their vegetation histories. Compared with CTs, MTs exhibited 50% more inhibition of almond (Prunus dulcis) BG activity and greater inhibition of the potential NAG activity in the gymnosperm-acclimatized soils. CTs exhibited lower BG inhibition in the angiosperm-acclimated soils, whereas both types of tannins exhibited higher peroxidase inhibition in the angiosperm soils than in gymnosperm soils. At all of the tested tannin concentrations, irrespective of the tannin type and site history, the potential peroxidase activity was inhibited two-fold more than the hydrolase activity and was positively associated with the redox-buffering efficiency of tannins. Our finding that the inhibitory activities and mechanisms of MTs and CTs are dependent on the vegetative history and enzyme class is novel and furthers our understanding of the role of tannins and soil isoenzymes in decomposition.

Increasing O-GlcNAc levels: An overview of small-molecule inhibitors of O-GlcNAcase.

The O-GlcNAc modification is found on many nucleocytoplasmic proteins. The dynamic nature of O-GlcNAc, which in some ways is reminiscent of phosphorylation, has enabled investigators to modulate the stoichiometry of O-GlcNAc on proteins in order to study its function. Although several genetic and pharmacological methods for manipulating O-GlcNAc levels have been described, one of the most direct approaches of increasing global O-GlcNAc levels is by using small-molecule inhibitors of O-GlcNAcase (OGA). As the interest in increasing O-GlcNAc levels has grown, so too has the number of OGA inhibitors. This review provides an overview of the available methods of increasing O-GlcNAc levels, with a special emphasis on inhibition of OGA by small molecules. Known inhibitors of OGA are discussed with particular attention on those most suitable for cell-based biological studies. Several examples in which OGA inhibitors have been used to study the functional role of the O-GlcNAc modification in biological systems are discussed, highlighting the pros and cons of different inhibitors.

MS/MS fragmentation-guided search of TMG-chitooligomycins and their structure-activity relationship in specific ß-N-acetylglucosaminidase inhibition.

The reducing tetrasaccharide TMG-chitotriomycin (1) is an inhibitor of ß-N-acetylglucosaminidase (GlcNAcase), produced by the actinomycete Streptomyces anulatus NBRC13369. The inhibitor shows a unique inhibitory spectrum, that is, selectivity toward enzymes from chitin-containing organisms such as insects and fungi. Nevertheless, its structure-selectivity relationship remains to be clarified. In this study, we conducted a structure-guided search of analogues of 1 in order to obtain diverse N,N,N-trimethylglucosaminium (TMG)-containing chitooligosaccharides. In this approach, the specific fragmentation profile of 1 on ESI-MS/MS analysis was used for the selective detection of desired compounds. As a result, two new analogues, named TMG-chitomonomycin (3) and TMG-chitobiomycin (2), were obtained from a culture filtrate of 1-producing Streptomyces. Their enzyme-inhibiting activity revealed that the potency and selectivity depended on the degree of polymerization of the reducing end GlcNAc units. Furthermore, a computational modeling study inspired the inhibitory mechanism of TMG-related compounds as a mimic of the substrate in the Michaelis complex of the GH20 enzyme. This study is an example of the successful application of a MS/MS experiment for structure-guided isolation of natural products.

Kinetic and thermodynamic insights into the inhibitory mechanism of TMG-chitotriomycin on Vibrio campbellii GH20 exo-ß-N-acetylglucosaminidase.

We investigated the inhibition kinetics of VhGlcNAcase, a GH20 exo-ß-N-acetylglucosaminidase (GlcNAcase) from the marine bacterium Vibrio campbellii (formerly V. harveyi) ATCC BAA-1116, using TMG-chitotriomycin, a natural enzyme inhibitor specific for GH20 GlcNAcases from chitin-processing organisms, with p-nitrophenyl N-acetyl-ß-d-glucosaminide (pNP-GlcNAc) as the substrate. TMG-chitotriomycin inhibited VhGlcNAcase with an IC50 of 3.0 ± 0.7 µM. Using Dixon plots, the inhibition kinetics indicated that TMG-chitotriomycin is a competitive inhibitor, with an inhibition constant Ki of 2.2 ± 0.3 µM. Isothermal titration calorimetry experiments provided the thermodynamic parameters for the binding of TMG-chitotriomycin to VhGlcNAcase and revealed that binding was driven by both favorable enthalpy and entropy changes (ΔH° = -2.5 ± 0.1 kcal/mol and -TΔS° = -5.8 ± 0.3 kcal/mol), resulting in a free energy change, ΔG°, of -8.2 ± 0.2 kcal/mol. Dissection of the entropic term showed that a favorable solvation entropy change (-TΔSsolv° = -16 ± 2 kcal/mol) is the main contributor to the entropic term.

Chemical approaches to understanding O-GlcNAc glycosylation in the brain.

O-GlcNAc glycosylation is a unique, dynamic form of glycosylation found on intracellular proteins of all multicellular organisms. Studies suggest that O-GlcNAc represents a key regulatory modification in the brain, contributing to transcriptional regulation, neuronal communication and neurodegenerative disease. Recently, several new chemical tools have been developed to detect and study the modification, including chemoenzymatic tagging methods, quantitative proteomics strategies and small-molecule inhibitors of O-GlcNAc enzymes. Here we highlight some of the emerging roles for O-GlcNAc in the nervous system and describe how chemical tools have significantly advanced our understanding of the scope, functional significance and cellular dynamics of this modification.

Novel GH-20 ß-N-acetylglucosaminidase inhibitors: Virtual screening, molecular docking, binding affinity, and anti-tumor activity.

ß-N-acetylglucosaminidases (GlcNAcases) play a crucial role in the metabolism of glycan-conjugated proteins/lipids in humans. Elevated levels of serum GlcNAcases have been associated with certain types of cancer, and GlcNAcases therefore serve as drug targets. Here, we employed virtual screening to identify two novel GlcNAcase inhibitors from the National Cancer Institute (NCI) Drug Library using a bacterial GH-20 GlcNAcase (VhGlcNAcase) as a search model. NSC73735 was shown to be most potent with IC50 of 12.7 ±â€¯1.2 µM, agreeing with Kd of 0.94 ±â€¯0.2 µM obtained by ITC. Molecular docking refinement indicated that Trp582 the key residue that interacted with all the inhibitor molecules. Docking NSC7373 into the active site of human O-GlcNAcase (hOGA) yielded reasonably good fit with the estimated Kd of 44.7 µM, indicating its possibility to be a true binding partner. NSC73735 was shown to significantly suppress both cell growth and GlcNAcase activity of five cancer cell lines (U937, THP-1, MCF-7, HepG2 and PC-3) that express endogenous GlcNAcases. The cell cytotoxicity assay indicated the inherent effects of the lead compound on GlcNAcase expression with cancer cell proliferation, and therefore this novel GlcNAcase inhibitor may serve as a virtuous candidate for further development of highly potent anti-tumor agents.

NAG-thiazoline is a potent inhibitor of the Vibrio campbellii GH20 ß-N-Acetylglucosaminidase.

Vibrio spp. play a vital role in the recycling of chitin in oceans, but several Vibrio strains are highly infectious to aquatic animals and humans. These bacteria require chitin for growth; thus, potent inhibitors of chitin-degrading enzymes could serve as candidate drugs against Vibrio infections. This study examined NAG-thiazoline (NGT)-mediated inhibition of a recombinantly expressed GH20 ß-N-acetylglucosaminidase, namely VhGlcNAcase from Vibrio campbellii (formerly V. harveyi) ATCC BAA-1116. NGT strongly inhibited VhGlcNAcase with an IC50 of 11.9 ± 1.0 µm and Ki 62 ± 3 µm, respectively. NGT was also found to completely inhibit the growth of V. campbellii strain 650 with an minimal inhibitory concentration value of 0.5 µm. ITC data analysis showed direct binding of NGT to VhGlcNAcase with a Kd of 32 ± 1.2 µm. The observed ΔG°binding of -7.56 kcal·mol-1 is the result of a large negative enthalpy change and a small positive entropic compensation, suggesting that NGT binding is enthalpy-driven. The structural complex shows that NGT fully occupies the substrate-binding pocket of VhGlcNAcase and makes an exclusive hydrogen bond network, as well as hydrophobic interactions with the conserved residues around the -1 subsite. Our results strongly suggest that NGT could serve as an excellent scaffold for further development of antimicrobial agents against Vibrio infections. DATABASE: Structural data are available in PDB database under the accession number 6K35.

Total synthesis and structural revision of TMG-chitotriomycin, a specific inhibitor of insect and fungal beta-N-acetylglucosaminidases.

TMG-chitotriomycin, a potent and selective inhibitor of the beta-N-acetylglucosaminidases that possesses an unique N,N,N-trimethyl-d-glucosamine (TMG) residue, is revised to be the TMG-beta-(1-->4)-chitotriose instead of the originally proposed alpha-anomer via its total synthesis, for which a highly convergent approach was developed in which the sterically demanding (1-->4)-glycosidic linkages are efficiently constructed by the Au(I)-catalyzed glycosylation protocol with glycosyl o-hexynylbenzoates as donors.