sp2-Iminosugars targeting human lysosomal β-hexosaminidase as pharmacological chaperone candidates for late-onset Tay-Sachs disease

The late-onset form of Tay-Sachs disease displays when the activity levels of human β-hexosaminidase A (HexA) fall below 10% of normal, due to mutations that destabilise the native folded form of the enzyme and impair its trafficking to the lysosome. Competitive inhibitors of HexA can rescue disease-causative mutant HexA, bearing potential as pharmacological chaperones, but often also inhibit the enzyme O-glucosaminidase (GlcNAcase; OGA), a serious drawback for translation into the clinic. We have designed sp2-iminosugar glycomimetics related to GalNAc that feature a neutral piperidine-derived thiourea or a basic piperidine-thiazolidine bicyclic core and behave as selective nanomolar competitive inhibitors of human Hex A at pH 7 with a ten-fold lower inhibitory potency at pH 5, a good indication for pharmacological chaperoning. They increased the levels of lysosomal HexA activity in Tay-Sachs patient fibroblasts having the G269S mutation, the highest prevalent in late-onset Tay-Sachs disease.Ministerio de Ciencia e Innovación 10.13039/50110001103Fondo Europeo de Desarrollo Regional PID2019-105858RB-I00, RTI2018-097609-B-C21Junta de Andalucía P20_00166Canadian Institutes of Health Research MOP-123341Natural Sciences and Engineering Research Council of Canada RGPIN-06466Japan Society for the Promotion of Science 17K10051Universidad de Sevilla BES-2017–079676, FPU17/0314

Reversed Immunoglycomics Identifies α-Galactosyl-Bearing Glycotopes Specific for Leishmania major Infection

All healthy humans have high levels of natural anti-α-galactosyl (α-Gal) antibodies (elicited by yet uncharacterized glycotopes), which may play important roles in immunoglycomics: (a) potential protection against certain parasitic and viral zoonotic infections; (b) targeting of α-Gal-engineered cancer cells; (c) aiding in tissue repair; and (d) serving as adjuvants in α-Gal-based vaccines. Patients with certain protozoan infections have specific anti-α-Gal antibodies, elicited against parasite-derived α-Gal-bearing glycotopes. These glycotopes, however, remain elusive except for the well-characterized glycotope Galα1,3Galβ1,4GlcNAcα, expressed by Trypanosoma cruzi. The discovery of new parasitic glycotopes is greatly hindered by the enormous structural diversity of cell-surface glycans and the technical challenges of classical immunoglycomics, a top-down approach from cultivated parasites to isolated glycans. Here, we demonstrate that reversed immunoglycomics, a bottom-up approach, can identify parasite species-specific α-Gal-bearing glycotopes by probing synthetic oligosaccharides on neoglycoproteins. This method was tested here seeking to identify as-yet unknown glycotopes specific for Leishmania major, the causative agent of Old-World cutaneous leishmaniasis (OWCL). Neoglycoproteins decorated with synthetic α-Gal-containing oligosaccharides derived from L. major glycoinositolphospholipids served as antigens in a chemiluminescent enzyme-linked immunosorbent assay using sera from OWCL patients and noninfected individuals. Receiver-operating characteristic analysis identified Galpα1,3Galfβ and Galpα1,3Galfβ1,3Manpα glycotopes as diagnostic biomarkers for L. major-caused OWCL, which can distinguish with 100% specificity from heterologous diseases and L. tropica-caused OWCL. These glycotopes could prove useful in the development of rapid α-Gal-based diagnostics and vaccines for OWCL. Furthermore, this method could help unravel cryptic α-Gal-glycotopes of other protozoan parasites and enterobacteria that elicit the natural human anti-α-Gal antibodies

Pharmacological Inhibition of O-GlcNAcase Enhances Autophagy in Brain through an mTOR-Independent Pathway

The glycosylation of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is conserved among metazoans and is particularly abundant within brain. O-GlcNAc is involved in diverse cellular processes ranging from the regulation of gene expression to stress response. Moreover, O-GlcNAc is implicated in various diseases including cancers, diabetes, cardiac dysfunction, and neurodegenerative diseases. Pharmacological inhibition of O-GlcNAcase (OGA), the sole enzyme that removes O-GlcNAc, reproducibly slows neurodegeneration in various Alzheimer’s disease (AD) mouse models manifesting either tau or amyloid pathology. These data have stimulated interest in the possibility of using OGA-selective inhibitors as pharmaceuticals to alter the progression of AD. The mechanisms mediating the neuroprotective effects of OGA inhibitors, however, remain poorly understood. Here we show, using a range of methods in neuroblastoma N2a cells, in primary rat neurons, and in mouse brain, that selective OGA inhibitors stimulate autophagy through an mTOR-independent pathway without obvious toxicity. Additionally, OGA inhibition significantly decreased the levels of toxic protein species associated with AD pathogenesis in the JNPL3 tauopathy mouse model as well as the 3×Tg-AD mouse model. These results strongly suggest that OGA inhibitors act within brain through a mechanism involving enhancement of autophagy, which aids the brain in combatting the accumulation of toxic protein species. Our study supports OGA inhibition being a feasible therapeutic strategy for hindering the progression of AD and other neurodegenerative diseases. Moreover, these data suggest more targeted strategies to stimulate autophagy in an mTOR-independent manner may be found within the O-GlcNAc pathway. These findings should aid the advancement of OGA inhibitors within the clinic

Synthesis of Uronic Acid 1-Azasugars as Putative Inhibitors of α-Iduronidase, β-Glucuronidase and Heparanase**

1-Azasugar analogues of l-iduronic acid (l-IdoA) and d-glucuronic acid (d-GlcA) and their corresponding enantiomers have been synthesized as potential pharmacological chaperones for mucopolysaccharidosis I (MPS I), a lysosomal storage disease caused by mutations in the gene encoding α-iduronidase (IDUA). The compounds were efficiently synthesized in nine or ten steps from d- or l-arabinose, and the structures were confirmed by X-ray crystallographic analysis of key intermediates. All compounds were inactive against IDUA, although l-IdoA-configured 8 moderately inhibited β-glucuronidase (β-GLU). The d-GlcA-configured 9 was a potent inhibitor of β-GLU and a moderate inhibitor of the endo-β-glucuronidase heparanase. Co-crystallization of 9 with heparanase revealed that the endocyclic nitrogen of 9 forms close interactions with both the catalytic acid and catalytic nucleophile

Biological Roles of the O-Methyl Phosphoramidate Capsule Modification in Campylobacter jejuni.

Campylobacter jejuni is a major cause of bacterial gastroenteritis worldwide, and the capsular polysaccharide (CPS) of this organism is required for persistence and disease. C. jejuni produces over 47 different capsular structures, including a unique O-methyl phosphoramidate (MeOPN) modification present on most C. jejuni isolates. Although the MeOPN structure is rare in nature it has structural similarity to some synthetic pesticides. In this study, we have demonstrated, by whole genome comparisons and high resolution magic angle spinning NMR, that MeOPN modifications are common to several Campylobacter species. Using MeOPN biosynthesis and transferase mutants generated in C. jejuni strain 81-176, we observed that loss of MeOPN from the cell surface correlated with increased invasion of Caco-2 epithelial cells and reduced resistance to killing by human serum. In C. jejuni, the observed serum mediated killing was determined to result primarily from activation of the classical complement pathway. The C. jejuni MeOPN transferase mutant showed similar levels of colonization relative to the wild-type in chickens, but showed a five-fold drop in colonization when co-infected with the wild-type in piglets. In Galleria mellonella waxmoth larvae, the MeOPN transferase mutant was able to kill the insects at wild-type levels. Furthermore, injection of the larvae with MeOPN-linked monosaccharides or CPS purified from the wild-type strain did not result in larval killing, indicating that MeOPN does not have inherent insecticidal activity

Synthesis of Carbohydrate Methyl Phosphoramidates

A two-step route for introducing methyl phosphoramidate moieties onto carbohydrates is reported. The approach uses methyl pivolyl <i>H</i>-phosphonate as the phosphorylating reagent to produce an isolable carbohydrate <i>H</i>-phosphonate intermediate that is then oxidized by a Todd–Atherton reaction. The stability of the product methyl phosphoramidates was subsequently evaluated using various deprotection strategies

<i>De Novo</i> Asymmetric Synthesis of a 6‑<i>O</i>‑Methyl‑d-<i>glycero</i>-l-<i>gluco</i>-heptopyranose-Derived Thioglycoside for the Preparation of <i>Campylobacter jejuni</i> NCTC11168 Capsular Polysaccharide Fragments

An enantioselective <i>de novo</i> synthesis of a thioglycoside derivative of the 6-<i>O</i>-methyl-d-<i>glycero</i>-l-<i>gluco</i>-heptopyranose residue found in the <i>Campylobacter jejuni</i> NCTC11168 (HS:2) capsular polysaccharide is reported. The compound is obtained from a furfural-derived chiral diol in 11 steps. Notably, compared to the only previous synthesis of this molecule, this approach significantly reduces the number of purification steps required to obtain the target

Trypanosoma cruzi - Derived Sugar Epitopes - Synthesis and Immunology

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease. As of today no effective vaccine has been developed for it. Certain developmental stages of T.cruzi express cell surface oligosaccharides with terminal alpha-galactosyl and rhamnosyl residues, which are believed to be highly immunogenic in humans. The exact structures and sizes of these epitopes are still unknown. Our quest is to shine light on the chemical structures of immunogenic alpha-Gal and alpha-Rha containing mono-, di-, and trisaccharides that are conjugated to a keyhole limpet hemocyanin (KLH) carrier protein through a combination of chemical synthesis and immunological studies. The compounds synthesized were screened for their ability to be recognized by Chagasic antibodies in an enzyme-linked immunosorbent assay (ELISA). The best recognized sugar-KLH conjugates were used to immunize alpha-1,3 Gal T-KO mice, which do not express cell surface proteins with terminal alpha-galactosides, and are therefore a suitable model for humans. We have successfully synthesized and conjugated a library of nine saccharides with terminal alpha-galactosyl or rhamnosyl moieties, which elicit various levels of antibody production in mice. Upon challenge of the immunized mice with lethal doses of live T. cruzi, prolonged survival was observed when compared to the control group. The work presented here has implications for the development of a carbohydrate-based vaccine for Chagas disease

Influence of MeOPN on colonization.

<p>(<b>A</b>) Colonization levels of <i>C. jejuni</i> 81-176 wild-type and <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> (MeOPN transferase mutant) in a chicken colonization model, 6 days post-infection. (<b>B</b>) Relative colonization levels of <i>C. jejuni</i> 81-176 wild-type and the MeOPN transferase mutant in a competitive piglet infection model. Note that no significant difference was observed between strains in the chicken colonization model. In the competitive piglet infection model, the MeOPN transferase mutant displays a 5-fold reduction in colonization relative to wild-type comparing the measured wild-type-to-mutant ratio of the inoculum to the ratio recovered from the piglet intestine following infection (p = 0.0005). Statistical significance was determined using a Mann−Whitney test. Horizontal bars represent median values.</p