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

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

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

A Branched and Double Alpha-Gal-Bearing Synthetic Neoglycoprotein as a Biomarker for Chagas Disease

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi and affects 6–7 million people worldwide. The diagnosis is still challenging, due to extensive parasite diversity encompassing seven genotypes (TcI-VI and Tcbat) with diverse ecoepidemiological, biological, and pathological traits. Chemotherapeutic intervention is usually effective but associated with severe adverse events. The development of safer, more effective therapies is hampered by the lack of biomarker(s) (BMKs) for the early assessment of therapeutic outcomes. The mammal-dwelling trypomastigote parasite stage expresses glycosylphosphatidylinositol-anchored mucins (tGPI-MUC), whose O-glycans are mostly branched with terminal, nonreducing α-galactopyranosyl (α-Gal) glycotopes. These are absent in humans, and thus highly immunogenic and inducers of specific CD anti-α-Gal antibodies. In search for α-Gal-based BMKs, here we describe the synthesis of neoglycoprotein NGP11b, comprised of a carrier protein decorated with the branched trisaccharide Galα(1,2)[Galα(1,6)]Galβ. By chemiluminescent immunoassay using sera/plasma from chronic CD (CCD) patients from Venezuela and Mexico and healthy controls, NGP11b exhibited sensitivity and specificity similar to that of tGPI-MUC from genotype TcI, predominant in those countries. Preliminary evaluation of CCD patients subjected to chemotherapy showed a significant reduction in anti-α-Gal antibody reactivity to NGP11b. Our data indicated that NGP11b is a potential BMK for diagnosis and treatment assessment in CCD patients

Loss of MeOPN results in a decrease in serum resistance.

<p>Survival of <i>C. jejuni</i> 81-176 wild-type, <i>cjj81176_1415</i>::<i>kan^r</i> (MeOPN biosynthesis mutant), <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> (MeOPN transferase mutant), and <i>kpsM</i>::<i>kan^r</i> (capsule mutant) in 10% natural human complement serum relative to survival in heat inactivated serum. Serum resistance was significantly different between <i>C. jejuni</i> 81-176 wild-type, the <i>cjj81176_1415</i>::<i>kan^r</i> (MeOPN biosynthesis mutant), and the <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> mutant at both 30 and 60 min (p<0.0001). Serum resistance was significantly different between <i>C. jejuni</i> 81-176 wild-type and the <i>kpsM</i>::Km^R mutant at both 30 and 60 min (p = 0.005 and p = 0.014, respectively) using an unpaired t-test. Results represent the mean (± SEM) of three independent experiments.</p

1D ¹H–³¹P HSQC spectra of select <i>Campylobacter</i> species containing orthologues of the <i>cj1416</i>-<i>cj1418</i> genes.

<p>Spectra contain the following resonances corresponding to MeOPN: <i>C. insulaenigrae</i> RM5435, peak at 3.74 ppm; <i>C. lari</i> UPTC NCTC 11845, peak at 3.70 ppm; <i>C. lari</i> subsp. <i>concheus</i> LMG 11760, peak at 3.70 ppm; <i>C. subantarcticus</i> RM8523, peak at 3.75 ppm; <i>C. cuniculorum</i> LMG 24588, peak at 3.76 ppm; <i>C. upsaliensis</i> RM3195, peak at 3.70 ppm; <i>C. upsaliensis</i> RM3940, peaks at 3.75 and 3.71 ppm; <i>C. helveticus</i> CCUG 30566, peak at 3.68 and 3.61 ppm; <i>C. jejuni</i> NCTC 11168, peaks at 3.71 and 3.68 ppm.</p

1D ¹H–³¹P HSQC analyses of <i>C. jejuni</i> 81-176 wild-type and the MeOPN biosynthesis and transferase mutants.

<p>Intact whole cells of <i>C. jejuni</i> were analysed by HR-MAS NMR after 48 h growth on MH agar. Depicted are the 1D ¹H–³¹P HSQC spectra, which specifically show the MeOPN resonance at 3.8 ppm in 81-176 wt (A), but not in the <i>cjj81176_1415</i>::<i>kan^r</i> MeOPN biosynthesis mutant (B), and <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> MeOPN transferase mutant (C).</p