13 research outputs found
Facile Solid-Phase Synthesis and Assessment of Nucleoside Analogs as Inhibitors of Bacterial UDP-Sugar Processing Enzymes
The privileged uptake of nucleosides into cells has generated interest in the development of nucleoside-analog libraries for mining new inhibitors. Of particular interest are applications in the discovery of substrate mimetic inhibitors for the growing number of identified glycan-processing enzymes in bacterial pathogens. However, the high polarity and the need for appropriate protecting group strategies for nucleosides challenges the development of synthetic approaches. Here, we report an accessible, user-friendly synthesis that branches from a common solid phase-immobilized uridinyl-amine intermediate, which can be used as a starting point for diversity-oriented synthesis. We demonstrate the generation of five series of uridinyl nucleoside analogs for investigating inhibitor structure-activity relationships. This library was screened for inhibition of representative enzymes from three functional families including a phosphoglycosyl transferase, a UDP-aminosugar acetyltransferase, and a glycosyltransferase. These candidates were taken from the Gram-negative bacteria Campylobacter concisus and Campylobacter jejuni and the Gram-positive bacterium Clostridium difficile, respectively. Inhibition studies show that specific compound series preferentially inhibit selected enzymes, with IC 50 values ranging from 35 ± 7 μM to 174 ± 21 μM. Insights from the screen provide a strong foundation for further structural elaboration, to improve potency, which will be enabled by the same synthetic strategy. The solid-phase strategy was also used to synthesize pseudouridine analogs of lead compounds. Finally, the compounds were found to be nontoxic to mammalian cells, further supporting the opportunities for future development.National Institutes of Health (Grant R01-GM097241
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
Synthesis of Galα(1,3)Galβ(1,4)GlcNAcα-, Galβ(1,4)GlcNAcα- and GlcNAc-containing neoglycoproteins and their immunological evaluation in the context of Chagas disease
α-Gal-NGPs as biomarkers of active CL in patients with <i>L</i>. <i>major</i> infection.
<p>Assessment by chemiluminescent ELISA of α-Gal-containing NGPs and controls (Cysteine-BSA and Galβ-BSA) were immobilized on a microplate and reacted with pools of sera (at 1:100 dilution) from patients with active or cured CL, or heterologous skin (non-CL) infections (n = 5 per group, randomly selected) from an endemic region (Saudi Arabia), as described [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006039#pntd.0006039.ref026" target="_blank">26</a>]. RLU, Relative luminescence units. Error bars indicate S.E.M. of triplicate determinations. The fold difference in reactivity between active CL vs. cured CL, and active CL vs. heterologous infection are indicated. Two-way ANOVA with Tukey’s multiple comparisons: ns, non-significant; (*), <i>P</i><0.05; (**), <i>P</i><0.01; (***), <i>P</i><0.001; (****), <i>P</i><0.0001.</p
α-Gal-NGPs as potential vaccine candidates against <i>L</i>. <i>major</i>.
<p>(A) Chemiluminescent ELISA to measure anti-α-Gal antibody levels in α1,3GalT-KO mice immunized with the α-Gal-containing NGP17B, NGP12B, or NGP5B. Immunizations groups are indicated in the legend, whereas NGPs and control (BSA) used as antigens in the chemiluminescent ELISA are shown in the Y-axis. Sera were used at 1:100 dilution. Two-way ANOVA with Tukey’s multiple comparisons: ns, non-significant; (***), <i>P<</i>0.001; (****), <i>P<</i>0.0001. (B) Lesion size (mm) in mice immunized with α-Gal-NGP (NGP12B, NGP17B, or NGP5B) or control (BSA), and then challenged with 1 x 10<sup>5</sup> <i>L</i>. <i>major-luc</i> metacyclic promastigotes. One-way ANOVA (compared with BSA control): ns, non-significant; (**), <i>P<</i>0.01. (A and B) Error bars indicate S.E.M. of triplicate determinations.</p
Anti-NGP5B antibody response is specific against terminal α-Gal residues.
<p>(A-B) Chemiluminescent ELISA reactivity of mouse serum pools obtained at Boost 3 (n = 6) and endpoint (n = 3) from α1,3GalT-KO mice vaccinated with NGP5B, NGP5B+CpG, CpG, or PBS. Immunized groups are indicated in the legend and antigens on the microplate are shown in the Y-axis. (C) Chemiluminescent ELISA reactivity of mouse serum obtained at Boost 2. NGP5B (125 ng/well) was treated or not with green-coffee bean α-galactosidase. One-way ANOVA (compared with untreated sample): (**), <i>P<</i>0.01; (***), <i>P</i><0.001; (****), <i>P</i><0.0001. (A-C) Error bars indicate S.E.M. of triplicate determinations.</p
Serum cytokine profile of NGP5B and NGP5B+CpG immunizations.
<p>(A-D) Th1 cytokines IL-12p40, IL-2, IFN-γ, and TNF-α. (E-G) Th2 cytokines IL-4, IL-10, and IL-5. (H) IFN-γ/IL4 ratio. (I) IFN-γ/IL10 ratio. Two-tailed unpaired Student’s <i>t</i>-test (compared with Naïve group): (*), <i>P</i><0.05; (***), <i>P</i><0.0001. (A-I) Error bars indicate S.E.M. of triplicate determinations.</p
Analysis of humoral immune response of NGP5B immunized mice.
<p>(A) Chemiluminescent ELISA reactivity against NGP5B of mouse sera obtained at prime (P), boost 1–3 (B1-B3), three weeks post-B3 (day 0) and at the endpoint (43 dpi) from α1,3GalT-KO mice vaccinated with NGP5B, NGP5B+CpG, CpG, or PBS. (B-C) Antibody isotyping (IgG1, IgG2a, IgG2b, IgG3, and IgE) of mouse sera obtained following boost 3 (B3) and at the experimental endpoint (43 dpi). Statistical analysis for A-C: Two-way ANOVA with Dunnett’s multiple comparison test: (*), <i>P</i><0.05; (****), <i>P</i><0.0001. (D) Percentage of lysis of <i>L</i>. <i>major-luc</i> metacyclic promastigotes incubated with sera from mice immunized with NGP5B, NGP5B+CpG, CpG or PBS. Control dead parasites: 10<sup>6</sup> <i>L</i>. <i>major</i>-<i>luc</i> metacyclic promastigotes, heat-killed at 100<sup>°</sup>C for 10 min, followed by 30-min incubation at RT with PI. Control live parasites: 10<sup>6</sup> <i>L</i>. <i>major</i>-<i>luc</i> metacyclic promastigotes in DMEM (no FBS) without any treatment. C, mouse serum with active complement; iC, mouse serum with heat-inactivated complement; NMS, normal (non-infected) mouse serum. One-way ANOVA with Tukey’s multiple comparisons (compared with NMS control): ns, non-significant; (**), <i>P</i><0.01; (***), <i>P</i><0.001. (A-D) Error bars indicate S.E.M. of triplicate determinations.</p
Vaccination with NGP5B or NGP5B+CpG induces antigen-specific CD4<sup>+</sup> and CD8<sup>+</sup> memory T cell after <i>L</i>. <i>major</i> challenge.
<p>(A and B) Percentage of antigen specific CD3<sup>+</sup>CD4<sup>+</sup> and CD3<sup>+</sup>CD8<sup>+</sup> T cells, respectively, from splenocytes of mice 3 weeks post-last immunization and at the endpoint (immunized→challenged). Splenocytes were cultured and stimulated <i>in vitro</i> with 20 μg/ml of antigen. (C) Splenocytes stimulated and gated on CD4<sup>+</sup>CD44<sup>+</sup>, CD4<sup>+</sup>CD69<sup>+</sup>, and CD4<sup>+</sup>CD44<sup>+</sup>CD69<sup>+</sup> T cell populations from immunized-challenged group, for the percentage of activated CD4<sup>+</sup> T cells in mice vaccinated with NGP5B or NGP5B+CpG. (D) Splenocytes stimulated and gated on CD8<sup>+</sup>CD44<sup>+</sup>, CD8<sup>+</sup>CD69<sup>+</sup>, and CD8<sup>+</sup>CD44<sup>+</sup>CD69<sup>+</sup> T cell populations from immunized-challenged group, for the percentage of activated CD8<sup>+</sup> T cells in mice vaccinated with NGP5B or NGP5B+CpG. Statistical analysis for A-D: Two-tailed unpaired multiple Student’s <i>t</i>-test: (*), <i>P</i><0.05; (**), <i>P</i><0.01. (A-D) Error bars indicate S.E.M. of triplicate determinations.</p