Modification of the degree of branching of a beta-(1,3)-glucan affects aggregation behavior and activity in an oxidative burst assay.

Scleroglucan is a ß-(1,3)-glucan which is highly branched at the 6-position with a single glucose residue. Acid hydrolysis of a high molecular weight scleroglucan gave a medium molecular weight, freely soluble material. Linkage analysis by the partially methylated alditol acetate method showed that the solubilized material had 30% branching. When the material was subjected to partial Smith degradations, the percent branching was reduced accordingly to 12% or 17%. After the percent branching was reduced, the average molecular weight of the samples increased considerably, indicating the assembly of higher ordered aggregate structures. An aggregate number distribution analysis was applied to confirm the higher aggregated structures. These aggregated structures gave the material significantly enhanced activity in an in vitro oxidative burst assay compared to the highly branched material.

Differential regulation of oxidative burst by distinct ß-glucan-binding receptors and signaling pathways in human peripheral blood mononuclear cells.

ß-Glucans possess broad immunomodulatory properties, including activation of innate immune functions such as oxidative burst activity. The differential roles of complement receptor type 3 (CR3) and Dectin-1, the known ß-glucan receptors, and their associated signaling pathways in the generation of oxidative burst induced by different physical forms of Saccharomyces cerevisiae-derived ß-glucan were examined in human peripheral blood mononuclear cells (PBMC). In this study whole glucan particle (WGP) or immobilized soluble ß-glucan (ISG) was used to represent the phagocytizable or the nonphagocytizable form of a fungus, respectively. Oxidative burst as measured by the formation of superoxide (SO) was detected in PBMC in response to WGP and ISG. SO induction with WGP was concluded to be Dectin-1-mediated and required Src family kinases, phosphatidylinositol-3 kinase and protein kinase B/Akt. In contrast, the SO induction generated by ISG was CR3-mediated and required focal adhesion kinase, spleen tyrosine kinase, phosphatidylinositol-3 kinase, Akt, p38 mitogen activated protein kinase, phospholipase C and protein kinase C. The study results support the hypothesis that human PBMC, specifically monocytes, utilize distinct receptors and overlapping, but distinct, signaling pathways for the oxidative burst in response to challenge by different physical forms of ß-glucan.

Chemoprotective antimalarials identified through quantitative high-throughput screening of Plasmodium blood and liver stage parasites.

The spread of Plasmodium falciparum parasites resistant to most first-line antimalarials creates an imperative to enrich the drug discovery pipeline, preferably with curative compounds that can also act prophylactically. We report a phenotypic quantitative high-throughput screen (qHTS), based on concentration-response curves, which was designed to identify compounds active against Plasmodium liver and asexual blood stage parasites. Our qHTS screened over 450,000 compounds, tested across a range of 5 to 11 concentrations, for activity against Plasmodium falciparum asexual blood stages. Active compounds were then filtered for unique structures and drug-like properties and subsequently screened in a P. berghei liver stage assay to identify novel dual-active antiplasmodial chemotypes. Hits from thiadiazine and pyrimidine azepine chemotypes were subsequently prioritized for resistance selection studies, yielding distinct mutations in P. falciparum cytochrome b, a validated antimalarial drug target. The thiadiazine chemotype was subjected to an initial medicinal chemistry campaign, yielding a metabolically stable analog with sub-micromolar potency. Our qHTS methodology and resulting dataset provides a large-scale resource to investigate Plasmodium liver and asexual blood stage parasite biology and inform further research to develop novel chemotypes as causal prophylactic antimalarials.

Enzymatic method to measure ß-1,3-ß-1,6-glucan content in extracts and formulated products (GEM assay).

An enzymatic method to measure ß-glucan content (GEM assay) is applicable in a variety of matrices. The method is composed of swelling the sample with KOH and initial digestion with a lyticase, which is followed by treatment with a mixture of exo-1,3-ß-d-glucanase and ß-glucosidase that converts the ß-glucan to glucose. The glucose generated by the enzymatic hydrolysis is measured by another enzymatic method. The method is shown to be accurate and precise. The method is selective and applicable to both highly branched and unbranched ß-1,3-glucans.