Human milk oligosaccharides (HMOs) are the third largest component of breast milk and have been described to convey developmental benefits to the newborn. HMOs may comprise a wealth of untapped therapeutic opportunities as this unique class of molecules can interact with, and thereby potentially modulate, mediators of inflammation. Unfortunately, investigation into the therapeutic utility of HMOs has been hindered by lack of access to individual structures in appropriate quantity and purity for thorough biological evaluation and preclinical development. Current methods for evaluating the biological role of HMOs rely on: 1) probing a heterogeneous mixture of HMOs bearing similar structural characteristics or 2) attempting to isolate individual structures from pooled milk using exhaustive chromatography techniques. Neither strategy provides a platform for readily examining individual HMOs against a panel of biological targets, nor an efficient strategy for target scale-up required for mode-of-action, structure-activity relationships, and pre-clinical development. There is an urgent need to develop a synthetic strategy that can access well-defined HMOs for high-throughput screening and is scalable to provide identified leads in appropriate quantity for biological evaluation and ligand optimization. To address the problem of compound availability, a chemoenzymatic synthetic strategy will be employed to develop a library comprising 100 HMOs. The strategy aims at chemically synthesizing a limited number of advanced intermediates which can be further modified by a panel of glycosyltransferases to provide a collection of targets with significant structural diversity. The library will be immobilized on microarray slides for high-throughput screening and in first instance, will be screened with carbohydrate recognizing proteins involved in intestinal inflammation. Identified leads will be examined for anti-inflammatory efficacy using an in vitro dendritic cell model. Finally, a complementary, convergent synthetic approach will be developed to provide large-scale access to efficacious hits required for in vivo animal studies and preclinical development. The short-term goal of this proposal is the successful synthesis of the largest collection of synthetic HMOs to date for assay implementation and early pharmacological screening. Additionally, our convergent synthetic methodology will provide a means to scale-up any HMO library member in appropriate quantity for preclinical development. The long-term goal is to use this methodology to facilitate the development of HMOs to address deficiencies in neonatal and infant formulas as well as investigate potential avenues for development of anti- inflammatory therapeutics.
Public Health Relevance Statement: Project narrative: Human milk oligosaccharides (HMOs) are an important class of biomolecules found in breast milk that provide developmental benefits to neonates. The lack of commercial access to individual HMOs for thorough biological evaluation has limited their incorporation into infant formula and nutritional supplements. To overcome this hurdle, we will employ a chemoenzymatic synthetic strategy which will develop a HMO screening platform and will permit large-scale synthesis of hits for follow-up studies.
Project Terms: Address; Animal Model; Animals; Anti-inflammatory; Binding; Binding Proteins; Biological; Biological Assay; Biological Markers; Carbohydrates; CD14 gene; Cell model; Cell surface; Characteristics; Chemicals; Chromatography; clinical development; Collection; course development; Dendritic Cells; Development; dietary supplements; Dose; Enterocytes; Epitopes; Evaluation; Event; exhaustion; Exhibits; follow-up; Follow-Up Studies; Glycoproteins; glycosyltransferase; Goals; high throughput screening; Histopathology; Human Milk; IL8 gene; Immobilization; Immune response; improved; In Vitro; in vivo; Individual; Infant formula; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory disease of the intestine; Inflammatory Response; intestinal epithelium; Intestines; Investigation; large scale production; Lead; Libraries; Ligands; member; Membrane Glycoproteins; Methodology; Methods; Microarray Analysis; Milk; milligram; Modeling; Necrotizing Enterocolitis; Neonatal; neonate; Neuraminidase; Newborn Infant; Oligosaccharides; Pathology; Pharmacology; Phase; Play; Polysaccharides; preclinical development; preclinical evaluation; prevent; Prevention; Production; Property; Proteins; Protocols documentation; Rattus; Role; scale up; screening; sialic acid binding Ig-like lectin; Sialic Acids; sialyl Lewis a; sialyl Lewis x; Signal Transduction; Slide; Structure; Structure-Activity Relationship; Techniques; Therapeutic
