Discovery of Natural Product Inhibitors of Fructose-1,6-bisphophatase for Obesity Management

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 DESCRIPTION (provided by applicant): Approximately one-third of Americans are obese resulting in over $1,400 extra health care costs per capita each year in the United States, which makes obesity a major public health and economics issue. Unabated high caloric diets can lead to obesity and associated medical. Anti-hyperglycemic agents such as sulfonylureas and biologicals such as insulin control free glucose levels but do not necessarily prevent obesity. There is vast potential to discover new therapeutic agents to help prevent excessive carbohydrate related obesity. Nature provides broad chemical diversity, and natural products continue to be sources of new chemical scaffolds and new classes of compounds for a wide range of therapeutic agents. The research of this predoctoral training grant will be to discover natural product inhibitors of the gluconeogenic enzyme, fructose-1,6- bisphosphatase (FBP). FBP functions as a gluconeogenic enzyme without involvement in any other biochemical or metabolic pathways; undesirable side effects that are common to less specific targets can be avoided. Although the pharmaceutical industry has validated FBP as a viable anti-obesity target, the only promising compounds weakly bind to a cofactor receptor site. Therefore, ligands to the active site of FBP and potent inhibitors of any type remain undiscovered; the diversity of natural products represents an unexplored resource for lead compounds. The primary impediment to the discovery of natural product lead compounds is that most natural products exist in complex mixtures and are not compatible with most high-throughput screening approaches. To overcome this impediment, I have been developing high-throughput mass spectrometry-based assays that are designed for identifying active compounds within complex mixtures. Specifically, my preliminary data show that microbead affinity selection screening (MMASS; invented in the laboratory of sponsor Dr. van Breemen), which uses high resolution UHPLC-MS and metabolomics-type data processing, provides the selectivity and speed required for screening complex natural product mixtures. Using the inflammation target 15-lipoxygenase (15-LOX) to establish proof of concept, I enhanced the throughput of MMASS by approximately 100-fold and then applied it to the discovery of a 15-LOX inhibitors in extracts of 15 North American prairie plants used by Native American women for their anti-inflammatory properties. These assays resulted in the identification of quercitrin as a 15-LOX inhibitor in an extract of mermaid weed. To complete my dissertation research, I will use MMASS to test botanical extracts provided by the UIC/NIH Center for Botanical Dietary Supplements, natural product libraries and extracts of microbial cultures available at UIC for potential new inhibitors of FBP. The IC50 values and mechanisms of inhibition of the hits will then be determined. There is significant need to identify new compounds that inhibit FBP and tapping the chemical diversity of natural products will provide lead compounds with perhaps unique scaffolds and novel binding mechanisms.