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

 描述(申请人提供)：大约三分之一的美国人患有肥胖症，导致美国每年人均医疗费用增加超过1,400美元，这使得肥胖症成为一个重大的公共卫生和经济问题。无节制的高卡路里饮食会导致肥胖和相关的医疗。抗高血糖药如磺脲类药物和生物制品如胰岛素可控制游离血糖水平，但不一定能预防肥胖。有巨大的潜力发现新的治疗药物来帮助防止过度碳水化合物相关的肥胖。大自然提供了广泛的化学多样性，天然产品继续成为新的化学支架和广泛治疗药物的新类别化合物的来源。这项博士前培训拨款的研究将是发现葡萄糖异生酶的天然产物抑制剂--果糖-1，6-二磷酸酶(FBP)。FBP作为一种糖异生酶发挥作用，不参与任何其他生化或代谢途径；可以避免不太特定的靶点常见的不良副作用。尽管制药业已经证实FBP是一种可行的减肥靶点，但唯一有希望的化合物是弱结合辅因子受体部位的。因此，FBP活性部位的配体和任何类型的有效抑制剂仍未被发现；天然产物的多样性代表着尚未开发的先导化合物资源。发现天然产物先导化合物的主要障碍是，大多数天然产物存在于复杂的混合物中，与大多数高通量筛选方法不兼容。为了克服这一障碍，我一直在开发基于高通量质谱学的分析方法，旨在识别复杂混合物中的活性化合物。具体地说，我的初步数据显示，微珠亲和选择筛选(MMASS；由赞助商van Breeman博士的实验室发明)使用高分辨率的UHPLC-MS和代谢组学类型的数据处理，提供了筛选复杂天然产品混合物所需的选择性和速度。使用炎症靶标15-脂氧合酶(15-LOX)来建立概念验证，我将MMASS的吞吐量提高了大约100倍，然后将其应用于在15种北美草原植物的提取物中发现15-LOX抑制剂，这些植物被美洲原住民妇女用于抗炎特性。这些分析结果表明，在美人鱼杂草提取物中，槲皮苷是一种15-LOX抑制物。为了完成我的论文研究，我将使用MMASS测试UIC/NIH植物膳食补充剂中心提供的植物提取物、天然产品库和UIC提供的微生物培养物提取物，以寻找潜在的FBP新抑制剂。然后将确定IC50值和抑制HITS的机制。很有必要确定 抑制FBP和利用天然产物的化学多样性的新化合物将为先导化合物提供可能独特的支架和新的结合机制。

 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.