The specific goal of this project is to develop a homogeneous high throughput screening assay to discover potent and selective, small molecule inhibitor(s) of fructoseamine-3-kinase (FN3K) the enzyme believed responsible for the majority of 3-deoxyglucosone (3DG) produced in vivo. The FN3K enzyme is widely distributed in the body, particularly in kidney, and represents a promising therapeutic target because of its catalytic role in the production of 3DG. The 3DG molecule is a highly reactive, dicarbonyl metabolite of glucose metabolism that induces oxidative stress and inflammation, and is a precursor to the formation of advanced glycation end products (AGEs), all of which contribute to the development of diabetic complications. Importantly, 3DG plasma levels have been directly linked recently to the development of diabetic complications in humans. We will establish and validate a HTS assay for FN3K with the aid of a novel, competitive, fluorescencepolarization immunoassay that measures ADP, which accumulates as a stochiometric consequence of phosphorylation of any substrate by any kinase. This novel, homogeneous assay technology is well suited to sugar kinases such as FN3K that require high concentrations of ATP and that have relatively low catalytic activity. The novel assay proposed has been made commercially available; however, it has not yet been subjected to rigorous validation for HTS. Our proposal presents an unusually strong approach to validate that the ADP competitive immunoassay assay reflects underlying mass-action kinetics; therefore, the work will benefit the HTS research community, particularly in the non-protein kinase field. Given the therapeutic relevance of our work, backup strategies are proposed to ensure that we achieve our main objective to deliver for HTS a robust, sensitive and valid assay to discover new tools and probes for the physiological importance of FN3K in health and disease. Developing and validating a high throughput screening assay to detect FN3K inhibitors in large, diverse, drug-like compound libraries is an important step to finding new tools to probe the physiological role of FN3K in health and disease, which will help lead to new therapies for both Type I and II diabetes. ? ? ?
