The glucose metabolite 3-deoxyglucosone (3DG) is a highly reactive, dicarbonyl, proinflammatory molecule that induces oxidative stress and is a precursor to the formation of advanced glycation end products (AGEs), which are believed to be important in mechanisms that directly contribute to the development of diabetic complications. Recent work indicates that 3DG levels are elevated in diabetics, and higher levels appear associated with more extensive retinopathy and neuropathy. The enzyme fructosamine-3-kinase (FN3K) produces 3DG, and our hypothesis is that the most direct way to lower 3DG is to inhibit this enzyme with a drug. This research will use high throughput screening (HTS) to find FN3K inhibitors that can be developed into drugs to treat people suffering from the debilitating complications of type II and type I diabetes. The HTS assay will use a novel, homogeneous method that measures the FN3K substrate-dependent enzymatic formation of ADP from ATP by a competitive immunoassay. To demonstrate that our HTS assay can reliably detect FN3K inhibitors, our SA1 objectives are to screen a 10,000 compound diversity subset of 'drug-like' small molecules. We will determine the potency (IC50) and selectivity values for any HTS 'hits'. Once we establish that the assay conditions are validated for HTS, the SA2 objective is to screen up to 100,000 diverse compounds, remove non-specific and cytotoxic inhibitors and assess the functional activity of selective inhibitors in cells over-expressing FN3K. Our project goal is to discover drug lead candidates amenable to optimization with medicinal chemistry and suitable for preclinical testing. Developing and validating an automated HTS 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 lead to new therapies to treat the complications of both Type I and II diabetes.
Public Health Relevance: The enzyme FN3K produces the toxic compound 3-deoxyglucosone (3DG), a highly reactive molecule that leads to the formation of advanced glycation end products (AGEs), which contribute to diabetic complications. This research will test compounds by HTS with an appropriate biochemical screen and perform appropriate follow-up assays to identify FN3K inhibitors that can be developed into drugs to treat people suffering from the debilitating complications of type II and type I diabetes.
Thesaurus Terms: "3-Deoxy-D-Glucosone; 3-Deoxyglucosone; 3-Deoxyhexosulose; Active Follow-Up; Advanced Glycosylation End Products; Animal Model; Animal Models And Related Studies; Assay; Bioassay; Biochemical; Biologic Assays; Biological Assay; Cells; Chemicals; Chemistry, Pharmaceutical; Chromatography, Gas-Liquid-Mass Spectrometry; Collection; Complications Of Diabetes Mellitus; D-Glucose; Development; Dextrose; Diabetes Complications; Diabetes Mellitus; Diabetes-Related Complications; Diabetic Complications; Disease; Disorder; Drug Evaluation, Preclinical; Drug Kinetics; Drug Screening; Drugs; Ec 2.7; Enzymes; Evaluation Studies, Drug, Pre-Clinical; Evaluation Studies, Drug, Preclinical; Gc Ms; Gcms; Glucose; Glycation End Products, Advanced; Glycosylation End Products, Advanced; Goals; Health; Hexoses; High Throughput Assay; Human; Human, General; Ic50; Immunoassay; Inhibitory Concentration 50; Kidney Diseases; Kinases; Lead; Libraries; Man (Taxonomy); Man, Modern; Mass Fragmentographies; Mass Fragmentography; Measures; Medication; Medicinal Chemistry; Methods; Monitor; Nephropathy; Neuropathy; Oxidative Stress; Pb Element; Pharmaceutic Chemistry; Pharmaceutic Preparations; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacokinetics; Phase; Phosphotransferases; Physiologic; Physiological; Poisons; Preclinical Drug Evaluation; Preclinical Testing; Production; Reaction; Renal Disease; Research; Retinal Diseases; Retinal Disorder; Role; Running; Safety; Spectrometry, Mass-Gas Chromatography; Spectrum Analysis, Mass-Gas Chromatography; Structure; Testing; Toxic Chemical; Toxic Substance; Toxic Effect; Toxicities; Transphosphorylases; Work; Advanced Glycation Endproduct; Counterscreen; Cytotoxic; Diabetes; Diabetic; Disease/Disorder; Drug Candidate; Drug/Agent; Follow-Up; Fructosamine-3-Kinase; Heavy Metal Pb; Heavy Metal Lead; High Throughput Screening; Inhibitor; Inhibitor/Antagonist; Ion Trap Mass Spectrometry; Kidney Disorder; Kinase Inhibitor; Mass Fragmentometry; Meetings; Model Organism; Neuropathic; Novel; Poison; Pre-Clinical; Preclinical; Public Health Relevance; Renal Disorder; Retina Disease; Retina Disorder; Retinopathy; Small Molecule; Social Role; Tool; Toxic Compound; Type I And Type Ii Diabetes"
