This proposal aims to develop a high throughput screening (HTS) assay that will identify small molecule inhibitors for the aldo-keto reductase AKR1C3, in hopes that they would lead to a more potent cellular response to insulin. Such inhibitors could be of great therapeutic potential to Type II diabetics, who suffer from insulin resistance in peripheral tissues such as muscle, adipose and liver. The selected compounds could also serve as useful tools for studying the insulin signal transduction pathway, especially concerning the nuclear transcription factor PPARgamma, which ultimately activates a set of insulin-responsive genes. The branch of the biosynthetic pathway addressed here starts with prostaglandin D2 (PGD2). This compound is normally converted to 15-deoxy-delta12,14-prostaglandin J2 - a potent PPARgamma ligand. However, especially in diabetic muscle tissue, AKR1C3 converts PGD2 along an alternative pathway to 9alpha11betaPGF2alpha, diminishing the amount of 15-deoxy-delta12,14-prostaglandin J2 available for PPARgamma activation. We have therefore proposed a model whereby the up-regulation of AKR1C3 increases the production of 9alpha11betaPGF2alpha at the expense of the production of 15-deoxy-delta12,14-prostaglandin J2. On the other hand, we believe that AKR1C3-specific inhibitors would attenuate the competing pathway, thus favoring production of 15-deoxy-delta12,14-prostaglandin J2 and the consequent stimulation of PPARgamma, leading to insulin sensitivity. To identify such inhibitors we will first establish a fluorescence-based assay for measuring AKR1C3 activity that will take advantage of the redox function of the enzyme. It will measure the decreasing fluorescence of NADPH as it is converted to NADP during the concomitant reduction of PGD2. A backup assay will follow the conversion of NADP to NADPH, as AKR1C3 oxidizes 1-acenapthenol. After the assay is developed, we will then adapt it for HTS format and screen a pilot library in search of inhibitors. Compounds identified as hits will then be tested in a dose response biochemical assay followed by a cell-based assay to confirm their inhibition of AKR1C3. Confirmed inhibitors may be of great value, both experimentally to researchers and therapeutically to diabetes patients. The research proposed here aims to identify compounds that are likely to have therapeutic potential for individuals suffering from insulin resistance and Type II diabetes. This ailment directly affects 20 million people in the United States, with only 42% of patients attaining sufficient glycemic control, as defined by the American Diabetes Association. An effective therapy could provide much relief to the patients. ?
