Currently, about 65% of U. S. adults are either overweight (= or >25 kg/m2) or obese (= or >30 kg/m2) and about 15% of U. S. children are similarly categorized. Excessive insulin secretion can lead to serious metabolic disorders, such as nesidioblastosis (insulin hyper secretion leading to severe persistent hypoglycemia), and may also be closely associated with development of obesity and type 2 diabetes. The beta-cell function of patients with type 2 diabetes progressively declines and the degree of hyperglycemia is increased. The beta-cell function and viability can be improved in preclinical models of diabetes by reducing insulin secretion to induce beta-cell secretory rest using the K(ATP) channel opener, diazoxide. Clinical studies indicate that inhibition of insulin release with diazoxide can also help treat or prevent diabetes. However, current K(ATP) channel openers possess low subunit-selectivity or have poor pharmacokinetic profiles. Evaluation of the full potential of beta-cell secretory rest attained by opening K(ATP) channels requires compounds with greater selectivity and fewer side effects. We propose to develop a high-throughput screening (HTS) assay for K(ATP) channels as a first step to develop such compounds. Our preliminary studies indicate that the potencies of 4 standard K(ATP)channel modulators determined by a fluorescence-based cell assay using a FlexStation correlate well with those measured using standard patch clamp recordings from the same pancreatic cell lines. Therefore, we propose to develop an HTS method using a fluorometric imaging plate reader (FLIPR(r)) to study native pancreatic K(ATP) channel function and to establish an HTS platform that can be used to discover novel subunit-selective K(ATP) channel modulators.
Our Specific Aims are to: 1. Develop a FLIPR(r) 96- or 384-well format HTS assay against the pancreatic form of K(ATP) channels(SUR1/Kir6. 2 subunits) for primary screening and against the muscle form (e. g. , SUR2/Kir6.1subunits) for selectivity counter-screening by optimization of assay conditions, automation of procedures, and data analysis. 2. Demonstrate the utility of the HTS assay by evaluating its selectivity and the reproducibility of the response to a structurally diverse panel of several hundred compounds, including commercially available K(ATP) modulators and a selection of small molecules from the SRI compound library. The proposed fluorometric method will provide a rapid, high-throughput functional assay for the primary screening of compounds against natural K(ATP) channels expressed in native pancreatic beta-cells. Ultimately, this assay should facilitate the discovery of novel therapeutics for the treatment of obesity and diabetes.
