A novel high-throughput screening assay for putative antidiabetic agents through PPARalpha interactions.

As natural peroxisome proliferator-activated receptor-alpha (PPARalpha) ligands, high levels of fatty acids and glucose could lead to hyperactivation of PPARalpha, like that seen in diabetes. Important diabetes research goals are to uncover new metabolic or signaling pathways involved in hyperglycemic cellular injury and to develop therapeutics for preventing or reversing this injury. Consequently, 1040 putative antidiabetic agents were screened for their ability to 1) affect PPARalpha lipid binding, 2) directly bind PPARalpha, and 3) alter PPARalpha transactivation in the presence of high glucose. A high-throughput fluorescent binding assay was developed to examine each compound's ability to restore fatty acyl-CoA binding to PPARalpha in the presence of high glucose concentrations. Approximately 1% of the compounds restored acyl-CoA binding by 60% or more. These compounds directly interacted with PPARalpha with high affinity (nM K(d)s), validating the primary screen. Furthermore, these compounds altered PPARalpha transactivation, and 1 strongly reversed the hyperactivation of PPARalpha found in the presence of clofibrate and high glucose levels.