Abstract
Hepatocyte nuclear factor 4α (HNF4α) is a liver-enriched transcription factor which belongs to the nuclear receptor family, and controls the expression of genes involved in various different functions such as nutrient transport, metabolism, growth and differentiation. Recently, fatty acids and/or fatty acyl CoAs as well as some xenobiotics (e.g. hypolipidemic drugs) have been suggested as HNF4α ligands. The aim of this study was to identify potential ligands for HNF4α, using a DAUDA fluorescence displacement assay. Attempts to purify the human HNF4α were not successful. However, the rat HNF4αLBD was successfully expressed and purified, therefore further work for ligand identification was performed using the rat HNF4α. LFABP was expressed and purified as a positive control, and used successfully to validate the DAUDA assay. Suitability of the DAUDA fluorescence displacement assay for screening of ligands for rat HNF4α was confirmed by a characteristic blue-shift in fluorescence emission upon protein binding, as previously reported. Ligands screened in this study included fatty acids (myristate, palmitate, oleate, linoleate, docosahexanoate), a fatty acyl CoA (palmitoyl CoA), and hypolipidemic xenobiotics of the fibrate family (fenofibrate, bezafibrate and clofibrate), as well as the thionolidinedione pioglitazone. All the fatty acids and palmitoyl CoA bound to rHNF4α, with Ki values between 122-161 nM, which is within the range of previous reports and probably of physiological relevance. Of the xenobiotics tested, only bezafibrate was a low-affnity ligand (Ki 1156±390nM) for rHNF4α. These results suggest that the DAUDA fluorescence displacement assay is suitable for the screening of HNF4α ligands, and expands on previous work suggesting that fatty acids and FACoAs are endogenous ligands for HNF4α.