UC San Diego

A number of diabetogenic stimuli interact to influence insulin promoter activity, making it an attractive target for both mechanistic studies and therapeutic interventions. Our lab developed a unique cell-based high- throughput screening assay for molecules that control insulin gene expression. The assay platform is based on an engineered cell line, T6PNE, derived from human fetal islets. T6PNE cells express insulin in response to inducible E47, an important insulin gene transactivator and were adapted for high-throughput assays using a lentiviral vector expressing the insulin-eGFP transgene. Through a screen of a subset of the ChemBridge Diverset small molecule library, we identified BIM5078, which represses insulin promoter activity. A cheminformatic analysis revealed structural similarity to a PPAR[gamma] ligand, FK614. Consistent with that result, BIM5078 was found to be a PPAR[gamma] agonist when tested in a PPAR response element reporter assay. However, FK614 was inactive when tested for its ability to modulate the insulin promoter. In a search for other potential targets of BIM5078, we found that it binds strongly to HNF4[alpha] (Kd: 11.9 ± 2.9nM) and modulates known HNF4[alpha] target genes. Like BIM5078, siRNA to HNF4[alpha] in T6PNE reduces insulin gene expression, suggesting that BIM5078 is an HNF4[alpha] antagonist. In order to advance mechanistic studies and in vivo delivery of this novel class of potent synthetic HNF4[alpha] ligands, we developed a structurally related small molecule (BI6015) that selectively inhibits HNF4[alpha] by eliminating off-target effects mediated through PPAR[gamma]. As predicted by the phenotype observed in the liver-specific HNF4[alpha] knockout, both pharmacologic inhibition with BI6015 and genetic silencing of HNF4[alpha] results in accelerated lipid accumulation. Interestingly, BIM5078 and BI6015 were noted to be selectively cytoxic to cancer cell lines in vitro. In vivo, BI6015 induces apopotosis of transplanted human hepatocellular carcinoma cells but not the normal liver in an orthotopic xenograft model, although potency was limited by suboptimal pharmacokinetic properties. The discovery of bioactive ligands for HNF4[alpha] raises the possibility that diseases involving HNF4[alpha], such as diabetes and cancer, might be amenable to pharmacologic intervention and provide a powerful tool to study the physiological role of HNF4[alpha]