A Chemical Screen Reveals New Modulators of Hepcidin Expression

Abstract

Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing the human Hepcidin promoter upstream of a firefly reporter gene. We then used high throughput methods to screen 10,360 chemicals in duplicate for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change >;3 standard deviations above or >;1.5 standard deviations below the mean of the other chemicals, while not adversely affecting cell viability. Using these criteria, we identified 32 small molecules that upregulated and 3 that downregulated Hepcidin expression. On retesting assays, we confirmed 22 of the initial positives (69%) and 1 of the initial negatives as regulators of Hepcidin expression. Functional classification of the positive regulators indicated: 4 anti‐inflammatory agents, 4 antimicrobials, 6 antineoplastic drugs, 4 kinase inhibitors, and 4 with other or unknown functions. We are now evaluating the mechanisms of action in human hepatocytes and in a zebrafish model. The best candidates will subsequently be tested in mouse models of iron overload syndromes. Funding: NIDDK R01 DK085250–01A1.