Development of Hepcidin Agonists and Antagonists.

Abstract SCI-27

Hepcidin binding to its receptor, the iron exporter ferroportin, leads to degradation of the ligand-receptor complex and decreased flow of iron into plasma from duodenum, macrophages and hepatocytes. Dysregulated production of hepcidin or its aberrant interaction with ferroportin causes a number of iron disorders. Hepcidin deficiency, either relative or absolute, underlies excessive iron loading in hereditary hemochromatosis, beta-thalassemia and hepatitis C. A rare type of hereditary hemochromatosis is also caused by ferroportin resistance to hepcidin. On the other side of the spectrum, hepcidin excess causes iron restriction and anemia, as is observed in patients with iron-refractory iron deficiency anemia. Hepcidin also contributes to development of anemia in different inflammatory disorders and chronic kidney disease, and may cause erythropoietin resistance. Hepcidin agonist and antagonists thus may be useful for treatment of different iron disorders. Antagonist strategies for lowering hepcidin levels have been reported, and include hepcidin-neutralizing monoclonal antibodies or agents interfering with the BMP-related hepcidin production (soluble hemojuvelin and dorsomorphin). To undertake the rational design of drug leads, we analyzed the critical structural features that determine the interaction of hepcidin and Fpn. We showed that the thiol form of Fpn residue Cys326 is essential for hepcidin binding, as is the presence of a disulfide bond on hepcidin. The mechanism of binding appears to involve disulfide exchange between the two molecules. Treatment of ferroportin-expressing cells with thiol-reactive agents prevented hepcidin binding and allowed continuous export of iron from cells. Systematic mutagenesis of additional residues on hepcidin showed that His3, Phe4 and Phe9 are also important for binding to ferroportin. To provide leads for hepcidin agonists, we then synthesized a mini-hepcidin containing only the 9 N-terminal hepcidin residues (DTHFPICIF), and showed that it displayed significant activity in vitro. Proteolysis-resistant analogs of this peptide were also active in vivo after intraperitoneal injection in mice. The small size of these peptides may allow oral bioavailability. Understanding the mechanisms of hepcidin regulation and its interaction with ferroportin will facilitate the development of new agents for the treatment of iron overload disorders and iron-restricted anemias.