Scientists have generated a series of small peptides that mimic the effects of the body’s iron regulatory protein hepcidin, which could represent a starting point for developing new treatments for disorders characterized by iron overload.
A team led by University of California, Los Angeles (UCLA) researchers used a site mutagenesis approach to identify which portion of the hepcidin protein binds to the transmembrane ferroportin receptor and in particular, to confirm which disulfide bonds generated by receptor-ligand docking are necessary to stabilize the interaction. They then generated and structurally optimized small (up to nine amino acid) peptides that were capable of acting as hepcidin agonists and tested their effects both in vitro and in vivo.
Reporting their findings in The Journal of Clinical Investigation, Elizabeta Nemeth, M.D., and colleagues, say parenteral and oral administration of the resulting mini-hepcidins both led to significant reductions in serum iron levels in mice, and when administered chronically, led to lower liver iron concentrations. The researchers describe their work in a paper titled “Minihepcidins are rationally designed small peptides that mimic hepcidin activity in mice and may be useful for the treatment of iron overload.”
Systemic iron regulation in vertebrates is mediated by the peptide hormone hepcidin, which inhibits dietary iron absorption and the release of recycled iron from macrophages. The hormone acts by inducing the endocytosis of its receptor ferroportin, which is the only known cellular exporter of iron, the researchers explain.
Hepcidin deficiency is a cause of or contributor to iron overload in a range of diseases inculding hereditary hemochromatosis, beta-thalassemia, and chronic hepatitis. Current treatments including phlebotomy and iron chelation, however, aren’t suitable for all patients and can lead to side effects.
While replacement hepcidin therapy would be the most effective approach to treating iron overload in relevant disorders, the 25-amino acid long protein has four disulphide bonds, which means it is prohibitively expensive to produce. The protein also has a very short half-life, which would necessitate frequent therapy, and is too large to make it efficiently available orally.
In the search for alternatives to full-length hepcidin, the UCLA team carried out a structure-function analysis of the hepcidin-ferroportin interface. The starting point was an extracellular ferroportin residue, C326, which the investigators had previously shown was essential for hepcidin binding, and is contained within an extracellular loop.
The team’s approach was to carry out site-directed mutagenesis first to identify the exact extent of the loop and then to identify which residues within it were crucial for hepcidin-ferroportin interaction. The results indicated that the extraceullar portion of this likely extended from amino acids G323 to S343. Further mutagenesis experiments within the loop portion identified two residues, F324 and Y333, as the sites that most likely make direct contact with hepcidin during ferroportin binding.
Their previous research had shown that deletion of the 5 N-terminal residues of hepcidin stops the protein from triggering ferroportin degradation and that H3, F4, and I6 were important for the peptide activity and required hydrophobic side chains for interaction with the ferroportin molecule. To see whether other N-terminal residues were also necessary, the team mutagenized the next large hydrophobic residue, F9. Substituting F9 with either alanine or cyclohexylalanine (a nonarmoatic hydrophobic residue), resulted in 100-fold and 10-fold reductions in activity, respectively, “suggesting that position 9 requires an aromatic side chain.”
To build a picture of the importance of disulfide bond formation during hepcidin-ferroportin interaction, the team carried out mutagenesis to explore the effects on peptide activity of preventing disulfide bond formation between the four disulfide bond-forming pairs of cysteines, based on the most recent data on the hepcidin structure. They found that substituting the native interacting pairs of cysteines with alanines in each case decreased activity by up to 100 fold, although none of the mutants displayed a complete loss of activity.
Their next stage was to carry out computer modeling to visualize hepcidin-ferroportin interaction. The input structures were the full-length hepcidin molecule (including the the most recent disulfide bond assignment), and a region of ferroportin (residues 306-362) that encompassed the C326 extracellular loop and flanking transmembrane helices. RosettaDock refinement generated 1,000 structures, but the 10 best scoring low-energy structures all had the N terminus of hepcidin forming the interface with ferroportin.
Of the 10 structures, the conformation most compatible with the mutagenesis data (actually second-lowest energy structure) was used as the final hypothetical model of the ferroportin-hepcidin complex. In this model, H3, F4, and I6 of hepcidin formed a hydrophobic pocket for the interaction with Y333 of ferroportin, and F9 of hepcidin interacted with F324 of ferroportin through pi-stacking, These structural points all agreed with the team’s mutagenesis data. The model also positioned the C326 residue in close proximity to the hepcidin disulfide framework.
The mutagenesis and RosettaDock modeling results both thus indicated that the N terminus of the hepcidin peptide, including H3, F4, I6, and F9, is involved in binding to ferroportin. To see whether they could generate a short peptide that mimicked this activity, the team synthesized mini-hepcidin peptides consisting of up to 9 N-terminal amino acids of hepcidin and tested the ability of each to cause ferroportin-GFP degradation in cellular bioassay.
These first-generation mini-hepcidins showed significant agonist activity, although not as strongly as the full length hepcidin molecule. Of the peptides generated, the 9 amino acid variant, hep9, demonstrated the greatest activity.
Additional modificationsto peptide structures were then carried out to address some of the undesirable physicochemical properties of the mini-hepcidin scaffold such as thiol instability, hydrophobicity, poor gastrointestinal absorption, susceptibility to proteolysis, and instability in the bloodstream. Modifications included generating circular peptides to increase stability, introducing unnatural amino acids to increase resistance to proteolysis in vivo, and creating pegylated variants.
A number of the resulting constructs that were most active in in vitro assays were then tested in mice (human hepcidin is active in the mouse presumably because the human and murine sequences for both hepcidin and ferroportin are very similar, the authors note). Selected analogs were injected intraperitoneally into mice, and serum iron levels were measured four hours after injection.
Of the tested mini-hepcidins, stabilized, retro-inverso hep9 showed significant activity, and minihepcidins that were conjugated to fatty or bile acids were even more active. In fact, the team notes, a palmitoyl-ri-hep9 construct led to almost as large a decrease in serum iron levels as the equivalent dose of a 25 amino acid hep25 peptide. Even a 7–amino acid retro-inverso peptide (ri-hep3-9) was bioactive after parenteral injection and led to a 70% decrease in serum iron when administered at very high concentrations.
Encouragingly, oral administration of high concentrations of retro-inverso hep9 that was palmitoylated or conjugated to bile acids also caused a significant decrease in serum iron. And in a proof-of-principle experiment, the researchers showed that chronic administration of mini-hepcidins significantly decreased iron loading in a mouse model of hereditary hemochromatosis.
“Hepcidin-1 knockout mice, which received intraperitoneal injections of a retro-inverso mini-hepcidin daily for two weeks, had significantly lower liver iron content that hepcidin-1 knockout mice injected with solvent,” they write. “Mini-hepcidins may be useful for the treatment of iron overload disorders. If hepcidin therapy proves to be effective and relatively free of side effects, it could represent a major improvement over existing therapies, either alone or in combination with current approaches, to allow modifications that would make the treatment less burdensome and better accepted by patients."