Improved iron chelators are required with the lifelong transfusion treatment of the genetic anemias, Sickle Cell Disease (SCD) and the Thalassemias (THL), because the regular blood transfusions, lead to accumulations of toxic, sometime fatal amounts of iron that must be removed. Transfusion treatment is also cost effective when compared to the many hospitalizations required with untreated cases. As access to medical treatment worldwide increases, so does the need for effective chelators. In SCD and THL, the extra iron derives from aged red cells, used in the transfusions, that cannot be effectively eliminated from the body, a contrast with other required metals and vitamins. The protection against malaria conferred by SCD and THL mutations increases the numbers of carriers. Combined with the small impact, currently, of prevention (genetic counseling) there are a large number of affected births worldwide. A WHO bulletin by Bernadette Modell, 2008 shows an estimated 330,000 affected births/year. The frequency in California, where newborn screening is in place, in a 2008 report, is 1/6600 (SCD) and 1/9000 1-THL. With transfusion treatment and daily chelation therapies, life expectancies increase to adulthood and beyond. Current chelator treatments use isolated chelators of various structures, that require either long intravenous exposure or, with newer oral chelators, daily consumption of large volumes of liquid. The chelators bind the small fraction of body iron that is in transit, in the """"""""labile iron pool"""""""", a heterogeneous mixture poorly defined at the molecular level. By contrast, the extra iron is mineralized and protected in the well-characterized protein nanocage, ferritin, or, after expansion of the ferritin mineralization capacity is exhausted, in hemosiderin, an insoluble material of damaged ferritin and iron mineral. Ferritin is a protein nanocage that synthesizes ferric oxide minerals from Fe (II) substrate, inside the cage. The iron oxide mineral is a cellular iron concentrate for protein synthesis and a scavenger of iron and oxidant during oxidant stress. Targeting iron chelators to ferritin itself should enhance iron removal during iron overload. This SBIR proposal seeks support for 'proof of principle"""""""" experiments that chelators, conjugated to ferritin binding peptides, will increase iron removal from cultured cells. The experiments rest on research supported by the NIH through the RO1 mechanism, the derived invention patented in 2007, and a contract between Ferrokin Biosciences (Rienhoff and Theil) and CHORI (Theil), a research institute owned by Children's Hospital and Research Center, Oakland. Rates of dissolving and chelating iron from ferritin iron mineral increase when the gated pores in the ferritin protein nanocage unfold, based on earlier experiments. The current model posits that closed pores in ferritin prevent reductant (in solution or the cytoplasm) from reacting with the ferritin mineral. Moreover, amino acid substitution, mild heat, physiological concentrations of urea (1 mM) all unfold the ferritin pores to increase iron mineral reduction/dissolution and chelation in solution;cultured cells containing ferritin protein with pores unfolded by amino acid substitution also released more iron to chelators in the medium. In addition, a hydrophobic peptide was recently identified in a group of five binding peptides isolated from a combinatorial (phage display) peptide library of 109, which increased chelation of iron from ferritin in solution. When the chelator, desferrioxamine B (DFO), was linked to the peptide, chelation of ferritin iron increased significantly over mixing the chelator with the peptide, and was eight times higher than with chelator alone. Whether the peptide-DFO complex will increase iron removal from cultured cells, animal models or humans remains to be determined. The strategy of targeting iron chelators to ferritin with a ferritin binding peptide is suitable for any iron chelator developed. We propose to determine safety and efficacy of iron removal by ferritin-targeted chelators in cultured human cells by: 1. Determining the toxicity and uptake of ferritin targeted peptides in cultured mammalian cells, using peptide linked to DFO or to a new oral chelator derived from deferrithiocin;if needed to enhance cell uptake, nona-arginine -linked peptide will be used. 2. Comparing the removal of 59Fe from cultured mammalian cells for chelator, chelator + peptide and chelator-peptide conjugates. We predict that the solution studies will be recapitulated in cultured cells, based on the similarity of effects of manipulating ferritin protein pores in solution and in cultured cells. The results will lay the foundation for iron chelation studies in animal models, wild type or SCD or THL, and later for clinical trials to improve the treatment of Sickle Cell Disease and Thalassemia and possibly for neurodegenerative diseases currently being considered for iron chelation therapy.
Current iron chelation therapy such as deferoxamine (DFO) relies on chelators inspired by bacterial siderophores intended to retrieve biologically available iron. As such, these compounds are not targeted to mammalian systems of iron transport or storage such as ferritin which might increase their potency or clinical efficiency. We have discovered a series of peptides that in vitro enhance the egress of stored iron from ferritin and that when covalently linked to deferoxamine further increase concentrations of chelatable iron. These conjugates have promise as new agents for the treatment of iron overload. This project aims to characterize such conjugates in cultured cells using specific peptide linked to DFO and to a new iron chealtor, FBS0701 measuring their uptake in cultured cells and their ability to effect the removal of 59Fe stored within cells.
