Enhanced iron removal with peptide linked chelators targeted to ferritin

描述（由申请人提供）：遗传性贫血、镰状细胞病（SCD）和地中海贫血（THL）的终身输血治疗需要改进的铁螯合剂，因为定期输血会导致有毒的、有时是致命的铁的积累，必须清除。与未经治疗的病例需要多次住院治疗相比，输血治疗也具有成本效益。随着全球医疗服务的普及，对有效螯合剂的需求也在增加。在SCD和THL中，额外的铁来自于输血中使用的老化的红细胞，不能有效地从体内清除，这与其他所需的金属和维生素形成对比。SCD和THL突变对疟疾的保护作用增加了携带者的数量。再加上目前预防（遗传咨询）的影响很小，全世界有大量受影响的新生儿。Bernadette Modell于2008年发布的世卫组织公报显示，每年估计有33万名受影响的新生儿。根据2008年的一份报告，在进行新生儿筛查的加州，该频率为1/6600 （SCD）和1/9000 （1-THL）。通过输血治疗和每日螯合治疗，预期寿命可延长至成年期及以后。目前的螯合剂治疗使用各种结构的分离螯合剂，需要长时间静脉注射，或者使用较新的口服螯合剂，每天消耗大量液体。螯合剂在“不稳定铁池”（一种在分子水平上定义不清的非均质混合物）中结合运输中的一小部分体内铁。相比之下，额外的铁被矿化和保护在表征良好的蛋白质纳米笼中，铁蛋白，或者，在铁蛋白矿化能力耗尽后，在铁血黄素中，一种受损铁蛋白和铁矿物的不溶性物质。铁蛋白是一种蛋白质纳米笼，在笼内由铁（II）底物合成氧化铁矿物。氧化铁矿物是一种细胞铁浓缩物，用于蛋白质合成和氧化应激时的铁和氧化剂的清除剂。靶向铁螯合剂铁蛋白本身应提高铁超载期间的铁去除。这项SBIR提案寻求支持“原理证明”实验，即螯合剂与铁蛋白结合肽结合，将增加培养细胞中的铁去除。该实验基于由美国国立卫生研究院通过RO1机制支持的研究，该衍生发明于2007年获得专利，并与Ferrokin Biosciences （Rienhoff and Theil）和CHORI (Theil)（奥克兰儿童医院和研究中心所属的研究机构）签订了合同。根据早期的实验，当铁蛋白纳米笼中的门控孔展开时，铁蛋白矿物中铁的溶解和螯合速率增加。目前的模型假设，铁蛋白中封闭的孔隙阻止还原剂（在溶液或细胞质中）与铁蛋白矿物发生反应。此外，氨基酸取代、温和加热、尿素生理浓度（1 mM）均能使铁蛋白孔展开，增加溶液中铁矿物的还原/溶解和螯合作用；含有铁蛋白的培养细胞通过氨基酸取代展开气孔，也向培养基中的螯合剂释放了更多的铁。此外，最近从109个组合肽库（噬菌体展示）中分离出的5个结合肽中发现了一个疏水肽，它增加了溶液中铁蛋白与铁的螯合作用。当螯合剂去铁胺B （DFO）与肽结合时，铁蛋白铁的螯合作用在与肽混合时显著增加，是单独螯合剂的8倍。肽- dfo复合物是否会增加培养细胞、动物模型或人类的铁去除仍有待确定。利用铁蛋白结合肽将铁螯合剂靶向铁蛋白的策略适用于任何开发的铁螯合剂。我们建议通过以下方法来确定铁蛋白靶向螯合剂在培养的人细胞中去除铁的安全性和有效性：测定培养的哺乳动物细胞中铁蛋白靶向肽的毒性和摄取，使用与DFO连接的肽或与铁铁硫蛋白衍生的新型口服螯合剂连接的肽；如果需要增强细胞摄取，将使用非精氨酸连接肽。2. 比较螯合剂、螯合剂+肽和螯合剂-肽偶联物对哺乳动物细胞中59Fe的去除效果。我们预测溶液研究将在培养细胞中重述，基于在溶液和培养细胞中操纵铁蛋白孔的效果的相似性。该结果将为铁螯合在动物模型、野生型或SCD或THL中的研究奠定基础，并为随后的临床试验奠定基础，以改善镰状细胞病和地中海贫血的治疗，并可能用于目前正在考虑铁螯合治疗的神经退行性疾病。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.