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、有时致命的铁积累，必须清除。与未经治疗的病例所需的许多住院治疗相比，输血治疗也具有成本效益。随着全球医疗服务的增加，对有效螯合剂的需求也在增加。在SCD和THL中，额外的铁来自老化的红细胞，用于输血，不能有效地从体内消除，与其他所需的金属和维生素形成对比。SCD和THL突变所赋予的对疟疾的保护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出生。Bernadette Modell在2008年发布的世卫组织公报显示，估计每年有330 000名新生儿受影响。在加州，新生儿筛查到位，在2008年的报告中，频率是1/6600（SCD）和1/9000 1-THL。通过输血治疗和每日螯合治疗，预期寿命增加到成年期及以后。目前的螯合剂治疗使用各种结构的分离的螯合剂，其需要长时间的静脉内暴露，或者对于较新的口服螯合剂，需要每天消耗大量的液体。螯合剂结合在“不稳定铁池”中运输的小部分体铁，所述不稳定铁池是在分子水平上定义不清楚的异质混合物。相比之下，额外的铁被矿化和保护在特征明确的蛋白质纳米笼铁蛋白中，或者在铁蛋白矿化能力膨胀耗尽后，在含铁血黄素中，含铁血黄素是一种受损铁蛋白和铁矿物的不溶性材料。铁蛋白是一种蛋白质纳米笼，在笼内从Fe（II）底物合成氧化铁矿物。氧化铁矿物质是用于蛋白质合成的细胞铁浓缩物，并且在氧化应激期间是铁和氧化剂的清除剂。将铁螯合剂靶向铁蛋白本身应在铁过载期间增强铁去除。这项SBIR提案寻求对“原理证明”实验的支持，即与铁蛋白结合肽缀合的螯合剂将增加培养细胞中的铁去除。这些实验基于NIH通过RO 1机制支持的研究，该机制是2007年获得专利的衍生发明，以及Ferrokin Biosciences（Rienhoff和Theil）与奥克兰儿童医院和研究中心拥有的研究机构CHORI（Theil）之间的合同。基于早期的实验，当铁蛋白纳米笼中的门控孔展开时，溶解和螯合铁的速率从铁蛋白铁矿物增加。目前的模型假定铁蛋白中的封闭孔阻止还原剂此外，氨基酸取代、温和加热、生理浓度的尿素（1 mM）都展开铁蛋白孔以增加铁矿物质还原/溶解和溶液中的螯合;含有具有通过氨基酸取代展开的孔的铁蛋白的培养细胞也向培养基中的螯合剂释放更多的铁。此外，最近在从109个组合（噬菌体展示）肽库分离的一组5个结合肽中鉴定了疏水肽，其增加了溶液中铁与铁蛋白的螯合。当螯合剂去铁胺B（DFO）与肽连接时，铁蛋白铁的螯合作用比螯合剂与肽混合时显著增加，并且比单独使用螯合剂高8倍。肽-DFO复合物是否会增加培养细胞、动物模型或人体的铁去除仍有待确定。用铁蛋白结合肽将铁螯合剂靶向铁蛋白的策略适用于开发的任何铁螯合剂。我们建议通过以下方式确定铁蛋白靶向螯合剂在培养的人类细胞中去除铁的安全性和有效性：1.使用与DFO或衍生自去铁硫菌素的新口服螯合剂连接的肽，测定培养的哺乳动物细胞中铁蛋白靶向肽的毒性和摄取;如果需要增强细胞摄取，将使用九精氨酸连接肽。2.比较螯合剂、螯合剂+肽和螯合剂-肽缀合物从培养的哺乳动物细胞中去除59 Fe。我们预测溶液研究将在培养细胞中重演，基于在溶液中和培养细胞中操纵铁蛋白蛋白孔的相似性。这些结果将为在野生型或SCD或THL动物模型中进行铁螯合研究奠定基础，并随后为临床试验奠定基础，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进行铁螯合治疗的神经退行性疾病。 公共卫生相关性：目前的铁螯合疗法如去铁胺（DFO）依赖于由细菌铁载体激发的螯合剂，旨在回收生物学上可利用的铁。因此，这些化合物不靶向哺乳动物的铁转运或储存系统，如铁蛋白，这可能会增加其效力或临床效率。我们已经发现了一系列肽，其在体外增强储存的铁从铁蛋白的排出，并且当共价连接到去铁胺时进一步增加可螯合铁的浓度。这些缀合物有希望作为治疗铁过载的新药剂。该项目旨在使用与DFO和新铁螯合剂FBS 0701连接的特定肽来表征培养细胞中的此类缀合物，测量其在培养细胞中的摄取及其影响细胞内储存的59 Fe的去除的能力。

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.