Targeting induction of calcium buffer proteins for treatment of viral encephaliti

描述(由申请人提供)：治疗病毒性脑炎的一个关键障碍是有效治疗患者的能力，当他们向医生提出病毒可能感染神经细胞的时候。不幸的是，神经元通常不是神经再生的，因此治疗方法应该专注于防止神经元的死亡，以及通过能够有效穿透血脑屏障的化合物来限制病毒复制。该项目将制定一项实现这一目标的战略。该项目R21阶段的目的是确定增加的钙缓冲蛋白是否可以确切地保护培养和动物的神经细胞免受西尼罗河病毒(West Nile Virus，WNV)诱导的死亡，并建立特定细胞代谢产物激活神经元细胞中钙缓冲蛋白的效用。R33阶段的目的是根据细胞培养效率和血脑屏障通透性鉴定铅代谢产物，确定铅化合物在啮齿动物中的活性，并探讨这种治疗乙型脑炎病毒(JEV)的方法。具体的目的是基于已发表的结果，即WNV在不同的细胞系中感染导致钙内流，这有利于复制，但诱导caspase 3裂解，增加细胞死亡的风险。相反，钙内流的抑制剂使病毒滴度降低2log10，并减少caspase3的裂解。在WN神经系统疾病的创新仓鼠模型中，Calbindin D28k的诱导与神经元存活相关。相反，低基础水平的Calbindin D28k与TUNEL染色增加和细胞损伤或死亡相关。此外，被研究的特定代谢物被很好地识别为与受体共转位到细胞核以激活钙缓冲蛋白的产生。具体目标是：R21 SA#1.测试钙缓冲蛋白可以保护培养的神经细胞和动物免受西尼罗河病毒诱导的死亡的假设。R21 SA#2。建立一种神经保护细胞培养试验，用于评估维生素D3代谢产物对钙缓冲蛋白的诱导，并用于监测西尼罗河病毒感染的神经细胞的存活。R33 SA#1。使用神经保护细胞培养试验和跨内皮通透性试验鉴定维生素D3代谢物的主要候选者。R33 SA#2.获得临床前数据，支持在不同给药途径和治疗计划的动物中使用主要候选药物治疗西尼罗河病毒脑炎。R33 SA#3.测定乙脑病毒的广谱治疗活性。由于在神经病毒学、抗病毒研究、维生素D代谢和钙信号方面的专业知识，我们在发现病毒性脑炎的治疗方法方面处于独特的地位。本项目可为WNV和JEV神经发病机制的基础研究提供新的途径，并加强临床实践对WNV、JEV和病毒性脑炎的治疗。 公共卫生相关性：R21阶段的目的是确定增加的钙缓冲蛋白是否可以确切地保护培养和动物的神经细胞免受西尼罗河病毒(WNV)诱导的死亡，并确定特定代谢物激活神经元细胞中钙缓冲蛋白的效用。R33阶段的目的是根据疗效和血脑屏障通透性确定铅代谢物，建立啮齿动物体内铅化合物对西尼罗河病毒(WNV)的活性，并探讨这种治疗日本脑炎病毒(JEV)的方法。为了验证钙缓冲蛋白可以保护培养的和动物的神经细胞免受西尼罗河病毒诱导的死亡的假设，我们将在仓鼠模型中将神经元存活与Calbindin D28k的诱导相关联。我们还将测试表达载体中钙结合蛋白水平的增加是否会增加 西尼罗河病毒感染的细胞和啮齿动物的存活。我们将建立一种神经保护细胞培养试验，用于评估诱导钙缓冲蛋白的选择性代谢物，并监测感染西尼罗河病毒的神经细胞的存活。候选者还将使用血脑屏障血管内皮细胞通透性试验进行筛选。这些分析对于选择最有效的代谢物是必要的。该先导测试剂将在仓鼠和基因敲除转基因小鼠中进行评估。我们将在动物模型中获得更合适的非肠道或口服治疗方案的临床前数据。如果有证据表明这种铅代谢物具有动物功效，那么饮食不足就有可能与严重的神经系统疾病相关。因此，将在仓鼠的饮食中进行缺乏、正常和超标代谢物的研究。此外，还将对100名西尼罗河病毒患者的血清样本进行代谢物检测。鉴于JEV与WNV的相似之处，以及JEV在RFA说明书中列出，JEV的治疗钙结合靶点也将被研究。由于在神经病毒学、抗病毒研究、维生素D代谢和钙信号方面的专业知识，我们在发现病毒性脑炎的治疗方法方面处于独特的地位。本项目可为WNV和JEV神经发病机制的基础研究提供新的途径，并加强临床实践对WNV、JEV和病毒性脑炎的治疗。

DESCRIPTION (provided by applicant): A critical barrier for the treatment of viral encephalitis is the ability to effectively treat patients after they present to their physicians when the virus as likely infected neuronal cells. Unfortunately, neurons are typically not neuro-regenerative, so therapeutic modalities should focus on preventing the death of neurons, as well as to limit virus replication with compounds that can effectively penetrate the blood brain barrier. This project will develop a strategy to accomplish this. The purpose of the R21 phase of the project is to determine if increased calcium buffer proteins can definitively protect neuronal cells in culture and animals from West Nile virus (WNV)-induced death, and to establish the utility of specific cellular metabolites to activate calcium buffer proteins in neuronal cells. The purpose of the R33 phase is to identify the lead metabolite based on cell culture-efficacy and blood brain barrier permeability, to establish activity of the lead compound in rodents, and to investigate this therapeutic approach with Japanese encephalitis virus (JEV). The specific aims are based on published results that WNV infection in different cell lines leads to Ca++ influx, which benefits is replication, but induces caspase 3 cleavage and increase the risk of cell death. Conversely, inhibitors of Ca++ influx decreases viral titer by >2 log10, and decreased caspase 3 cleavage. In an innovative hamster model of WN neurological disease, induction of calbindin D28k correlates with neuronal survival. Conversely, low basal levels of calbindin D28k correlates with increased TUNEL staining, and cell injury or death. Moreover, the specific metabolites to be investigated are well recognized to co-translocate to the nucleus with the receptor to activate production of calcium buffer proteins. The Specific Aims are: R21 SA #1. Test the hypothesis that calcium buffer proteins can protect neuronal cells in culture and animals from WNV-induced death. R21 SA #2. Establish a neuro- protection cell culture assay for evaluation of vitamin D3 metabolites for induction of calcium buffer proteins, and for monitoring the survival of WNV-infected neuronal cells. R33 SA #1. Identify lead candidate of vitamin D3 metabolites using the neuro-protective cell culture assay and a transendothelial permeability assay. R33 SA #2. Obtain preclinical data supporting the treatment of WNV encephalitis with lead candidate in animals with different routes of administration and treatment schedules. R33 SA #3. Determine the broad-spectrum therapeutic activity in JEV. Because of expertise in neurovirology, antiviral research, vitamin D metabolism, and calcium signaling, we are uniquely positioned to discover therapy for viral encephalitis. This project could provide new avenues of basic research for WNV and JEV neuro- pathogenesis, and enhance clinical practice for the management of WNV, JEV, and viral encephalitis in general. PUBLIC HEALTH RELEVANCE: The purpose of the R21 phase is to determine if increased calcium buffer proteins can definitively protect neuronal cells in culture and animals from West Nile virus (WNV)- induced death and to determine the utility of specific metabolites to activate calcium buffer proteins in neuronal cells. The purpose of the R33 phase is to identify the lead metabolite based on efficacy and blood brain barrier permeability, to establish activity of the lead compound in rodents for West Nile virus (WNV), and to investigate this therapeutic approach with Japanese encephalitis virus (JEV). To test the hypothesis that calcium buffer proteins can protect neuronal cells in culture and animals from WNV- induced death, we will correlation of neuronal survival with induction of calbindin D28k in the hamster model. We will also test if increased levels of calcium binding proteins from expression vectors will increase the survival of WNV-infected cells and rodents. We will establish a neuro-protection cell culture assay for evaluation of selective metabolites for induction of calcium buffer proteins, and for monitoring the survival of WNV-infected neuronal cells. Candidates will also be screened using a BBB transendothelial permeability assay. These assays will be necessary to select the most efficacious metabolite. The lead test agent will be evaluated in hamsters and in knockout transgenic mice. We will obtain preclinical data on more appropriate parenteral or oral treatment regimens in animal models. If there is evidence for animal efficacy of the lead metabolite, there is a possibility that dietary deficiency could correlate with severe neurological disease. Therefore, studies with deficient, normal and super levels of the metabolite in the diet of hamsters will be conducted. Also, serum samples from a cohort of 100 WNV patients will be assayed for the surrogate metabolite. In light of similarities of JEV with WNV, and that JEV is listed in the RFA instructions, the therapeutic calcium binding target will also be investigated fo JEV. Because of expertise in neurovirology, antiviral research, vitamin D metabolism, and calcium signaling, we are uniquely positioned to discover a therapy for viral encephalitis. This project could provide new avenues of basic research for WNV and JEV neuro-pathogenesis, and enhance clinical practice for the management of WNV, JEV, and viral encephalitis in general.