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，当病毒可能感染神经元细胞时，患者向医生报告后，有效治疗患者的能力。不幸的是，神经元通常不是神经再生的，因此治疗方式应该集中在防止神经元死亡，以及限制病毒复制的化合物，可以有效地穿透血脑屏障。该项目将制定一项战略来实现这一目标。该项目的R21阶段的目的是确定增加的钙缓冲蛋白是否可以明确地保护培养物和动物中的神经元细胞免受西尼罗河病毒（WNV）诱导的死亡，并确定特定细胞代谢物在激活神经元细胞中的钙缓冲蛋白方面的效用。R33阶段的目的是基于细胞培养功效和血脑屏障渗透性来鉴定先导代谢物，以确定先导化合物在啮齿动物中的活性，并研究日本脑炎病毒（JEV）的这种治疗方法。具体目标是基于已发表的结果，即不同细胞系中的WNV感染导致Ca++内流，这有利于复制，但诱导半胱天冬酶3切割并增加细胞死亡的风险。相反，Ca++内流抑制剂使病毒滴度降低>2 log10，并降低半胱天冬酶3裂解。在WN神经系统疾病的创新仓鼠模型中，钙结合蛋白D28k的诱导与神经元存活相关。相反，钙结合蛋白D28k的低基础水平与增加的TUNEL染色和细胞损伤或死亡相关。此外，待研究的特定代谢物被充分认识到与受体共易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物和动物中的神经元细胞免于WNV诱导的死亡的假设。R21 SA #2。建立神经保护细胞培养试验，用于评价维生素D3代谢产物诱导钙缓冲蛋白，并监测WNV感染的神经元细胞的存活。R33 SA #1。使用神经保护细胞培养试验和跨内皮渗透性试验鉴定维生素D3代谢物的主要候选物。R33 SA #2。获得临床前数据，支持在不同给药途径和治疗方案的动物中使用先导候选药物治疗西尼罗河病毒脑炎。R33 SA #3。确定对JEV的广谱治疗活性。由于在神经病毒学、抗病毒研究、维生素D代谢和钙信号传导方面的专业知识，我们在发现病毒性脑炎的治疗方法方面处于独特的地位。本研究为研究西尼罗河病毒和乙脑病毒的神经发病机制提供了新的基础研究途径，也为西尼罗河病毒、乙脑病毒和病毒性脑炎的临床治疗提供了新的思路。

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.