Enteric Virus Calcium Channel Inhibitors

肠道病毒钙通道抑制剂

• 批准号: 8663901
• 负责人: Joseph M. Hyser
• 金额: $ 12.29万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-15 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8663901
• 关键词: Acute; Address; Advisory Committees; Animals; Antigens; Antiviral Agents; Area; Attenuated; Award; Biochemical; Biological Assay; Biomedical Research; Calcium; Calcium Channel; Cell Death; Cell membrane; Cell physiology; Cells; Cessation of life; Characteristics; Childhood; Chronic; Complex; Cytoplasmic Inclusion; Data; Dehydration; Development; Diarrhea; Disease; Drug Targeting; Electrophysiology (science); Endoplasmic Reticulum; Enteral; Enterocytes; Enterovirus; Fostering; Future; Genes; Goals; HIV; Hepatitis C virus; Hepatitis C virus p7 protein; Histopathology; Homeostasis; Human; Human Papillomavirus; Immunosuppression; Intestines; Ion Channel; Ions; K-Series Research Career Programs; Life; Lipid Bilayers; Mediating; Membrane; Mentors; Methods; Molecular; Molecular Virology; Morphogenesis; Mus; Nonstructural Protein; Nuclear Envelope; Pathogenesis; Pharmaceutical Preparations; Play; Probability; Property; Proteins; RNA Interference; Research; Role; Rotavirus; Rotavirus Infections; Rotavirus disease; Scientist; Severities; Signal Pathway; Small Intestines; Techniques; Testing; Training; Translational Research; Villus; Viral; Viral Gastroenteritis; Virulence Factors; Virus; Virus Diseases; Virus Replication; Vomiting; base; career; cell injury; cytotoxicity; drug development; in vivo; inhibitor/antagonist; insight; member; mouse model; novel; novel therapeutics; patch clamp; pathogen; porin; prevent; programs; prototype; pup; research study; skills; small molecule libraries; synthetic peptide; tool; virus pathogenesis

DESCRIPTION (provided by applicant): Viroporins are a diverse class of small hydrophobic proteins that insert into intracellular or the plasma membrane and form pores to disrupt cellular ion homeostasis. Chief among the biologically relevant ions is calcium, a ubiquitous secondary messenger that is involved in many signaling pathways. Rotaviruses (RV) are the leading cause of childhood viral gastroenteritis, resulting in diarrhea, vomiting and life-threatening dehydratio. One mechanism for the destruction of RV-infected enterocytes is the dramatic elevation in cytoplasmic calcium, which is triggered by the rotavirus nonstructural protein 4 (NSP4). Recent data show that NSP4 viroporin activity releases endoplasmic reticulum (ER) calcium stores and causes a steady-state elevation in the concentration of cytoplasmic calcium. Elevated cytoplasmic calcium is also necessary for RV replication, so NSP4 viroporin activity is likely a critical aspect of the RV replication strategy and an attractive target for inhibitors. This proposl seeks to define the functional characteristics of the NSP4 viroporin, demonstrate the importance NSP4 viroporin activity has for RV replication, and assess the ability of putative NSP4 viroporin inhibitors to reduce RV replication and pathogenesis. Aim 1 will use lipid bilayer and patch clamp electrophysiology experiments to define NSP4 ion channel activity in both artificial and native ER membranes. Putative inhibitors of NSP4 viroporin activity will be also tested for NSP4 ion channel inhibition. Aim 2 seeks to demonstrate the importance of NSP4 viroporin activity and viroporin-mediated elevation of cytoplasmic calcium for the assembly of RV replication complexes, called viroplasms. NSP4 ion channel inhibitors will be tested for their ability to block the NSP4-induced elevation in cytoplasmic calcium, and block RV replication and viroplasm assembly in RV-infected cells. Aim 3 will test the putative NSP4 inhibitors in a mouse model of RV diarrhea, to determine whether blocking NSP4 viroporin activity will protect animals from diarrhea. Completion of these studies adds to our fundamental understanding of viroporin ion channel activity, and constitutes the first electrophysiology studies on a viral calcium channel. These experiments also will determine whether inhibition of the rotavirus NSP4 viroporin effectively blocks virus replication, demonstrating that NSP4 is a good antiviral drug target. The data and techniques in these studies will form the basis of my independent research program on viral calcium channels and development of viroporin inhibitors as tools for molecular virology studies and leads for antiviral drug development. My two mentors, Dr. Frank Horrigan and Dr. Timothy Palzkill, are experts in electrophysiology and drug development, respectively. The members of my Advisory Committee each have unique expertise in areas relevant to the proposed studies. The advanced training I receive through this award will foster the development of skills and new expertise necessary for an independent biomedical research career focused on viral calcium channels, particularly those of important human pathogens.

描述（由申请方提供）：病毒孔蛋白是一类不同的小疏水蛋白，插入细胞内或质膜并形成孔以破坏细胞离子稳态。生物相关离子中最主要的是钙，它是一种普遍存在的第二信使，参与许多信号通路。轮状病毒（RV）是引起儿童病毒性胃肠炎的主要原因，可导致腹泻、呕吐和危及生命的脱水。RV感染的肠上皮细胞的破坏机制之一是细胞质钙的急剧升高，这是由轮状病毒非结构蛋白4（NSP4）引发的。最近的数据表明，NSP4病毒孔蛋白活性释放内质网（ER）钙储存，并导致细胞质钙浓度的稳态升高。升高的细胞质钙也是RV复制所必需的，因此NSP4病毒孔蛋白活性可能是RV复制策略的关键方面，也是抑制剂的有吸引力的靶点。该提议试图定义NSP4病毒孔蛋白的功能特征，证明NSP4病毒孔蛋白活性对RV复制的重要性，并评估推定的NSP4病毒孔蛋白抑制剂减少RV复制和发病机制的能力。目的1将使用脂质双层和膜片钳电生理学实验来确定人工和天然ER膜中的NSP4离子通道活性。还将测试NSP4病毒孔蛋白活性的推定抑制剂的NSP4离子通道抑制。目的2试图证明NSP4病毒孔蛋白活性和病毒孔蛋白介导的细胞质钙离子升高对于RV复制复合物（称为病毒载体）组装的重要性。将测试NSP4离子通道抑制剂阻断 NSP4诱导细胞质钙升高，并阻断RV感染细胞中的RV复制和病毒质组装。目的3将在RV腹泻的小鼠模型中测试推定的NSP4抑制剂，以确定阻断NSP4病毒孔蛋白活性是否会保护动物免于腹泻。这些研究的完成增加了我们对病毒孔蛋白离子通道活性的基本理解，并构成了对病毒钙通道的第一个电生理学研究。这些实验还将确定轮状病毒NSP4病毒孔蛋白的抑制是否有效地阻断病毒复制，证明NSP4是良好的抗病毒药物靶标。这些研究中的数据和技术将构成我关于病毒钙通道的独立研究计划的基础，并开发病毒孔蛋白抑制剂作为分子病毒学研究的工具和抗病毒药物开发的先导。我的两位导师弗兰克·霍里根博士和蒂莫西·帕兹基尔博士分别是电生理学和药物开发方面的专家。我的咨询委员会成员在与拟议研究相关的领域都拥有独特的专业知识。我通过这个奖项获得的高级培训将促进技能和新的专业知识的发展，这些技能和新的专业知识对于专注于病毒钙通道的独立生物医学研究职业生涯是必要的，特别是那些重要的人类病原体。