Enteric Virus Calcium Channel Inhibitors

描述（由申请人提供）：病毒孔蛋白是一种不同类型的小疏水蛋白，它们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离子，它是一种无处不在的次级信使，参与许多信号传导途径。轮状病毒（RV）是儿童病毒性胃肠炎的主要病因，可导致腹泻、呕吐和危及生命的脱水。轮状病毒非结构蛋白4 （NSP4）引发细胞质钙的急剧升高，是病毒感染肠细胞被破坏的机制之一。最近的数据表明，NSP4病毒孔蛋白活性释放内质网（ER）钙储存，并引起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病毒蛋白活性可能是RV复制策略的一个关键方面，也是抑制剂的一个有吸引力的靶点。本研究旨在定义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，并评估假定的NSP4病毒孔蛋白抑制剂减少RV复制和发病的能力。目的1将使用脂质双分子层和膜片钳电生理实验来定义人工和天然内质网膜中NSP4离子通道的活性。假定的NSP4病毒孔蛋白活性抑制剂也将测试NSP4离子通道抑制。目的2旨在证明NSP4病毒孔蛋白活性和病毒孔蛋白介导的细胞质钙升高对RV复制复合体（称为病毒质）组装的重要性。将测试NSP4离子通道抑制剂的阻断能力

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.