The PIP2-virus interface and PI 4-kinase: novel biology and validation targets

描述(由申请人提供)：我们的长期目标有两个：1)使用一套新的化学工具来研究关键的宿主-病原体界面及其在病毒生命周期中的作用；2)我们还寻求验证一组可以作为未来基础的有针对性的靶点 化学筛选，以确定候选药物，以及注释所有可用病原体基因组，这些基因组对这些靶标的化学抑制敏感。磷脂酰肌醇(PI)4-激酶活性对于产生特定的磷脂酰肌醇是必不可少的，如PI-4，5-二磷酸(PI(4，5)P2，或PIP2)。我们在丙型肝炎病毒NS5A蛋白中发现了一个新的PIP2结合基序。这个基序被称为碱性氨基酸PIP2钳子(或“BAAPP结构域”)，介导与PIP2的特异性相互作用，BAAPP结构域上的点突变取消了PIP2的结合和丙型肝炎病毒的RNA复制。在鼻病毒和恶性疟原虫等不同的病原体中也发现了类似的BAAPP结构域。最后，最近开发的小分子通过靶向特定的PI 4-激酶(PI4K)来抑制PIP2的产生而不会对宿主产生毒性 家族成员--PI4KIIIα(PI4KIII？)和beta(PI4KIII？)亚型--在体外有效地抑制丙型肝炎病毒的复制(例如，抑制剂PT423的IC50为365 NM；CC50&GT；10微米)，并对几种迄今测试过的含有BAAPP结构域的病原体显示出类似的活性。我们现在寻求：1)进一步探索和开发PI4KIII在丙型肝炎病毒生命周期中的作用，方法是：a)利用生化分级和膜相关复制酶分析，确定PT423处理对丙型肝炎病毒复制复合体完整性和酶活性的影响；b)评估PI4KIII抑制剂与其他类型针对复制酶复合体组装和功能的抗丙型肝炎病毒药物的协同作用；c)通过确定对各种丙型肝炎病毒抗病毒药物耐药的丙型肝炎病毒变异体对PT423的敏感性，提供PI4KIII抑制剂可以抑制多药耐药病毒的概念的证据；2)进一步验证PI4KIII作为未来药物筛选工作的靶点：a)在存在或不存在丙型肝炎病毒的情况下，使用PT423连接的珠子与质谱仪进行细胞下拉试验，以确定候选的脱靶蛋白以及面对病毒感染时光谱可能如何改变；b)通过研究在存在针对PI4KIII的siRNA或具有导致PT423抗性的工程候选突变的PI4KIII突变体的情况下抑制丙型肝炎所需的PT423水平，进一步验证我们的模型PI4KIII抑制剂的作用机制；3)通过以下方法确定定义PI4KIII对化学抑制剂的敏感性的关键BAAPP结构域特征：a)确定全长NS5A的晶体结构，以及是否有PIP2；b)进行BAAPP结构域的结构/功能分析，以使用石英晶体微天平、荧光偏振和多肽的核磁共振结构来确定它们与PIP2结合的关键决定因素；c)开发一种算法，在所有现有病原体基因组的数据库中自动识别上述确定的与PIP2结合的BAAPP结构域的关键特征；d)提供已识别的病原体容易受到PI4KIII抑制剂抑制的概念证据。

DESCRIPTION (provided by applicant): Our long term objectives are twofold: 1) to use a novel chemical tool set to study a critical host-pathogen inter- face and its role in virus life cycles; 2) We also seek to validate a focused set of targets that can serve as the basis for future chemical screens to identify drug candidates, as well as an annotate all available pathogen genomes that are susceptible to chemical inhibition of these targets. Phosphoinositide (PI) 4-kinase activity is essential for generating specific phosphoinositides such as PI-4,5-bisphosphate (PI(4,5)P2, or "PIP2"). We discovered a novel PIP2 binding motif within the hepatitis C virus (HCV) NS5A protein. This motif, termed a Basic Amino Acid PIP2 Pincer (or "BAAPP domain"), mediates specific interaction with PIP2, and point mutations in the BAAPP domain abrogate PIP2 binding and HCV RNA replication. Similar BAAPP domains are found in pathogens as diverse as rhinovirus and P. falciparum. Finally, small molecules recently developed to inhibit PIP2 production without host toxicity by targeting specific PI 4-kinase (PI4K) family members-the PI4KIII alpha (PI4KIII¿) and beta (PI4KIII¿) isoforms--potently inhibit HCV replication in vitro (e.g. inhibitor PT423 exhibits IC50 365 nM; CC50 > 10microM), and display comparable activity against several BAAPP domain-containing pathogens tested to date. We now seek to: 1) Further probe and exploit the role of PI4KIII in the HCV life cycle by: a) determining the effect of PT423 treatment on HCV replication complex integrity and enzymatic activity using biochemical fractionation and membrane-associated replicase assays; b) evaluating the potential for inhibitors of PI4KIII to synergize with other classes of anti-HCV agents targeting replicase complex assembly and function; c) providing proof-of-concept that PI4KIII inhibitors can inhibit multidrug resistant viruses by determining the susceptibility to PT423 of HCV variants resistant to various classes of HCV antivirals; 2) Further validate PI4KIII as a target for future drug screening efforts by: a) performing cellular pull-down assays, in the presence or absence of HCV, with PT423-linked beads coupled with mass spectrometry to identify candidate off-target proteins and how that spectrum might change in the face of viral infection; b) further validating the mechanism of action of our model PI4KIII inhibitors by studying PT423 levels required for HCV inhibition in the presence of either siRNA against PI4KIII¿, or PI4KIII¿ mutants with engineered candidate mutations conferring resistance to PT423; 3) Determine the critical BAAPP domain features that define susceptibility to chemical inhibitors of PI4KIII by: a) determining the crystal structure of full length NS5A, with and withou PIP2; b) performing a structure/function analysis of BAAPP domain peptides to define their key determinants of PIP2 binding using quartz crystal microbalance, fluorescence polarization, and the nuclear magnetic resonance structures of the peptides; c) developing an algorithm to automatically identify the above determined key features of a PIP2 binding BAAPP domain within databases of all available pathogen genomes; d) providing proof-of-concept that identified pathogens are susceptible to inhibition with PI4KIII inhibitors.