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

描述（由申请人提供）：我们的长期目标是双重的：1）使用新的化学工具集来研究关键的宿主-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集中的靶标，这些靶标可以作为未来研究的基础。 化学筛选，以确定候选药物，以及注释对这些靶标的化学抑制敏感的所有可用病原体基因组。磷酸肌醇（PI）4-激酶活性对于产生特异性磷酸肌醇如PI-4，5-二磷酸（PI（4，5）P2或“PIP 2”）是必需的。我们在丙型肝炎病毒（HCV）NS 5A蛋白中发现了一个新的PIP 2结合基序。该基序称为碱性氨基酸PIP 2钳（或“BAAPP结构域”），介导与PIP 2的特异性相互作用，并且BAAPP结构域中的点突变消除PIP 2结合和HCV RNA复制。在鼻病毒和恶性疟原虫等多种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异性PI 4-激酶（PI 4K）来抑制PIP 2产生而没有宿主毒性。 家族成员--PI 4 KIII α（PI 4 KIII <$）和β（PI 4 KIII <$）亚型--在体外有效抑制HCV复制（例如抑制剂PT 423表现出IC 50 365 nM; CC 50> 10 μ M），并且对迄今为止测试的几种含有BAAPP结构域的病原体表现出相当的活性。我们现在力求：1）通过以下方式进一步探测和利用PI 4 KIII在HCV生命周期中的作用：a）使用生物化学分级分离和膜相关复制酶测定来确定PT 423处理对HCV复制复合物完整性和酶活性的影响; B）评估PI 4 KIII抑制剂与靶向复制酶复合物组装和功能的其它类别的抗HCV剂协同作用的潜力; c）通过确定对各种HCV抗病毒药具有抗性的HCV变体对PT 423的易感性，提供PI 4KIII抑制剂可以抑制多药耐药病毒的概念验证; a）在存在或不存在HCV的情况下，用与质谱法偶联的PT 423连接的珠进行细胞下拉测定，以鉴定候选脱靶蛋白质以及该谱在面对病毒感染时可能如何变化; B）通过研究在针对PI 4 KIII的siRNA或具有赋予对PT 423的抗性的工程化候选突变的PI 4 KIII突变体存在下HCV抑制所需的PT 423水平，进一步验证我们的模型PI 4 KIII抑制剂的作用机制; 3）通过以下方式确定定义对PI 4KIII的化学抑制剂的敏感性的关键BAAPP结构域特征：a）确定具有和不具有PIP 2的全长NS 5A的晶体结构; B）使用石英晶体微天平、荧光偏振和肽的核磁共振结构进行BAAPP结构域肽的结构/功能分析以确定其PIP 2结合的关键决定因素; c）开发算法以在所有可用的病原体基因组的数据库中自动鉴定PIP 2结合BAAPP结构域的上述确定的关键特征; d）提供鉴定的病原体对PI 4KIII抑制剂的抑制敏感的概念验证。

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.