ICAM-1 RECEPTOR ANALOGUES AS ANTIRHINOVIRUS AGENTS

人类鼻病毒(HRV)的主要群体占36%至45% 在所有普通感冒中，数千万人失去了上学和工作时间， 数十亿美元的医生就诊和非处方药 补救措施、继发性细菌感染和病情恶化 呼吸道疾病。我们将使用细胞受体来治疗主要的 鼻病毒组ICAM-L研制新型嵌合ICAM-L 具有更好的抗病毒活性的类似物。此外，我们还将研究 细胞间黏附分子-L结合位点的三维结构 鼻病毒，以及中和和破坏病毒的机制 鼻病毒通过ICAM-L类似物，使药物小分子具有抗病毒作用 病毒活性可以被开发出来。我们将准备改良的ICAM-L 含有ICAM-L的Ig SF结构域1和2的片段，并具有 对鼻病毒的亲和力与ICAM-L相同，具有全部5个IgSF结构域，AS 用Biacore的表面等离子体共振光谱进行了验证。 在CHO细胞凝集素抗性突变体中表达的纯化片段 有均相的糖基化会直接结晶或之后结晶 结构域2和结构域3之间被蛋白酶切割。几种不同的 已经获得了晶体的三维结构 将与斯蒂芬·哈里森博士和贾博士合作确定- 怀旺。长期目标是获得ICAM-1的结构 与鼻病毒复合的片段和较长的ICAM-L片段。我们 将制备含有细胞间黏附分子-L免疫球蛋白的改良多价嵌合体 结构域1-5或1-2融合成铰链和/或Ig G、Ig A和 免疫球蛋白。将测试这些药物是否能抑制鼻病毒与 细胞、斑块减少和心率变异性的破坏。我们将测试 嵌合体将表现出不同的疗效的假设 不同的检测方法，效果更好的嵌合体将是 通过使用完全激活的2结构域ICAM-L片段生成，并包括 细胞间黏附分子5个结构域分析中的两个亚类(Ig G和Ig M)--L 嵌合体。我们将测量所有嵌合体的有效亲和力 心率变异性与Biacore。最后，我们将测量亲和度和 细胞间黏附分子-L在被破坏的病毒粒子和天然空衣壳上的结合位点， 并通过检测RNA的释放和破坏机制来研究 假设病毒粒子作为一个整体的分裂不是一致的， 但可以以循序渐进的方式发生，可能是五聚体对五聚体。这 假说将通过测量ICAM-1结合和破坏来检验 在BlAcore中实时，并通过测量构象特定的结合 假设不同的MAB到80S颗粒制剂的百分比 被破坏的五聚体。

The major group of human rhinovirus (HRV) are responsible for 36 to 45% of all common colds, tens of millions of lost school and work days, billions of dollars of doctor's office visits and over-the-counter remedies, secondary bacterial infections, and exacerbations of respiratory diseases. We will use the cellular receptor for the major group of rhinoviruses, ICAM-l, to develop novel chimeric ICAM-l analogues with improved anti-viral activity. Furthermore, we will study the 3-dimensional structure of the binding site on ICAM- l for rhinovirus, and the mechanism of neutralization and disruption of rhinovirus by ICAM-l analogues, so that small drug molecules with anti- viral activity can be developed. We will prepare improved ICAM-l fragments that contain IgSF domains 1 and 2 of ICAM- l and that have the same affinity for rhinovirus as ICAM-l with all 5 IgSF domains, as demonstrated by surface plasmon resonance spectroscopy with BIAcore. Purified fragments expressed in CHO cell lectin-resistant mutants that have homogenous glycosylation will be crystallized directly or after cleavage between domains 2 and 3 with proteases. Several different kinds of crystals have already been obtained and the 3-dimensional structure will be determined in collaboration with Drs. Stephen Harrison and Jia- huai Wang. Longer term goals are to obtain the structure of an ICAM- 1 fragment complexed with rhinovirus and of a longer ICAM- l fragment. We will prepare improved multivalent chimeras that contain ICAM- l IgSF domains 1-5 or 1-2 fused to hinge and/or Fc regions of IgG, IgA, and IgM. These will be tested for inhibition of binding of rhinovirus to cells, plaque reduction, and disruption of HRV. We will test the hypothesis that chimeras will demonstrate differing efficacy in different assays, and that chimeras with improved efficacy will be generated by using fully active 2 domain ICAM- l fragments and including two more subclasses (IgG and IgM) in the analysis of 5 domain ICAM- l chimeras. We will measure the effective affinity of all the chimeras for HRV with BIAcore. Finally, we will measure affinity and number of binding sites for ICAM-l on disrupted virions and natural empty capsids, and examine the mechanism of RNA release and disruption by testing the hypothesis that disruption is not concerted for the virion as a whole, but can occur in a stepwise fashion, possibly pentamer-by-pentamer. This hypothesis will be tested by measuring ICAM-1 binding and disruption in real time in BlAcore, and by measuring binding of conformation-specific mAb to 80S particle preparations that are hypothesized to differ in % of disrupted pentamers.