NOX1 and NOX2 as Therapeutic Targets in Influenza

说明(申请人提供)：流感病毒表现出很高的变异率和重组率，很快就会使免疫无效，需要每年生产疫苗。此外，高达85%的分离物对针对病毒本身的现有抗病毒分子具有抗药性。这些因素表明，针对宿主编码功能的药物的医疗需求尚未得到满足，因此不受病毒选择的影响。此外，高致命性流感病毒株(如1918年流感、禽流感)偶尔会出现，这些病毒往往会引发“细胞因子风暴”，导致肺细胞功能障碍和损害(急性呼吸窘迫综合征或ARDS)，从而导致发病率/死亡率。这些致病改变包括肺泡液转运中断、肺泡上皮细胞凋亡、中性粒细胞和单核细胞对肺组织的渗透/破坏。目前，尚不存在预防肺损伤的有效治疗方法。类似的变化也发生在其他病原体上，包括SARS冠状病毒和炭疽病，在这些地方，针对流感开发的宿主导向疗法预计也将有效。NOx酶是一种NADPH-氧化酶，能产生超氧化物和次生活性氧簇(ROS)，作为信号分子，在高浓度时直接损害生物分子。我们提出了一个涉及上皮细胞NOX1和单核细胞/PMN NOX2产生的ROS的信号级联反应，作为关键步骤：a)促进病毒复制和/或传播，b)介导肺损伤。Nox1产生的上皮ROS是触发细胞因子风暴和细胞功能变化的最早事件之一。利用NOX基因敲除小鼠和抑制剂，初步证据表明，抑制NOX1和NOX2将在治疗流感感染方面受益。这项申请代表了Emory和疾病控制中心(CDC)之间的合作努力，在那里可以进行使用高毒力流感(PR8，H5N1禽流感)毒株的研究。兰贝斯实验室以其在氮氧化物发现、酶学和细胞生物学方面的专业知识而闻名，它已经发现(使用高通量和低通量筛选)四种化学系列的小分子氮氧化物抑制剂。在与埃默里药物发现研究所的合作下，我们的团队将进一步开发这些抑制剂，提高它们的效力、异构体选择性、代谢稳定性和药理学特性，并将作为候选药物协调临床前开发。Gangappa实验室是疾控中心流感小组的一部分，将：1)将活病毒感染到基因缺失的小鼠中，以证明NOX1和/或NOX2作为治疗目标的概念证明；以及2)在感染和未感染的WT小鼠中测试候选药物。总体目标是开发针对NOx产生的ROS的新型临床前候选药物，从而阻止导致肺组织损伤和病毒复制/传播的宿主信号通路。这些化合物一般用于治疗所有流感病毒株和可能导致严重肺功能障碍/病理的其他病原体(SARS冠状病毒、炭疽病)。 公共卫生相关性：高致命性类型的流感和其他病毒(如SARS冠状病毒)偶尔出现(禽流感，1918年流感)，缺乏有效的疫苗可导致大流行 造成大量人员死亡的事故。这些病毒是致命的，因为它们倾向于引起“细胞因子风暴”，从而对肺部造成损害，并改变肺部防止积液的方式。这项拨款申请的目标是开发和在动物模型中测试针对肺细胞中的酶的化合物，称为NADPH-氧化酶(NOx酶)，这种化合物可以进一步开发成抑制病毒复制和治疗或预防大流行流感中发生的肺损伤的药物。

Description (as provided by the applicant): Influenza virus shows high rates of mutation and recombination that soon renders immunization ineffective and requires yearly production of vaccines. In addition, up to 85% of isolates are resistant to available antiviral molecules targete against the virus itself. These factors demonstrate an unmet medical need for drugs that target host-encoded functions and that are therefore not subject to viral selection. Furthermore, highly lethal strains of influenza (e.g. 1918 flu, bird flu) occasionally arise, causing morbidity/mortaliy through the propensity of these viruses to induce a "cytokine storm" that mediates lung cell dysfunction and damage (acute respiratory distress syndrome or ARDS). Such pathogenic changes include disruptions in alveolar fluid transport, apoptosis of alveolar epithelial cells, an infiltration/destruction of lung tissue by neutrophils and monocytes. Currently, effective treatments to prevent lung damage do not exist. Similar changes occur with other pathogens including SARS-Coronavirus and anthrax, where host-directed therapies developed for influenza are also expected to be effective. Nox enzymes are NADPH-oxidases that generate superoxide and secondary reactive oxygen species (ROS) that act as signaling molecules, and in high concentrations directly damage biomolecules. We propose a signaling cascade involving both epithelial NOX1- and monocyte/PMN Nox2-generated ROS as key steps that: a) facilitate viral replication and/or spread and b) mediate lung damage. The generation of epithelial ROS by Nox1 is among the earliest events that trigger the cytokine storm and cellular functional changes. Using Nox knockout mice and inhibitors, preliminary evidence suggests that inhibition of Nox1 and Nox2 will be therapeutically beneficial in influenza infection. This application represents a collaborative effort between Emory and the Centers for Disease Control (CDC), where studies using highly virulent strains of influenza (PR8, H5N1 bird flu) can be carried out. The Lambeth Laboratory, known for its expertise in Nox discovery, enzymology and cell biology, has discovered (using high- and low-throughput screens) four chemical series of small molecule Nox inhibitors. In collaboration with the Emory Institute for Drug Discovery, our group will furthe develop these inhibitors, improving their potency, isoform selectivity, metabolic stability, and pharmacological properties and will coordinate preclinical development as candidate drugs. The Gangappa Laboratory, part of the Influenza group at the CDC will: 1) infect live virus into genetically deleted mice to demonstrate proof-of-concept for Nox1 and/or Nox2 as therapeutic targets, and 2) test drug candidates in infected and non-infected WT mice. The overall goal is to develop novel pre-clinical drug candidates that target Nox-generated ROS, thereby blocking host signaling pathways that lead to lung tissue damage and viral replication/spread. Such compounds will be generally useful in the treatment of all strains of influenza and likely other pathogens (SARS-CoV, anthrax) that result in severe lung dysfunction/pathology. Public Health Relevance: Highly lethal types of flu and other viruses (e.g. SARS-coronavirus) arise occasionally (bird flu, 1918 flu), and the lack of effective vaccines can result in pandemics that cause large numbers of fatalities. These viruses are lethal because of their tendency to cause a "cytokine storm" resulting in damage to the lung as well as changes in the way the lungs keep fluids from accumulating. The goal of this grant application is to develop and test in animal models compounds that target enzymes in lung cells called NADPH-oxidases (Nox enzymes) and that can be further developed into drugs that inhibit viral replication and treat or prevent the lung damage that occurs in pandemic influenza.