NOX1 and NOX2 as Therapeutic Targets in Influenza

描述（由申请人提供）：流感病毒表现出很高的突变和重组率，很快使免疫无效，需要每年生产疫苗。此外，高达85%的分离株对针对病毒本身的现有抗病毒分子具有耐药性。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，偶尔会出现高致命性流感（如1918年流感、禽流感），通过这些病毒诱导“细胞因子风暴”的倾向，介导肺细胞功能障碍和损伤（急性呼吸窘迫综合征或ARDS），导致发病/死亡。这些致病性改变包括肺泡液运输的中断、肺泡上皮细胞的凋亡、中性粒细胞和单核细胞对肺组织的浸润/破坏。目前，预防肺损伤的有效治疗方法尚不存在。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，在这些病原体上，针对流感开发的宿主导向疗法预计也会有效。Nox酶是nadph氧化酶，产生超氧化物和次级活性氧（ROS），作为信号分子，高浓度时直接破坏生物分子。我们提出了一个涉及上皮细胞NOX1-和单核细胞/PMN nox2生成的ROS的信号级联，作为a)促进病毒复制和/或传播和b)介导肺损伤的关键步骤。上皮ROS由Nox1产生是触发细胞因子风暴和细胞功能改变的最早事件之一。使用Nox敲除小鼠和抑制剂，初步证据表明抑制Nox1和Nox2将对流感感染有益。这一申请代表了埃默里大学和疾病控制中心（CDC）之间的合作努力，CDC可以使用高毒性流感毒株（PR8， H5N1禽流感）进行研究。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，已经发现（使用高通量和低通量筛选）四个化学系列的小分子氮氧化物抑制剂。通过与埃默里药物发现研究所的合作，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，并将协调临床前开发作为候选药物。Gangappa实验室是CDC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作为治疗靶点的概念验证，2)在感染和未感染的WT小鼠中测试候选药物。总体目标是开发新的临床前候选药物，靶向nox产生的ROS，从而阻断导致肺组织损伤和病毒复制/传播的宿主信号通路。这些化合物通常可用于治疗所有流感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）。

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.