Development of a high-throughput screen for inhibitors of water permeability thro

描述(申请人提供)：脑水肿(和脊髓中的相应部分)是中风、创伤性脑损伤、创伤性脊髓损伤和细菌性脑膜炎造成损害的主要原因。在美国，仅中风一项就是第三大致死原因和第一大致残原因。不幸的是，只有为数不多的几种治疗方案可用于治疗脑和脊髓水肿，而且疗效有限。脑和脊髓水肿的中心是星形细胞末端的H2O通道水通道蛋白-4(AQP4)，它包裹着血脑屏障(BBB)处的CNS血管。AQP4在缺血、创伤性脑损伤和其他一系列与炎症相关的中枢神经系统病理中表达上调。但是，矛盾的是，在缺血性中风、视网膜缺血和细菌性脑膜炎的实验模型中，AQP4缺失小鼠的表现明显好于它们的野生型小鼠，这使AQP4成为一个有吸引力的药物靶点。本提案的一个独特方面是认识到AQP是双功能蛋白质，不仅对水渗透，而且对溶解的气体O2和CO2也是如此。事实上，在上述病理中，AQP4的上调可能具有使血脑屏障最大限度地提高O2通透性的有益效果，同时也有增加渗透水通透性和易于发生脑水肿的副作用。Aerology的长期目标是开发一种药物，通过阻断AQP4的透水性，保持气体通透性不变，治疗脑水肿。这项SBIR应用的目标是：(1)开发用于AQP4透水性抑制剂的高通量筛选(HTS)，使用光学方法评估渗透诱导的细胞肿胀的后果。(2)利用建立在电生理学和光学基础上的生物物理方法，建立水和气体渗透性的对应屏。这些反筛选将识别：(A)HTS的假阳性，(B)阻止气体和H2O渗透性的化合物，以及(C)降低AQP4以外的AQP透水性的化合物。(3)使用MicroSource GenPlus 960和Maybridge 20k文库进行高通量筛选试验，并确定一些核心结构。这项拟议的工作将为第二阶段的研究做准备，HTS将大大扩展，以确定几个核心结构，这些结构将被开发为治疗脑水肿的新药的先导。脑肿胀(脑水肿)和脊髓肿胀是中风、创伤性脑损伤、创伤性脊髓损伤和细菌性脑膜炎造成损害的主要原因。不幸的是，只有为数不多的几种治疗方案可用于治疗脑和脊髓水肿，而且疗效有限。该项目的目标是开发技术，以确定治疗脑水肿的新药，同时保持氧气向大脑的输送。

DESCRIPTION (provided by applicant): Cerebral edema (and its counterpart in the spinal cord) is a major contributor to the damage caused by stroke, traumatic brain injury, traumatic spinal-cord injury, and bacterial meningitis. Stroke alone is the third leading cause of death in the USA and the leading cause of disability. Unfortunately, only a few treatment options are available for cerebral and spinal-cord edema, and these are of limited efficacy. Central in the development of cerebral and spinal-cord edema is the H2O channel aquaporin-4 (AQP4) in the astrocytic endfeet that envelope CNS blood vessels at the blood-brain barrier (BBB). AQP4 is upregulated by ischemia, traumatic brain injury, and a diverse array of other CNS pathologies associated with inflammation. But, paradoxically, AQP4-null mice do substantially better than their wild-type counterparts in experimental models of ischemic stroke, retinal ischemia, and bacterial meningitis, making AQP4 an attractive drug target. A unique aspect of the present proposal is the recognition that the AQPs are bifunctional proteins that are not only permeable to water, but also to the dissolved gases O2 and CO2. Indeed, the upregulation of AQP4 in the aforementioned pathologies may have the beneficial effect of maximizing O2 permeability across the BBB, with the side effect of increasing osmotic water permeability and predisposing to cerebral edema. The long-term goal of Aeromics is to develop a drug that treats cerebral edema by blocking the water permeability of AQP4, leaving the gas permeability intact. The goals of this SBIR application are to: (1) Develop high-throughput screens (HTSs) for inhibitors of the water permeability of AQP4, using optical methods to assess the consequences of osmotically induced cell swelling. (2) Establish counterscreens for water and gas permeabilities, using biophysical approaches founded on electrophysiology and optics. These counterscreens will identify: (a) false positives from the HTS, (b) compounds that block gas as well as H2O permeability, and (c) compounds that reduce the water permeability of AQPs other than AQP4. (3) Perform pilot high-throughput screens using the MicroSource GenPlus 960 and the Maybridge 20k libraries, and identify some core structures. The proposed work will prepare Aeromics for a Phase II study, in which the HTS would be greatly expanded to identify several core structures that would be developed into leads for new drugs to treat cerebral edema. Swelling of the brain (cerebral edema) and spinal cord contributes in a major way to the damage caused by stroke, traumatic brain injury, traumatic spinal-cord injury, and bacterial meningitis. Unfortunately, only a few treatment options are available for cerebral and spinal-cord edema, and these are of limited efficacy. The goal of this project is to develop the technology to identify new drugs for treating cerebral edema while maintaining the delivery of oxygen to the brain.