Scale-Up from Pilot To High-Throughput Screen for Inhibitors of Aquaporin-4

描述（由申请人提供）：脑水肿（CE或脑肿胀）是中风、创伤性脑损伤和细菌性脑膜炎引起的损伤的主要原因。中风是美国第三大死亡原因，也是导致残疾的主要原因。不幸的是，很少有治疗方案可用于CE，这些治疗方案的疗效有限。星形细胞终足中的水通道水通道蛋白-4 （AQP4）在CE的发展中起着核心作用，该通道在血脑屏障（BBB）处包裹着中枢神经系统血管。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优于野生型小鼠。过去，CE的许多靶点都未能产生有效的治疗方法。然而，由于AQP4是血脑屏障中水摄取的关键把关人，它是一个有希望的新药物靶点，得到了遗传原理证明的支持。本提案的一个独特方面是认识到aqp是双功能蛋白，不仅可以渗透到H2O，还可以渗透到溶解气体（CO2, O2， NO）。尽管针对这种新靶点开发第一代药物存在风险，但对脑水肿进行有效且无创治疗的潜在益处要大得多。用George Richerson博士（爱荷华大学神经学主任）的话来说，“作为一名神经学家，我真的想不出一种药物能比治疗脑水肿的药物对神经学的影响更大。”Aeromics的长期目标是开发一种药物，通过阻断AQP4的水渗透性来治疗脑水肿，使气体渗透性保持不变。在其第一个I期SBIR （NIH/NINDS 1R43NS060199）中，Aeromics (a)开发了一种成功的高通量测定方法和适当的计数器筛选，(b)在21k化合物中试筛选中测试了该测定方法。这个试点屏幕从四个不同的结构类别中识别了6个命中。这些热门药物目前正在作为正在进行的II期项目（NIH/NINDS 2R44NS060199）的一部分进行开发。我们这项新应用的目标是从试点筛选扩大到高通量筛选（HTS），拥有10万种化合物的非重叠组，以探索更广泛的化学空间，从而确保足够数量的先导物通过我们药物发现项目的下游元素过渡，最大限度地降低整体项目风险。我们将：(1)在辛辛那提大学药物发现中心（University of Cincinnati’s Drug Discovery Center）进行100k化合物HTS，确定更多阻断AQP4的新结构类；(2)使用基于细胞的敏感光散射试验（部分由NIH/NINDS 1R43NS060199下的Aeromics开发）进行计数器筛选；(3)测试与AQP4直接相互作用的命中点（AQP4蛋白脂质体收缩试验）；(4)通过细胞体积细胞术验证这些结果。最后，我们将评估水通道蛋白家族成员的每一次撞击，以确定特异性。拟议的工作将使Aeromics为新的II期SBIR做准备，其中包括药物化学和药理学概念验证，并将确保新药候选物从临床前工作成功过渡到提交IND申请，以治疗中风性脑水肿。

DESCRIPTION (provided by applicant): Cerebral edema (CE, or brain swelling) is a major contributor to the damage caused by stroke, traumatic brain injury, and bacterial meningitis. Stroke alone is the third leading cause of death in the USA and the leading cause of disability. Unfortunately, few treatment options are available for CE, and these are of limited efficacy. Central in the development of CE is the H2O channel aquaporin-4 (AQP4) in the astrocytic endfeet that envelope CNS blood vessels at the blood-brain barrier (BBB). AQP4-null mice do substantially better than their wild-type counterparts in experimental models of ischemic stroke, retinal ischemia, and bacterial meningitis. In the past, many targets of CE have failed to produce effective therapeutics. However, because AQP4 is the key gatekeeper for H2O uptake across the BBB, it is a promising new drug target, supported by a genetic proof-of-principle. A unique aspect of the present proposal is the recognition that the AQPs are bifunctional proteins that are permeable not only to H2O, but also to dissolved gases (CO2, O2, NO). Although developing a 1st generation drug for such a novel target entails risk, the potential benefits of an effective and noninvasive treatment for cerebral edema are far greater. In the words of Dr. George Richerson (Chief of Neurology at the University of Iowa), "As a neurologist, I really can't think of a drug that would have more of an impact in neurology than one that treated 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 gas permeability intact. In its first Phase I SBIR (NIH/NINDS 1R43NS060199), Aeromics (a) developed a successful high-throughput assay and appropriate counter screens, and (b) tested this assay in a 21k-compound pilot screen. This pilot screen identified 6 hits from four different structural classes. These hits are currently being developed as part of an ongoing Phase II program (NIH/NINDS 2R44NS060199). Our goal for this new application is to scale-up from pilot screen to a high-throughput screen (HTS) with a non- overlapping group of 100k compounds to explore a wider chemical space, thereby ensuring that a sufficient number of leads transition through the downstream elements of our drug-discovery program, minimizing overall program risk. We will: (1) perform a 100k-compound HTS at the University of Cincinnati's Drug Discovery Center, identifying more novel structural classes that block AQP4, (2) counter screen using a sensitive cell-based light scattering assay (developed in part at Aeromics under NIH/NINDS 1R43NS060199), (3) test hits for direct interaction with AQP4 (AQP4 proteoliposomes-shrinking assay), and (4) verify these results by cell volume cytometry. Finally, we will evaluate each hit across a panel of aquaporin family members to ascertain specificity. The proposed work will prepare Aeromics for a new Phase II SBIR that will include medicinal chemistry and a pharmacological proof-of-concept, and will ensure the successful transition of new drug candidates from preclinical work to filing an IND application for treating stroke-induced cerebral edema. PUBLIC HEALTH RELEVANCE: Stroke is the third leading cause of death in the United States, resulting in 137,000 annual fatalities. Many stroke survivors are disabled, leading to a combined annual medical cost of ~$70 billion. Brain swelling (cerebral edema, CE) is a major contributor to the damage caused by stroke, traumatic brain injury (TBI), and bacterial meningitis. Unfortunately, only a few treatment options are available for CE, and these are of limited efficacy. Recent studies have shown that the protein AQP4 is the primary route for water uptake by the brain, making it a promising new target for CE. In a previous Phase I SBIR, Aeromics developed technologies to identify drug candidates for treating CE through inhibition of AQP4, and discovered 6 new compounds in a pilot program. These compounds are currently under development in a Phase II SBIR. With the current screening technologies in place, we are ready to scale-up our drug discovery program to complete a more thorough search for new drug candidates.