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，很少有治疗选择可用于CE，这些都是有限的疗效。CE发展的中心是在血脑屏障（BBB）处包裹CNS血管的星形胶质细胞终足中的H2O通道水通道蛋白-4（AQP 4）。在缺血性中风、视网膜缺血和细菌性脑膜炎的实验模型中，AQP 4缺失小鼠的表现明显优于野生型小鼠。在过去，CE的许多靶点未能产生有效的治疗方法。然而，由于AQP 4是跨越BBB的H2O摄取的关键守门人，因此它是一个有希望的新药物靶点，得到遗传学原理证明的支持。本建议的一个独特的方面是认识到水通道蛋白是双功能蛋白质，不仅对H2O，而且对溶解的气体（CO2，O2，NO）是可渗透的。尽管为这种新靶点开发第一代药物会带来风险，但有效和非侵入性治疗脑水肿的潜在益处要大得多。用爱荷华州大学神经病学主任乔治·里奇森博士的话来说，“作为一名神经学家，我真的想不出有什么药物比治疗脑水肿的药物对神经病学的影响更大。“Aeromics的长期目标是开发一种药物，通过阻断AQP 4的水渗透性来治疗脑水肿，使气体渗透性保持完整。在其第一个I期SBIR（NIH/NINDS 1 R43 NS 060199）中，Aeromics（a）开发了一种成功的高通量测定和适当的计数器筛选，（B）在21 k化合物中试筛选中测试了该测定。该初步筛选从四种不同的结构类别中鉴定出6个命中物。这些命中目前正在开发作为一个正在进行的第二阶段计划（NIH/NINDS 2 R44 NS 060199）的一部分。我们对这一新应用的目标是从中试筛选扩大到高通量筛选（HTS），其中包含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化合物通过我们的药物发现计划的下游元素过渡，最大限度地降低整体计划风险。我们将：（1）在辛辛那提大学的药物发现中心进行100 k化合物HTS，鉴定阻断AQP 4的更多新结构类别，（2）使用灵敏的基于细胞的光散射测定进行反筛选（部分由Aeromics在NIH/NINDS 1 R43 NS 060199下开发），（3）与AQP 4直接相互作用的测试命中（AQP 4蛋白脂质体收缩测定），和（4）通过细胞体积细胞术验证这些结果。最后，我们将通过一组水通道蛋白家族成员评估每个命中，以确定特异性。拟议的工作将为Aeromics准备新的II期SBIR，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申请用于治疗中风引起的脑水肿。 公共卫生相关性：中风是美国第三大死亡原因，每年导致137，000人死亡。许多中风幸存者都是残疾人，导致每年约700亿美元的医疗费用。脑肿胀（脑水肿，CE）是中风、创伤性脑损伤（TBI）和细菌性脑膜炎造成的损害的主要原因。不幸的是，只有少数治疗方案可用于CE，这些都是有限的疗效。最近的研究表明，蛋白质AQP 4是大脑摄取水分的主要途径，使其成为CE的一个有前途的新靶点。在之前的I期SBIR中，Aeromics开发了通过抑制AQP 4来确定治疗CE的候选药物的技术，并在试点项目中发现了6种新化合物。这些化合物目前正在II期SBIR中开发。随着目前的筛选技术的到位，我们准备扩大我们的药物发现计划，以完成对新候选药物的更彻底的搜索。

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.