Molecular Basis for Inhibition of Edema Factor

描述(由申请人提供)：本项目的目标是寻找炭疽蛋白水肿因子(EF)的小分子抑制剂。这种毒素是由炭疽芽孢杆菌在催化失活状态下分泌的。当毒素通过炭疽来源的转运蛋白保护性抗原输送到炭疽患者的细胞质中时，它与脊椎动物关键的细胞内钙结合蛋白钙调蛋白(CAM)形成复合体。这种结合激活EF，并将其转化为腺苷环化酶，其催化活性是受害者自己的环状AMP产生酶的1000倍。传统的炭疽病疗法(针对炭疽菌，而不是炭疽毒素)非常有效，但不足以挽救所有炭疽病受害者的生命。EF抑制剂代表了一种完全不同的、完全互补的抗击炭疽病的方法。这些抑制剂将通过治疗疾病的下游影响，帮助恢复细胞平衡，并可能减缓疾病的进程。通过让常规治疗和免疫系统有更多的时间发挥作用，这些抑制剂可能会显著提高炭疽病的存活率。 我们解决了EF(非活化态)和EF/CaM络合物(活化态)的晶体结构。以这些结构为指导，并与制药研究人员密切磋商，我们将使用基于结构的计算药物发现方法来识别代表两种不同类型的EF抑制剂的小分子；类型A是封闭活性部位的抑制剂，类型B是那些保持EF处于无CAM、催化非活性状态的抑制剂。我们已经有了对A型抑制剂的分析。我们将完成对B型抑制物的简单分析的开发，然后将这些分析应用于我们的计算机药物筛选建议的大约1000种化合物。我们还将确定EF与通过该筛选程序鉴定的小分子形成的络合物的晶体结构，以便通过合理的药物设计方法来改善这些抑制剂的药学性质。

DESCRIPTION (provided by applicant): The objective of this project is to find small molecule inhibitors of the anthrax protein Edema Factor (EF). This toxin is secreted by Bacillus anthracis in a catalytically inactive state. When the toxin is transported into the cellular cytoplasm of anthrax victims by the anthrax-derived transporter, Protective Antigen, it forms a complex with calmodulin (CAM) - the key intracellular calcium-binding protein in vertebrates. This association activates EF, and converts it into an adenylyl cyclase with 1000 times greater catalytic activity than the victim's own cyclic AMP-producing enzymes. Conventional anthrax therapies (which target the anthrax bacterium, not the anthrax toxins) are highly effective, but are insufficient to save all anthrax victims. EF inhibitors represent a completely distinct, and wholly complementary approach to combating anthrax. Such inhibitors will, by treating the downstream effects of the disease, help restore cellular equilibrium, and are likely to slow the course disease. By allowing more time for conventional treatments and the immune system to work, these inhibitors may significantly increase anthrax survival rates. We have solved crystal structures of EF (the inactive state) and the EF/CaM complex (the active state). Using these structures as a guide, and in close consultation with pharmaceutical researchers, we will use structure-based, computational drug discovery methods to identify small molecules representing two distinct types of EF inhibitors; Type A, inhibitors that occlude the active site, and Type B, those that maintain EF in its CAM-free, catalytically inactive state. We already have assays for inhibitors of Type A. We will complete the development of a simple assay for inhibitors of Type B, and then apply these assays to the top approximately 1000 compounds suggested by our computational drug screens. We will also determine the crystal structures of EF in complex with those small molecules identified by this screening procedure, so that the pharmaceutical properties of these inhibitors may be improved through rational drug design methods.