Molecular Basis for Inhibition of Edema Factor

描述（由申请人提供）：本项目的目的是寻找炭疽蛋白水肿因子（EF）的小分子抑制剂。这种毒素是由炭疽杆菌以无催化活性状态分泌的。当毒素通过炭疽衍生的转运蛋白（保护性抗原）转运到炭疽受害者的细胞质中时，它与脊椎动物中关键的细胞内钙结合蛋白钙调蛋白（CAM）形成复合物。这种关联激活EF，并将其转化为腺苷酸环化酶，其催化活性比受害者自身的环AMP产生酶高1000倍。传统的炭疽疗法（针对炭疽细菌，而不是炭疽毒素）非常有效，但不足以拯救所有炭疽受害者。EF抑制剂代表了一种完全不同的、完全互补的对抗炭疽的方法。这种抑制剂将通过治疗疾病的下游效应，帮助恢复细胞平衡，并可能减缓疾病的进程。通过允许更多的时间进行常规治疗和免疫系统的工作，这些抑制剂可以显着提高炭疽的存活率。 我们已经解决了EF（非活性状态）和EF/CaM复合物（活性状态）的晶体结构。使用这些结构作为指导，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EF抑制剂的小分子; A型，封闭活性位点的抑制剂，和B型，那些保持EF在其CAM自由，催化非活性状态的抑制剂。我们已经有了A型抑制剂的检测方法。我们将完成B型抑制剂的简单检测方法的开发，然后将这些检测方法应用于我们的计算药物筛选建议的前约1000种化合物。我们还将确定EF的晶体结构与这些小分子通过这种筛选程序确定的复合物，使这些抑制剂的药物性能可以通过合理的药物设计方法来改善。

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.