Molecular Basis for Inhibition of Edema Factor

描述（由申请人提供）：该项目的目的是寻找炭疽蛋白水肿因子（EF）的小分子抑制剂。这种毒素由炭疽芽孢杆菌在催化无活性状态下分泌。当这种毒素通过炭疽源性转运蛋白保护性抗原进入炭疽受害者的细胞质时，它会与钙调蛋白（CAM）形成复合物，CAM是脊椎动物细胞内钙结合蛋白的关键。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状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患者。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。通过给予常规治疗和免疫系统更多的时间来发挥作用，这些抑制剂可能会显著提高炭疽的存活率。

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.