Intervention against anthrax edema factor (EF)

2001年发生的生物恐怖主义炭疽事件表明，炭疽孢子可以成为一种有效的生物恐怖主义制剂，造成大规模的破坏和伤亡。如果炭疽孢子再次被使用，我们需要所有可能的治疗方法来保护受影响的个人免受这种可怕的生物战剂的伤害。炭疽病是由革兰氏阳性细菌炭疽杆菌的生长和炭疽细菌分泌的毒素引起的。两种主要的炭疽毒素，水肿毒素（EdTx）和致死毒素（LeTx）改变细胞内信号传导。EdTx的催化组分水肿因子（EF）具有钙调蛋白激活的腺苷酸环化酶活性，以提高不受控制的细胞内cAMP水平。越来越多的证据表明，EF是一个关键的毒力因子， 炭疽病发病机理我们已经确定了EF单独和EF与钙调蛋白复合物的分子结构，以表明EF与钙调蛋白的相互作用与细胞靶点与钙调蛋白结合的方式明显不同。我们假设，可以特异性结合EF并破坏与钙调蛋白的相互作用的小分子量化合物可以用于对抗EF作用的潜在疗法。 我们已经使用串联的基于细胞和基于蛋白结合的10，000个化合物文库的筛选以及先导物优化来鉴定可以阻断TdR的先导物（4-[4-（4-硝基苯基）-噻唑基氨基]-苯-磺酰胺）。 EF与其细胞激活剂钙调素的相互作用具有5 μ M的亲和力和最小的细胞毒性。在本申请中，我们将结合联合收割机化学合成、基于细胞的测定和体外测定，以将该磺酰胺化合物的亲和力优化至低至中nM水平。我们还将使用X-射线晶体学来确定EF的分子结构，并与优化的磺酰胺铅复合，以解决该铅如何中断钙调蛋白与EF的结合。我们已经发现阿德福韦酯，一种临床批准的抗B型肝炎病毒药物，可以有效地抑制组织培养细胞中EF的活性。阿德福韦酯与EF、ATP的细胞底物竞争，减少cAMP的形成。我们亦会研究 优化的磺酰胺化合物可以与阿德福韦协同工作，以实现对EF-中毒的更大抑制。我们的提案的成功将为产生有效的EF抑制剂作为炭疽感染的辅助治疗提供蓝图。

The bioterrorism-related anthrax in 2001 has shown that anthrax spores can be an effective bioterrorism agent to cause massive disruption and causalities. In the event that anthrax spores are used again, we need all possible therapeutics to protect affected individuals from this terrible bio-warfare agent. Anthrax is caused by the growth of the gram-positive bacteria, Bacillus anthracis and by toxins secreted by anthrax bacteria. Two major anthrax toxins, edema toxin (EdTx) and lethal toxin (LeTx) alter intracellular signaling. Edema factor (EF), the catalytic component of EdTx has calmodulin-activated adenylyl cyclase activity to raise the uncontrolled intracellular cAMP level. The growing evidences suggest that EF is a key virulence factor for anthrax pathogenesis. We have determined the molecular structure of EF alone and EF in complex with calmodulin to show that the interaction of EF with calmodulin is distinctly different from how cellular targets bind to calmodulin. We hypothesize that small molecular weight compound that can specifically bind EF and disrupt the interaction with calmodulin could be exploited for potential therapeutics against the action of EF. We have used a tandem cell-based and protein-binding based screen of a 10,000 compound library as well as lead optimization to identify a lead (4-[4-(4-nitrophenyl)-thiazolylamino]-benzene-sulfonamide) that can block the interaction of EF with its cellular activator, calmodulin with 5 microM affinity and the minimal cell toxicity. In this application, we will combine chemical synthesis, cell-based assays, and in vitro assay to optimize the affinity of this sulfonamide compound to the low to mid-nM level. We will also use X-ray crystallography to determine the molecular structure of EF in complex with the optimized sulfonamide lead to address how this lead interrupts the binding of calmodulin to EF. We have found adefovir, a clinically approved anti-hepatitis B virus drug that can potently inhibit the activity of EF in tissue culture cells. Adefovir dipivoxil works to compete with the cellular substrate of EF, ATP to reduce the cAMP formation. We will also examine whether the optimized sulfonamide compound can work in concert with adefovir to achieve the greater inhibition of EF-intoxication. Success in our proposal will provide the blueprint to generate the effective EF inhibitors as an adjunct therapeutic against anthrax infection.