Intervention against anthrax edema factor (EF)

2001年与生物恐怖主义有关的炭疽表明，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人免受这种可怕的生物战剂的伤害。炭疽是由革兰氏阳性细菌——炭疽芽孢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。水肿因子（EF）， EdTx的催化成分，具有钙调素激活腺苷酸环化酶活性，提高细胞内不受控制的cAMP水平。越来越多的证据表明EF是一个关键的毒力因子

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.