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.

R01AI066503

2005-02-15

2006-07-31