One effective way to counter bioterrorism is to stockpile inhibitors that can selectively cripple enzymes pivotal to pathogens. Anthrax can be detoxified by selective inhibitors targeting anthrax's lethal factor (LF) that acts as a zinc protease. Botulinum toxin can be detoxified by selective inhibitors targeting its zinc endopeptidase. If such inhibitors were readily available, anthrax and botulinum would no longer be life threatening. The effectiveness of this approach rests on the fact that pathogens rely on enzymes. Furthermore, viral and bacterial enzymes have high substrate selectivity and can therefore be inhibited by selective inhibitors without interfering with other enzymes required for normal functions. To expeditiously develop selective enzyme inhibitors as countermeasures to biological weapons, we have recently developed i) an effective method for computationally simulating zinc proteases such as LF and botulinum neurotoxin serotype A (BoNTA), ii) an advanced computer program and a 1.1 teraflop supercomputer optimized for rapidly identifying enzyme inhibitors in silico, and iii) a database of 2.5 million unique chemical structures. In addition, we have computationally refined the 3D structures of LF and BoNTA. Our pilot study using these new technologies and the refined structures has already culminated in a small molecule that selectively inhibits BoNTA with an estimated IC (50) of 4 ?M and a weak small-molecule inhibitor of LF. Here we propose to develop potent and selective small-molecule inhibitors of LF and BoNTA as countermeasures to anthrax and botulinum toxin. We will develop the desired LF inhibitors by i) computationally identifying selective LF inhibitors from 2.5 million chemical structures, ii) synthesizing top 500 LF inhibitor candidates, iii) performing in vitro studies of 500 compounds, ex vivo studies of top 50 compounds, and in vivo studies of top 5 compounds, and iv) structural optimization of top 5 compounds with a combinatorial chemistry approach to obtain inhibitors with chemical and biological properties satisfying the criteria for clinic drugs. We will use the same strategy to develop BoNTA inhibitors as the one used for LF inhibitors. Successful completion of this proposal will lead to a timely treatment for intoxication caused by anthrax or botulinum toxin. It complements the efforts of other workers in developing methods for rapidly identifying anthrax and botulinum toxin.
