EXCEED THE SPACE PROVIDED. 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). 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 unrelated enzymes. 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 IC50 of 4 uM and a nonpeptidic LF inhibitor with an estimiated ICs0 of 150 uM. Here we propose to develop improved small-molecule inhibitors of LF and BoNTA as countermeasures to anthrax and botulinum toxin. We will develop improved LF inhibitors by i) computationally identifying LF inhibitors from 2.5 million chemical structures, ii) purchasing or synthesizing the top 500 LF inhibitor candidates, iii) performing in vitro studies of the 500 compounds, ex vivo studies of the top 50 compounds, and in vivo studies of the top 5 compounds, and iv) structural optimization of the top 5 compounds with combinatorial chemistry 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. The generic approach demonstrated in this proposal is useful to the search of countermeasures to other biological weapons and to the search of therapeutics for various human diseases. PERFORMANCE SiTE ========================================Section End===========================================
