Directed evolution of inhibitors of anthrax toxin

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DESCRIPTION (provided by applicant): The need for a rapid response to new bioterror threats and emerging infectious diseases is frustrated by the slow pace of drug discovery. We hypothesize that the protein synthetic machinery can be engineered for the rapid evolution in vitro of high-affinity peptide and peptide analog inhibitors of any target molecule. The goal here is to develop drug candidates for disseminated anthrax, a disease incurable with antibiotics. With anthrax toxin as the target, both peptide and protease-resistant, membrane-permeable, N-methyl-peptide analog ligands will be produced by our "pure translation display" technology. This technology allows synthesis of polymers of both natural and unnatural amino acids and, by virtue of genetic encoding, the screening of millions of times more polymer variants than chemically-synthesized libraries. Thus, our libraries will be much more diverse in structure and will contain binders that are much more potent. Specific Aim 1. To test the hypothesis that pure translation display with natural aminoacyl-tRNA substrates ill yield higher-affinity peptide binders for anthrax toxin's heptameric protective antigen fragment than prior methods. Specific Aim 2. To test the hypothesis that pure translation display with N-methylated arhinoacyl-tRNA substrates will yield binders for anthrax toxin that are resistant to proteases. Specific Aim 3. To test the hypothesis that the binders inhibit anthrax toxin function and have pharmacologically-desirable properties. Peptide and N-methyl-peptide analog binders will be tested in cellular assays of toxin function and for transport across Caco cells. Relevance of this research to public health. Anthrax is one of the biological weapons most likely to be used with devastating effect on a large population, and improved prophylactic and treatment options are urgently needed. Work in this proposal should yield several compounds that will have come a long way towards candidates suitable for testing for clinical use, either as injectables or pills. Furthermore, the technology developed will have potential utility in diagnosis, target validation and treatment of any disease.