Directed evolution of inhibitors of anthrax toxin

描述(由申请人提供)：药物发现的缓慢速度阻碍了对新的生物恐怖威胁和新出现的传染病做出快速反应的需要。我们假设，蛋白质合成设备可以设计用于任何目标分子的高亲和力多肽和多肽类似物的体外快速进化。这里的目标是开发治疗播散性炭疽病的候选药物，这是一种用抗生素无法治愈的疾病。以炭疽毒素为靶标，利用我们的“纯翻译展示”技术，将生产出多肽和蛋白水解酶抗性、膜透过性、N-甲基肽类似物配体。这项技术允许合成天然和非天然氨基酸的聚合物，并通过遗传编码筛选出比化学合成文库多数百万倍的聚合物变体。因此，我们的图书馆将在结构上更加多样化，并将包含更有效的活页夹。 具体目的1.验证与天然氨基酰-tRNA底物纯翻译展示的炭疽毒素七聚体保护性抗原片段的亲和力比以往方法更高的假设。 具体目的2.验证与N-甲基化的Arhinoacyl-tRNA底物的纯翻译展示将产生抗蛋白酶的炭疽毒素结合子的假设。 具体目的3.检验粘合剂抑制炭疽毒素功能并具有药理上理想的特性的假设。多肽和N-甲基多肽类似物结合剂将在毒素功能的细胞分析中进行测试，并用于跨Caco细胞的传输。 这项研究与公共卫生的相关性。炭疽病是最有可能对大量人口造成毁灭性影响的生物武器之一，迫切需要改进预防和治疗方案。这项提案的工作应该会产生几种化合物，这些化合物将在很长一段时间内成为适合临床测试的候选化合物，无论是注射剂还是药丸。此外，所开发的技术将在任何疾病的诊断、目标确认和治疗方面具有潜在的实用价值。

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.