Directed evolution of inhibitors of anthrax toxin

描述（由申请人提供）：药物发现的缓慢步伐阻碍了对新的生物恐怖威胁和新出现的传染病的快速反应的需求。我们假设蛋白质合成机制可以被设计用于任何靶分子的高亲和力肽和肽类似物抑制剂的体外快速进化。这里的目标是开发用于传播性炭疽病的候选药物，这种疾病无法用抗生素治愈。以炭疽毒素为靶点，我们的“纯翻译展示”技术将产生肽和蛋白酶抗性的、膜可渗透的N-甲基肽类似物配体。该技术允许合成天然和非天然氨基酸的聚合物，并且凭借遗传编码，筛选比化学合成文库多数百万倍的聚合物变体。因此，我们的库在结构上将更加多样化，并且将包含更有效的粘合剂。 具体目标1。为了检验假设，纯翻译展示与天然氨酰-tRNA底物将产生更高的亲和力的肽结合剂炭疽毒素的七聚体保护性抗原片段比以前的方法。 具体目标2。为了检验以下假设，即用N-甲基化的arhinoacyl-tRNA底物进行纯翻译展示将产生对蛋白酶具有抗性的炭疽毒素结合剂。 具体目标3。检验结合剂抑制炭疽毒素功能并具有药理学理想性质的假设。肽和N-甲基肽类似物结合剂将在毒素功能的细胞试验中进行检测，并用于跨Caco细胞的转运。 这项研究与公共卫生的相关性。炭疽是最有可能对广大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办法。这项提案中的工作应该产生几种化合物，这些化合物将在很长一段时间内成为适合临床使用的候选药物，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、靶点验证和治疗中具有潜在的实用性。

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.