Integrating Chemistry and Evolution to Illuminate Biology and Enable Novel Therapeutics

 描述（由申请人提供）：我们的实验室已经进行了两个研究计划，在化学和进化的接口。在第一个项目中，我们开发了蛋白质进化和蛋白质递送的新方法，大大提高了它们的有效性。在第二个项目中，我们开发了一种新的方法来合成和发现生物活性小分子，将生物进化的强大方面与合成有机化学相结合。由此产生的DNA模板合成方法使编码合成分子的DNA序列能够经历与生物进化平行的翻译、选择和扩增。该提案旨在将这两个研究项目整合为最大化研究者研究奖（MIRA）下的单一努力。 在第一个项目中，我们开发了一个系统，使蛋白质能够在实验室中不断进化，几乎不需要研究人员的干预。由此产生的系统，噬菌体辅助连续进化（PACE），允许蛋白质以比传统方法快100倍的速度进行定向进化。我们建议将这些发展应用于不断进化四类蛋白质或RNA，每一类都具有操纵基因或基因产物共价结构的能力，并且每一类都与下一代人类疗法的开发具有潜在的相关性：将目的DNA插入人类基因组中的安全港基因座的重组酶，特异性切割疾病相关蛋白的蛋白酶，具有改变的PAM特异性和增强的活性的正交Cas9（CRISPR）核酸酶，以及仅在处于特定疾病相关细胞状态的那些细胞中介导基因组工程的“智能”Cas9指导RNA。成功将建立这些蛋白质和RNA的新的治疗潜力，以解决广泛的人类疾病，包括许多人类遗传疾病。 在第二个项目中，我们开发了DNA模板合成的基础，生成了DNA模板小分子文库，并对这些文库进行了体外选择，以获得对生物医学感兴趣的初始靶点的亲和力。这些结果导致了具有显著选择性和效力的新型激酶和蛋白酶抑制剂的发现，包括胰岛素降解酶（IDE）的第一个生理抑制剂。我们在小鼠中使用这种化合物来验证抑制胰岛素降解作为改善葡萄糖耐量的潜在治疗策略。我们建议开发具有更高治疗潜力的第二代IDE抑制剂，以探索我们最近发现的IDE抑制可以降低体内血压，并通过创建一个新的DNA模板化的> 250，000个大环化合物文库来扩展这种方法的应用，并选择该文库与涉及人类疾病的>150个蛋白质靶点结合。这些努力将共同评估超过30，000，000种潜在的小分子-蛋白质相互作用，其方式利用了体外选择，PCR和现代DNA测序的显着效率。

 DESCRIPTION (provided by applicant): Our laboratory has conducted two research programs at the interface of chemistry and evolution. In the first program, we developed new approaches to protein evolution and protein delivery that have dramatically increased their effectiveness. In the second program, we developed a new approach to the synthesis and discovery of bioactive small molecules that combines powerful aspects of biological evolution with synthetic organic chemistry. The resulting method of DNA-templated synthesis has enabled DNA sequences encoding synthetic molecules to undergo translation, selection, and amplification paralleling biological evolution. This proposal seeks to integrate these two research programs into a single effort under a Maximizing Investigators' Research Award (MIRA). In the first program, we developed a system that enables proteins to evolve continuously in the laboratory, requiring virtually no researcher intervention. The resulting system, phage-assisted continuous evolution (PACE), allows proteins to undergo directed evolution at a rate ~100-fold faster than conventional methods. We propose to apply these developments to continuously evolve four classes of proteins or RNAs, each with the ability to manipulate the covalent structure of genes or gene products, and each with potential relevance to the development of next-generation human therapeutics: recombinase enzymes that insert DNA of interest into safe-harbor loci in the human genome, proteases that specifically cleave disease-associated proteins, orthogonal Cas9 (CRISPR) nucleases with altered PAM specificities and enhanced activities, and "smart" Cas9 guide RNAs that mediate genome engineering only in those cells that are in specific disease-associated cell states. Success would establish the novel therapeutic potential of these proteins and RNAs to address a wide range of human diseases, including many human genetic disorders. In the second program, we developed the foundations of DNA-templated synthesis, generated libraries of DNA-templated small molecules, and performed in vitro selections on these libraries for affinity to an initial set of targets of biomedical interest. The results led t the discovery of novel kinase and protease inhibitors with remarkable selectivity and potency, including the first physiological inhibitor of insulin-degrading enzyme (IDE). We used this compound in mice to validate inhibition of insulin degradation as a potential therapeutic strategy for improving glucose tolerance. We propose to develop second-generation IDE inhibitors with increased therapeutic potential, to explore our recent discovery that IDE inhibition can lower blood pressure in vivo, and to expand the application of this approach by creating a new DNA-templated library of >250,000 macrocycles and to selecting this library for binding to >150 protein targets implicated in human disease. These efforts will collectively result in the evaluation of more than 30,000,000 potential small molecule-protein interactions in a manner that leverages the remarkable efficiency of in vitro selection, PCR, and modern DNA sequencing.