Developing a synthetic evolution approach to create de novo enzymes

描述（由申请人提供）：控制化学反应的立体化学是小分子药物发现和开发过程中的重要技能。促进所需立体中心形成的催化剂的鉴定是至关重要的，但在当今药物中发现的复杂化学结构的合成中一直是一个限制性步骤。我们建议建立一个通用的方法，使从头生物催化剂的广泛的化学反应的创建。我们的策略是不同的，因为它远远超出了目前的酶工程方法，这些方法仅限于优化现有的酶。我们将通过我们最近开创的体外选择和进化技术，从1013个随机蛋白质的组合文库中分离出活性生物催化剂。关键的优势是使用的文库包含比其他蛋白质工程方法多几个数量级的蛋白质变体。我们的具体目标是：1）从基于自然界最成功的酶支架（8桶折叠）的文库中分离从头生物催化剂。2)通过比较（�/�）8桶酶文库与非催化锌指支架文库，确定构建最有效生物催化剂所需的特征，以选择新的活性。3)通过体外进化优化人工生物催化剂的活性和稳定性，确定其进化潜力。为了证明我们的选择子方法的广泛适用性，我们将集中在两个重要的，但截然不同的键形成反应：小分子之间的碳-碳键形成和两个RNA寡核苷酸之间的磷酸二酯键形成。我们将分离狄尔斯-桤木反应的生物催化剂，这是有机化学中的中心反应之一，它可以产生多达四个新的立体中心，因此对于药物化学中许多高价值的多环天然产物的合成至关重要。由于Diels-Alder反应的重要性，过去有几种催化剂设计工作针对这种化学反应。因此，该反应可作为评估我们广泛适用的技术的基准。此外，我们将产生一种人工RNA连接酶，该酶具有作为医学研究工具的独特潜力，可用于推进某些类别RNA的RNA测序应用。初步研究表明，我们的方法是可行的。在概念验证实验中，我们创造了一种人工生物催化剂，可以催化一种没有已知天然酶的反应。这种高选择性的从头催化剂使反应加速超过200万倍。我们研究的长期目标是创造生物催化剂，促进药物合成过程中没有催化剂的关键反应。我们设想我们的一般方法，用于生产设计催化剂的化学反应的利益，以显着影响药物合成的方式接近。

DESCRIPTION (provided by applicant): Controlling the stereochemistry of chemical reactions is a vital skill in the small molecule drug discovery and development process. The identification of catalysts that promote the formation of the desired stereo centers is paramount, yet has been a limiting step in the synthesis of the complex chemical structures found in drugs today. We propose to establish a general approach that will enable the creation of de novo biocatalysts for a wide range of chemical reactions. Our strategy is different as it goes well beyond current enzyme engineering methods that are limited to the optimization of existing enzymes. We will isolate active biocatalysts from combinatorial libraries of 1013 randomized proteins through an in vitro selection and evolution technique that we have recently pioneered. The key advantage is the use of libraries that contain several orders of magnitude more protein variants than other protein engineering methods. Our specific aims are: 1) to isolate de novo biocatalysts from a library based on nature's most successful enzyme scaffold, the (�/�)8 barrel fold. 2) To identify features necessary to build the most efficient biocatalysts by comparing the (�/�)8 barrel enzyme library with a non-catalytic zinc finger scaffold library for the selection of novel activites. 3) To identify the evolutionary potential of an artificial biocatalyst by optimizing its activity ad stability through in vitro evolution. In order to demonstrate the broad applicability of our selecton approach, we will focus on two important, but vastly different bond formation reactions: a carbon-carbon bond formation between small molecules and a phosphodiester bond formation between two RNA oligonucleotides. We will isolate biocatalysts for a Diels- Alder reaction, one of the central reactions in organic chemistry that creates up to four new stereo centers and is therefore critical for the synthesis of many high-value polycyclic natural products in pharmaceutical chemistry. Because of the importance of the Diels-Alder reaction, several catalyst design efforts have targeted this chemistry in the past. Therefore, this reaction serves as a benchmark to evaluate our broadly applicable technology. Furthermore, we will generate an artificial RNA ligase enzyme that has unique potential as a medical research tool to advance RNA sequencing applications for certain classes of RNA. Preliminary studies show that our approach is feasible. In a proof of concept experiment, we created an artificial biocatalyst that catalyzes a reaction for which there are no known natural enzymes. This highly selective de novo catalyst accelerates the reaction more than two-million-fold. The long-term goal of our research is to create biocatalysts that facilitate key reactions in the drug synthesis process for which no catalyst is available. We envision our general method for producing designer catalysts for chemical reactions of interest to substantially impact the way drug synthesis is approached.