Developing a synthetic evolution approach to create de novo enzymes

开发合成进化方法来从头创造酶

• 批准号: 9054135
• 负责人: Burckhard Seelig
• 金额: $ 28.61万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9054135
• 关键词: Area; Benchmarking; Benign; Binding; Biology; Biomedical Research; Breeding; Carbon; Catalysis; Chemical Structure; Chemicals; Chemistry; Complex; Development; Diels Alder reaction; Elements; Engineering; Enzymes; Evolution; Generations; Goals; Health; In Vitro; Laboratories; Lead; Learning; Libraries; Ligase; Ligation; Medical Research; Methods; Natural Products; Natural Selections; Nature; Oligonucleotides; Organic Chemistry; Organic Synthesis; Organic solvent product; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacotherapy; Process; Property; Protein Engineering; Proteins; RNA; RNA Ligase (ATP); Randomized; Reaction; Research; Structure; Structure-Activity Relationship; Techniques; Technology; Testing; Therapeutic; Tube; Variant; Work; Zinc; Zinc Fingers; base; catalyst; chemical reaction; combinatorial; design; drug development; drug discovery; drug synthesis; human disease; innovation; insight; interest; novel; phosphodiester; polypeptide; protein folding; research study; scaffold; skills; small molecule; stereochemistry; success; tool; transcriptome sequencing

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.

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一项重要技能，促进所需立体中心形成的催化剂的鉴定至关重要，但一直是小分子药物发现和开发过程中的限制步骤。我们建议建立一种通用方法，能够为广泛的化学反应创建从头生物催化剂，因为它远远超出了现有的酶工程方法。是仅限于现有酶的优化。我们将通过我们最近开创的体外选择和进化技术，从 1013 种随机蛋白质的组合文库中分离出活性生物催化剂，其关键优势是使用含有多个数量级蛋白质的文库。我们的具体目标是：1) 从基于自然界最成功的酶支架 (�/�)8 桶折叠的库中分离出新的生物催化剂 2) 确定必要的特征。通过比较 (�/�)8 桶酶库与非催化锌指支架库来选择新活性，构建最有效的生物催化剂 3) 通过优化其活性来确定人工生物催化剂的进化潜力。为了证明我们的选择方法的广泛适用性，我们将重点关注两个重要但截然不同的键形成反应：小分子之间的碳-碳键形成和小分子之间的磷酸二酯键形成。我们将分离用于 Diels-Alder 反应的生物催化剂，这是有机化学的核心反应之一，可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，过去的一些催化剂设计工作都针对这种化学反应，因此，该反应可作为评估我们广泛适用的技术的基准。此外，我们将生成一种具有以下特性的人工 RNA 连接酶。作为医学的独特潜力初步研究表明，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，可以催化没有已知天然酶的反应。从头催化剂将反应加速超过两百万倍 我们研究的长期目标是创造出促进药物合成过程中关键反应的生物催化剂，而我们设想了生产设计师的通用方法。感兴趣的化学反应的催化剂对药物合成的方式产生重大影响。