Characterizing and engineering toluene o-xylene monooxygenase for the synthesis of common drug metabolites

用于合成常见药物代谢物的甲苯邻二甲苯单加氧酶的表征和工程设计

• 批准号: 10674949
• 负责人: Gonul Schara
• 金额: $ 10.73万
• 依托单位: CALIFORNIA STATE UNIVERSITY-STANISLAUS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-04 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10674949
• 关键词: Active Sites; Agar; Aniline; Area; Biological Assay; Bioremediations; Bupropion; Catalysis; Cells; Chemistry; Clinical Research; Codon Nucleotides; Color; Cytochrome P450; Development; Disadvantaged; Disease; Docking; Drug Kinetics; Engineering; Ensure; Enzymes; Escherichia coli; Family; Friends; Funding; Generations; Goals; Grant; Health; High Pressure Liquid Chromatography; Human; In Vitro; Investigation; Investigational New Drug Application; Libraries; Light; Liver; Membrane; Mixed Function Oxygenases; Modeling; Motivation; Mutagenesis; NADH; Nuclear Magnetic Resonance; Nylons; Omeprazole; Oxygenases; Pathway interactions; Performance; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Plasmids; Positioning Attribute; Preclinical Drug Development; Preparation; Probability; Process; Production; Property; Protein Engineering; Pseudomonas; Research; Research Project Grants; Research Proposals; Role; Science; Site-Directed Mutagenesis; Specificity; Spottings; Structure; Structure-Activity Relationship; Students; Study models; Substrate Specificity; Techniques; Ticlopidine; Time; Toxic effect; Toxicity Tests; Toxicology; Underrepresented Populations; United States Food and Drug Administration; United States National Institutes of Health; Variant; career; career development; catalyst; chemical synthesis; cofactor; computerized tools; cost; drug biological activity; drug development; drug synthesis; drug-like compound; enzyme activity; experience; experimental study; exposed human population; improved; in vivo; interest; liquid chromatography mass spectroscopy; new chemical entity; oxidation; pre-clinical; preclinical study; programs; research and development; scale up; simulation; success; toluene 2-xylene monooxygenase

Project Summary/Abstract As drugs are metabolized in the body, their metabolites represent new chemical entities to which humans are exposed. Thus, investigating the toxicities and characterizing biological activities of drug metabolites is crucial for the development of safe, effective drugs. In fact, pharmacokinetic studies are critical components of investigational new drug applications to the US Food and Drug Administration. As a part of these studies, however, large quantities of pure metabolites are needed to characterize them in vitro and, especially in vivo. The chemical synthesis of drug metabolites is problematic in terms of yield, selectivity, and can be a major cost- driver in preclinical studies. Direct enzymatic synthesis of drug metabolites is an attractive alternative. Although P450 monooxygenase enzymes, found in human liver, have been in the spot light as drug metabolizing biocatalysts, many challenges still remain such as low biocatalytic activity, limited drug substrate specificity and product range. The investigation of alternate enzymes or biocatalysts may lead to a diversified metabolites product spectra and open up new horizons for efficient metabolite production. Due to its excellent chemistry, wide substrate range, and malleable catalytic activity, we propose to investigate the biocatalyst toluene o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1 as a drug metabolizing enzyme. Using protein engineering techniques, we plan to create ToMO variants with enhanced drug oxidation activity and fine-tuned specificity. Using protein engineering, we previously created several variants of ToMO for green chemistry and bioremediation applications. The prospect of using ToMO and its engineered variants for the synthesis of drug metabolites presents a new and exciting approach. Our long-term goal is to improve the versatility of ToMO even further by exploring and expanding its substrate repertoire for drug development and pharmaceutical production. The specific aims of this project are to characterize wild-type ToMO for drug oxidation activity with 6 different drug substrates and drug-like candidates including aniline, acetanilide, bupropion, ticlopidine, chlorphenamine, and omeprazole (Aim 1), construct ToMO variant libraries using protein engineering and screen for further improvements (Aim 2), and sequence, model, and characterize positive ToMO variants for drug oxidation activity (Aim 3).

项目摘要/摘要 由于药物在体内代谢，其代谢物代表了人类所在的新化学实体 裸露。因此，研究药物代谢产物的毒性和表征生物学活性至关重要 为了开发安全有效的药物。实际上，药代动力学研究是关键组成部分 美国食品药品监督管理局的调查新药应用。作为这些研究的一部分， 但是，需要大量的纯代谢物来在体外，尤其是体内表征它们。 药物代谢物的化学合成在产量，选择性方面是有问题的，并且可能是主要成本 - 临床前研究的驱动力。药物代谢产物的直接酶促合成是一种有吸引力的替代方法。虽然 在人肝脏中发现的P450单加氧酶酶已在药物代谢中处于现场光线状态 生物催化剂，许多挑战仍然存在，例如低生物催化活性，有限的药物底物特异性和 产品范围。对替代酶或生物催化剂的研究可能导致多样化的代谢产物 产品光谱并为有效的代谢产物打开新的视野。由于其出色的化学反应 较宽的底物范围和可延展的催化活性，我们建议研究甲苯O-二甲苯的生物催化剂 假单胞菌的单加氧酶（Tomo）。 OX1作为药物代谢酶。使用蛋白质工程 技术，我们计划创建具有增强药物氧化活性和微调特异性的Tomo变体。 使用蛋白质工程，我们以前创建了几种用于绿色化学和的Tomo变体 生物修复应用。使用Tomo及其工程变体合成药物的前景 代谢产物提出了一种新的令人兴奋的方法。我们的长期目标是提高Tomo的多功能性 进一步探索和扩大其底物库，以进行药物开发和药物生产。 该项目的具体目的是表征野生型Tomo，用于6种不同的药物氧化活性 药物底物和类似药物样的候选物，包括苯胺，乙酰胺，安非他酮，惠吡啶，氯苯甲胺， 和奥美拉唑（AIM 1），使用蛋白质工程和屏幕构建Tomo变体库以进一步 改进（AIM 2）以及序列，模型和表征药物氧化活性的阳性Tomo变体 （目标3）。