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

基本信息

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
项目类别：
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

项目摘要

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).

项目概要/摘要当药物在体内代谢时，其代谢物代表了人类所接触的新化学实体。裸露。因此，研究药物代谢物的毒性和表征生物活性至关重要以开发安全、有效的药物。事实上，药代动力学研究是向美国食品和药物管理局提交研究性新药申请。作为这些研究的一部分，然而，需要大量的纯代谢物来在体外，特别是在体内表征它们。药物代谢物的化学合成在产量、选择性方面存在问题，并且可能是主要的成本临床前研究的驱动力。药物代谢物的直接酶法合成是一种有吸引力的替代方案。虽然人类肝脏中发现的 P450 单加氧酶作为药物代谢而受到关注生物催化剂仍然存在许多挑战，例如生物催化活性低、药物底物特异性有限和产品范围。对替代酶或生物催化剂的研究可能会产生多样化的代谢物产品谱并为高效代谢物生产开辟新视野。由于其优异的化学性质，广泛的底物范围和可延展的催化活性，我们建议研究生物催化剂甲苯邻二甲苯假单胞菌属的单加氧酶 (ToMO)。 OX1作为药物代谢酶。利用蛋白质工程技术，我们计划创建具有增强的药物氧化活性和微调特异性的 ToMO 变体。我们之前利用蛋白质工程创建了几种用于绿色化学的 ToMO 变体，生物修复应用。使用ToMO及其工程变体用于药物合成的前景代谢物提出了一种新的、令人兴奋的方法。我们的长期目标是提高 ToMO 的多功能性，甚至进一步探索和扩展其用于药物开发和药品生产的底物库。该项目的具体目标是表征野生型 ToMO 的药物氧化活性，具有 6 种不同的活性药物底物和类药物候选物，包括苯胺、乙酰苯胺、安非他酮、噻氯匹定、扑尔敏、和奥美拉唑（目标 1），使用蛋白质工程构建 ToMO 变体库并筛选进一步改进（目标 2），以及药物氧化活性的正 ToMO 变体的序列、模型和表征（目标 3）。