Novel Strategies in Light-driven P450 Enzymes

光驱动 P450 酶的新策略

• 批准号: 10410303
• 负责人: Lionel E. Cheruzel
• 金额: $ 14.65万
• 依托单位: SAN JOSE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-05 至 2022-06-03
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10410303
• 关键词: Accounting; Acids; Address; Anabolism; Area; Automation; Biochemical; Biodegradation; Biological Assay; Catalysis; Chemicals; Computers; Cyclooxygenase Inhibitors; Cysteine; Cytochrome P450; Development; Docking; Electrons; Engineering; Enzymes; General Population; Goals; Grant; Heme; High Pressure Liquid Chromatography; Human; Hybrids; Hydrogen Peroxide; In Situ; Institution; Investigation; Knowledge; Laboratories; Lead; Libraries; Light; Liquid substance; Metabolism; Methodology; Minority; Mixed Function Oxygenases; Modeling; Non-Steroidal Anti-Inflammatory Agents; Organism; Pharmaceutical Preparations; Pharmacologic Substance; Photosensitizing Agents; Play; Positioning Attribute; Property; Protein Engineering; Reaction; Research; Robot; Role; Stomach; System; Testing; TimeLine; Toxic effect; Universities; Visible Radiation; base; drug candidate; drug metabolism; experimental study; fatty acid amide hydrolase; gastrointestinal; hybrid enzyme; innovation; interest; mutant; non-Native; novel; novel drug class; novel strategies; novel therapeutics; scaffold; simulation; small molecule; undergraduate student

Project Summary: Cytochromes P450 are a superfamily of heme-thiolate monooxygenases, found in almost all living organisms, that play an important role in the biosynthesis and biodegradation of endogenous compounds. In humans, P450s are the major enzymes involved in drug metabolism and bioactivation, accounting for 75% of the total metabolism. Harnessing the synthetic potential of these monooxygenases has also led to the recent use of P450 enzymes in late-stage functionalization of various compounds. Our laboratory has developed an efficient light-driven P450 hybrid enzyme system that utilizes the photochemical properties of Ru(II)-diimine photosensitizers covalently attached to strategically positioned non-native single cysteine mutants of their heme domain to perform P450 reactions upon visible light excitation. We recently started to investigate a complementary bimolecular approach to activate P450 enzymes with peroxygenase activity, which is the focus of Aim 1. Using various protein engineering approaches, we are assembling a large and diverse library of light- initiated P450 enzymes with unique photocatalytic activity towards a range of substrates and with human P450-like activities. We have also recently focused on developing chemoenzymatic approaches by marrying the advantages of chemical catalysis with the selectivity of the P450 biocatalysis. We identified a promising substrate anchoring approach to enhance the catalytic activity of P450 enzymes and alter their regioselectivity. In Aim 2, we will capitalize on those strategies to synthesize a library of biphenylalkanoic acid and diphenylamine derivatives, as potential dual fatty acid amide hydrolase (FAAH)/cyclooxygenase (COX) inhibitors to mitigate gastric damages, currently plaguing the use of nonsteroidal anti-inflammatory drugs (NSAIDs). A combination of computer docking simulation and biochemical assays will be used to ascertain their inhibitory activity. In Aim 3, we will apply concepts identified in the substrate engineering approach to develop a new platform for late stage substrate functionalization. Our long-term goal is to employ the libraries of light-driven P450 enzymes combined with the chemoenzymatic and substrate engineering strategies for the development of novel drugs and late stage diversification of lead compounds.

项目摘要： 细胞色素P450是血红素硫酸硫酸盐单加氧酶的超家族，几乎在所有活生物体中发现， 该在内源性化合物的生物合成和生物降解中起着重要作用。在人类中 P450是参与药物代谢和生物活化的主要酶，占总数的75％ 代谢。利用这些单加氧酶的合成潜力也导致了最近的使用 P450酶在各种化合物的后期功能化中。我们的实验室发展了一个高效的 光驱动的P450混合酶系统利用Ru（II） - 二胺的光化学特性 光敏剂共价连接到其战略性位置的非本地单半胱氨酸突变体 血红素结构域在可见光激发后执行P450反应。我们最近开始调查一个 用过氧酶活性激活P450酶的互补双分子方法，这是重点 AIM 1。使用各种蛋白质工程方法，我们组装了一个大而多样的光库 具有独特的光催化活性对一系列底物的启动P450酶，人类 类似P450的活动。我们最近还致力于通过结婚来开发化学酶方法 化学催化的优势与P450生物催化的选择性。我们确定了一个有前途的 底物锚定方法增强P450酶的催化活性并改变其区域选择性。 在AIM 2中，我们将利用这些策略，以合成双苯基甲酸和 二苯胺衍生物，作为潜在的双脂肪酸酰胺水解酶（FAAH）/环氧合酶（COX） 减轻胃损伤的抑制剂，目前困扰使用非甾体类抗炎药 （NSAIDS）。计算机对接模拟和生化测定的结合将用于确定 他们的抑制活性。在AIM 3中，我们将应用在基板工程方法中确定的概念 为晚期底物功能化开发一个新的平台。 我们的长期目标是采用与化学酶的轻驱动P450酶的库 以及用于开发新药物和铅的晚期多样化的底物工程策略 化合物。