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.

项目概要： 细胞色素 P450 是血红素硫醇单加氧酶的超家族，几乎存在于所有生物体中， 在内源性化合物的生物合成和生物降解中发挥重要作用。在人类中， P450是参与药物代谢和生物活性的主要酶，占总量的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 酶参与各种化合物的后期功能化。我们的实验室开发了一种高效的 利用 Ru(II)-二亚胺光化学特性的光驱动 P450 杂合酶系统 光敏剂共价连接到其战略定位的非天然单半胱氨酸突变体 血红素域在可见光激发下执行 P450 反应。我们最近开始调查一个 互补双分子方法激活具有过氧合酶活性的P450酶，这是重点 目标 1. 使用各种蛋白质工程方法，我们正在组装一个大型且多样化的光库 首创的 P450 酶对一系列底物和人类具有独特的光催化活性 P450 样活动。我们最近还致力于通过结合开发化学酶方法 化学催化的优点与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。 在目标 2 中，我们将利用这些策略来合成联苯链烷酸和 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，目前困扰着非甾体类抗炎药的使用 （非甾体抗炎药）。将使用计算机对接模拟和生化检测相结合来确定 他们的抑制活性。在目标 3 中，我们将应用基板工程方法中确定的概念 开发用于后期基板功能化的新平台。 我们的长期目标是利用光驱动 P450 酶库与化学酶相结合 用于新药开发和先导化合物后期多样化的底物工程策略 化合物。