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

项目总结/摘要 当药物在体内代谢时，它们的代谢物代表了新的化学实体， 暴露了因此，研究药物代谢产物的毒性和表征其生物活性至关重要 来研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率、选择性方面是有问题的，并且可能是主要的成本- 临床前研究的驱动因素。药物代谢物的直接酶促合成是一种有吸引力的替代方法。虽然 P450单加氧酶是在人体肝脏中发现的一种重要的药物代谢酶， 尽管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， 的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，并开辟了新的视野，有效的代谢产物生产。由于其优异的化学性质， 广泛的底物范围，和可塑性的催化活性，我们提出了研究生物催化剂甲苯邻二甲苯 作为药物代谢酶的假单胞菌属物种OX 1的单加氧酶（ToMO）。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。 使用蛋白质工程，我们先前为绿色化学创造了几种ToMO变体， 生物修复应用。ToMO及其工程变体在药物合成中的应用前景 代谢物提出了一个新的和令人兴奋的方法。我们的长期目标是提高ToMO的多功能性， 进一步通过探索和扩大其用于药物开发和药物生产的底物库。 本项目的具体目的是用6种不同的方法表征野生型ToMO的药物氧化活性， 药物底物和药物样候选物，包括苯胺、乙酰苯胺、安非他酮、噻氯匹定、氯苯那敏， 和奥美拉唑（Aim 1），使用蛋白质工程构建ToMO变体文库，并筛选进一步的 改进（目标2），并测序，建模和表征药物氧化活性的阳性ToMO变体 (Aim 3）。

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).