Non-cytochrome P450 enzymes involved in the oxidative metabolism of xenobiotics: Focus on the regulation of gene expression and enzyme activity

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Abstract

Oxidative metabolism is one of the major biotransformation reactions that regulates the exposure of xenobiotics and their metabolites in the circulatory system and local tissues and organs, and influences their efficacy and toxicity. Although cytochrome (CY)P450s play critical roles in the oxidative reaction, extensive CYP450-independent oxidative metabolism also occurs in some xenobiotics, such as aldehyde oxidase, xanthine oxidoreductase, flavin-containing monooxygenase, monoamine oxidase, alcohol dehydrogenase, or aldehyde dehydrogenase-dependent oxidative metabolism. Drugs form a large portion of xenobiotics and are the primary target of this review. The common reaction mechanisms and roles of non-CYP450 enzymes in metabolism, factors affecting the expression and activity of non-CYP450 enzymes in terms of inhibition, induction, regulation, and species differences in pharmaceutical research and development have been summarized. These non-CYP450 enzymes are detoxifying enzymes, although sometimes they mediate severe toxicity. Synthetic or natural chemicals serve as inhibitors for these non-CYP450 enzymes. However, pharmacokinetic-based drug interactions through these inhibitors have rarely been reported in vivo. Although multiple mechanisms participate in the basal expression and regulation of non-CYP450 enzymes, only a limited number of inducers upregulate their expression. Therefore, these enzymes are considered non-inducible or less inducible. Overall, this review focuses on the potential xenobiotic factors that contribute to variations in gene expression levels and the activities of non-CYP450 enzymes.

Introduction

Oxidative metabolism is the most common and important type of biotransformation in drug phase I metabolism, which includes oxidation, reduction, and hydrolysis. Cytochrome P450s (CYP450s) are responsible for the oxidation of more than 90% of marketed drugs (Williams et al., 2004). Therefore, comprehensive knowledge of the structure-metabolism relationship, inhibition, induction, and polymorphisms of CYP450s has been obtained through numerous studies. With this knowledge, scientists can design structures for novel drug candidates that avoid CYP450 metabolism to improve drug stability, gain favorable pharmacokinetic properties with suitable half-life and sufficient exposure, and prevent drug-drug interactions (de Groot, 2006).

However, the development of combinatorial chemistry and rational drug design has allowed an increasing number of drugs with novel structures to emerge in recent years that are metabolized independently by CYP450s. In our research, we found that cyanopyrrolidine dipeptidyl peptidase-4 (DPP-4) inhibitors, the epidermal growth factor receptor tyrosine kinase inhibitor-containing alkynyl group, the triazolopyridazine tyrosine-protein kinase c-Met inhibitor, oxazolidinone antibiotics, and drugs with an aliphatic carbon chain underwent non-P450-mediated metabolism (Diao et al., 2013; Kong et al., 2019; Lin, Xie, Gao, Chen, & Zhong, 2014; Meng et al., 2015; Xu et al., 2017). Also, the well-established metabolic stability screening system occasionally fails to identify the involvement of non-P450 enzymes. The best-known example of this phenomenon is the induction of nephrotoxicity by SGX523, a c-Met inhibitor (Diamond et al., 2010). Based on previous in vitro microsome metabolism results, rats and dogs were used in non-clinical toxicity studies. Both species lack the enzyme aldehyde oxidase (AOX) that is, however, present in humans and results in the formation of a major metabolite (2-quinolinone-SGX523) in humans but was minimally detected or not detected at all in the non-human preclinical studies carried out in rats and dogs. This metabolite could cause crystal nephropathy and obstructive renal failure in human patients due to its low water solubility and ease of precipitation in renal tubules. Another example is the development of Lu AF09535, a negative allosteric modulator of human metabotropic glutamate 5 receptors. In vitro preclinical studies failed to identify the main enzyme of AOX in the metabolism of Lu AF09535, and an unexpected low exposure of Lu AF09535 was observed in the first clinical trial in humans.

Establishing metabolic phenotyping and investigating factors that inhibit or induce metabolic enzyme(s) activity for a specific drug are essential steps during drug development. These steps facilitate selecting appropriate species for preclinical toxicity studies and accurate prediction of metabolite-mediated adverse effects and potential clinical drug interactions in humans. They also help eliminate inappropriate candidate compounds such as Lu AF09535 early in the process of drug development.

This review focuses on several common non-cytochrome P450 enzymes involved in the oxidative metabolism of xenobiotics, including flavin-containing monooxygenase (FMO), AOX, xanthine oxidoreductase (XOR), monoamine oxidase (MAO), alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDH). It summarizes the current knowledge concerning factors that influence the non-cytochrome P450 enzyme activities, such as species differences, inhibitors, inducers, and gene regulation. Genetic polymorphisms, while an important aspect, are beyond the scope of this review.

Section snippets

General overview

FMOs are a subfamily of class B external flavoprotein monooxygenases located in the endoplasmic reticulum membranes. The FMO-mediated reaction is initiated in the presence of NADPH where flavin adenine dinucleotide (FAD) obtains two electrons (FADH2) then is oxidized into peroxyflavin (FADH-OOH) by oxygen. In the presence of a soft nucleophilic substrate, one oxygen atom from peroxyflavin transfers to the substrate, and another oxygen atom leaves FADH-OH in the form of water (Fig. 1) (Ziegler,

General overview

AOX belongs to the xanthine oxidase (XO) family of molybdo-flavoenzymes and exists in the cellular cytosol. AOX catalyzes the oxidation of a broad range of endogenous and exogenous substances in the presence of oxygen and water. The molybdenum (Mo) center of AOX attacks the electron-deficient site of the substrate through a nucleophilic mechanism, and Mo (VI) is reduced to Mo (IV). The electron transfer via two Fe-S redox centers converts it into molecular oxygen. Subsequently, molecular oxygen

General overview

XOR is another sub-group in the molybdo-flavoenzyme family that has been widely researched for over 100 years. AOX evolved from the XOR family, and they have many similarities in terms of structure and mechanisms of action. XOR is not limited as a cytosolic enzyme, as it is also located on the outer surface of the cell membrane and is present in milk and the circulation system (Harrison, 2002). XOR exhibits two forms based on its different electron acceptors, including xanthine dehydrogenase

General overview

Monoamine oxidases A and B are flavoprotein monooxygenases located in the mitochondria's outer membrane, and a small fraction adjuvant to the microsomal fraction (Youdim, Edmondson, & Tipton, 2006). The two isoforms demonstrate a similarity of approximately 70% in their amino acid sequences and play a central role in the metabolism of monoamine neurotransmitters and exogenous drug oxidation, such as antidepressant sertraline and anti-migraine drug sumatriptan (Dixon, Park, & Tarbit, 1994;

General overview

Mammalian ADHs are cytosolic enzymes that belong to the medium-chain dehydrogenases/reductases (MDR) superfamily. The catalytic mechanism of ADH is depicted in Fig. 5. In addition to the most well-known ethanol oxidation process, ADH is also involved in some aspects of exogenous drug metabolism. The role of ADH in drug metabolism has been described in a newly published review (Di, Balesano, Jordan, & Shi, 2021), in which the catalytic mechanism, tissue distribution, species differences,

General overview

ALDHs are distributed in every subcellular organelle, including the cytosol, mitochondria, endoplasmic reticulum, and nucleus (Marchitti, Brocker, Stagos, & Vasiliou, 2008). In a typical ALDH-catalysis, the thiol of cysteine in the active site attacks the carbonyl carbon of aldehyde substrate through a nucleophilic reaction and forms an oxyanion thiohemiacetal intermediate. Hydride transfers from the carbon of the oxyanion thiohemiacetal intermediate to the cofactor NAD(P)+, forming the

Concluding remarks

This review summarizes the xenobiotic factors that influence the activities of non-CYP450 enzymes to understand better and evaluate non-CYP450-mediated potential drug interactions in drug development and applications. Non-CYP450 enzymes participate in the primary or secondary oxidative metabolism of xenobiotics or metabolic intermediates in the broad substrate spectrum, such as aldehyde, hydroxyl, heterocycle, and amines. These metabolic enzymes play different roles in the body. Generally, the

Declaration of Competing Interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This research was financially supported by the National Natural Science Foundation of China [81573500].

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    Xiaoyan Pang, Chongzhuang Tang, and Runcong Guo contributed equally to this work.

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