Biochemistry of Drug Metabolizing Enzymes: Trends and Challenges

By Muhammad Sajid Hamid Akash and Kanwal Rehman

Biochemistry of Drug Metabolizing Enzymes: Trends and Challenges is a complete and well-integrated reference on their mechanisms of action, their role in diseases, agents responsible for their deactivation, and their malfunction. Chapters explain the biochemistry of DMEs, including biochemical activation, functions, computational approaches, different contaminants on the action and function of DMEs, and describe the importance of DMEs in the drug development process. Conditions covered include metabolic diseases, cardiovascular diseases, neurological diseases, physiological diseases, xenobiotics and inflammatory responses, and their contribution in the malfunctioning of drug metabolizing enzymes.

This book is the perfect resource for pharmacology and biochemistry researchers to understand the principles of DMEs. Researchers in the corporate environment will also benefit from the comprehensive list of diseases associated with malfunction of DMEs.

• Includes extensive classification of DMEs, their mechanism of action and computational analysis
• Covers the biotransformation of drug by DMEs and the possible impact of environmental contaminants
• Discusses the activity of DMEs in different clinical conditions such as cardiovascular disease, metabolic disorders, inflammation and neurotoxicity
• Includes modern and novel bioanalytical techniques to predict the effect of DMEs

• Language: English
• Publisher: Academic Press
• Release date: May 28, 2022
• ISBN: 9780323951210

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Chapter 1: Biochemical activation and functions of drug-metabolizing enzymes

Anam Shabbira,*; Kamran Haiderb,*; Kanwal Rehmanc; Muhammad Sajid Hamid Akashd; Shuqing Chene    a LIAS College of Pharmacy, Faisalabad, Pakistan

b Department of Pharmacy, University of Agriculture, Faisalabad, Pakistan

c Department of Pharmacy, The Women University, Multan, Pakistan

d Department of Pharmaceutical Chemistry, Government College University, Faisalabad, Pakistan

e Department of Precision Medicine and Biopharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China

* These authors contributed equally in this work.

Abstract

Drug-metabolizing enzymes (DMEs) perform a crucial role in the metabolism of xenobiotics, especially the drugs. These DMEs also lead to the conversion of prodrugs into their active form, which mediates more pharmacological activity. DMEs are categorized based on their method of metabolizing the drugs such as oxidative, reductive, and/or conjugative enzymes. These DMEs have their bio-activation pathways as well as the specific mechanism of action to perform their function. The biotransformation pathway is classified into three phases and produces a metabolite that is pharmacologically inactive and excreted from the body in nontoxic and/or relatively less toxic form. Among the DMEs, cytochrome P450 enzymes (CYP450s) are the predominant players. Certain drugs or chemical substances exhibit inhibitory or stimulatory effects on DMEs, thus altering the normal metabolism of other drugs. Thus, the knowledge of DMEs is quite essential for achieving the maximum therapeutic effects and safeguarding the undesirable attributes like subtherapeutic or toxic effects.

Keywords

Biochemical activation; Drug-metabolizing enzymes; Cytochrome P450; Oxidative enzymes; Conjugative enzymes

Conflict of interest

All the authors declare that they do not have any conflict of interest for this article.

1: Introduction

Drug-metabolizing enzymes (DMEs) are the diverse group of enzymes that are involved in the biotransformation reactions of a vast variety of xenobiotics (Penner, Woodward, & Prakash, 2012a). Biotransformation reactions mainly take place in the liver that accelerate the excretion process of exogenous as well as endogenous substances (Garza, Park, & Kocz, 2019). It is not mandatory that all the biotransformation reactions occur in the liver; however, kidney, adipose tissues, skin, intestine, and lungs may also involve in the biotransformation reactions. The chemical structure of drugs is altered by an array of biotransformation reactions. The enzymes that are involved in catalyzing such reactions can convert the active drug into inactive metabolite, inactive drug to active metabolite, or active drug to active metabolite or cause active drug to become even more toxic (biotoxification). These active metabolites produced after metabolism exhibit the desired therapeutic effect (Coleman, 2020). These biotransformation reactions are categorized into three phases, i.e., phase I, phase II, and phase III reactions (Phang-Lyn & Llerena, 2019). Generally, phase I reaction involves the hydrolysis, oxidation, and reduction, while conjugation reactions come under phase II reactions (Corsini & Bortolini, 2013) as shown in Fig. 1. Phase III indicates the removal of drug or its metabolites mediated by membrane transporters from the body usually via gut, liver kidneys, or lungs (Almazroo, Miah, & Venkataramanan, 2017). Among the DMEs, cytochrome P450 (CYPs) supervise the metabolic process in comparison to other metabolizing enzymes. Subsequently, DMEs also provide the protection against environmental toxins (Atkins, 2020). Additionally, other than CYPs, flavin monooxygenases (FMOs) also contribute to biotransformation via oxidation of drugs. Among the five isoforms of FMOs, FMO3 is also present in the adult liver, while FMO1 is present in the fetal liver (Siddens, Krueger, Henderson, & Williams, 2014). Other than these enzymes, aldehyde oxidases are also involved in mediating the various biotransformation reactions (Romão et al., 2017; Terao et al., 2016).

Fig. 1

Fig. 1 Phases of drug metabolism. Phase I involves oxidation, reduction and hydrolysis whereas phase II involves conjugation reaction.

The major contribution in the metabolism of a drug is made by oxidative metabolic enzymes, which may be involved in carrying out the metabolism of about 75% of the total marketed drugs. Oxidative enzymes include CYP450, aldehyde dehydrogenase, alcohol dehydrogenase, prostaglandin H synthase, and molybdenum hydroxylase (Penner, Woodward, & Prakash, 2012b). Alternatively, polymorphism in DMEs has also a significant impact with respect to pharmacological contexts, for instance a decrease in function variants provokes the minimum clearance and elevates the plasma concentration of drug to be metabolized. Whereas an increase of functional variation of DMEs gives rise to maximum clearance and minimum drug plasma concentration (Ortiz de Montellano, 2013).

In this chapter, we have discussed in detail the contribution of DMEs in the biotransformation of drugs. These DMEs are generally specific in their nature as well as functions. As these may be oxidative, hydrolytic, reductive, and conjugative, depending upon their activity. Furthermore, we have also focused on the effect of genetic and environmental factors on the biochemical activation of DMEs, as these are involved in the activation and/or inhibition of DMEs (Penner et al., 2012a).

2: Phase I drug-metabolizing enzymes

2.1: Oxidative enzymes

These enzymes are CYP450, alcohol dehydrogenase, prostaglandin H synthase, molybdenum hydroxylases, and aldehyde dehydrogenases. It is estimated that more than 75% of the marketed drugs are metabolized by these enzymes (Guengerich & Rendic, 2010).

2.1.1: Cytochrome P450

Cytochrome P450 (CYP450) enzymes have diverse distribution in the living organisms as they perform several important functions such as drug metabolism, steroid biosynthesis, along with degradation of xenobiotics (Gilani & Cassagnol, 2020; Li et al., 2020). Their activity and distributions are not only limited in the human but also present in the animals, microbes, viruses along with plants, suggesting their unique and specific distribution in nature (Nelson, 2018; Zhang & Li, 2017). Additionally, CYP450 enzymes are considered more significant oxidative enzymes compared to other oxidative metabolizing enzymes, as these enzymes contributed to a majority of oxidative reactions. These reactions may involve the epoxidation, hydroxylation, sulfoxidation, and deamination (Guengerich & Munro, 2013; Rudolf, Chang, Ma, & Shen, 2017; Zhang & Li, 2017).

Biochemical activation of CYP450 enzymes

Biochemical activation of CYP450 enzymes necessitates two electrons for the event of per-substrate oxidation. These electrons are generally supplied by NADPH reductase in all mammalian CYP450 enzymes (Eq. 1) (Iyanagi, Xia, & Kim, 2012). During the biochemical activation of CYP450 reductase, the flavins prosthetic group uncouples these two electrons that are supplied by NADPH and transfer them toward the heme group of CYP450. Similarly, electrons donated by CYP450 reductase can be accepted by CYP450 in hypoxic conditions and utilized for catalyzing the reduction reactions (Ortiz de Montellano, 2013).

si1_e

   (1)

However, there are some undesirable factors associated with CYP450 that include less stability, increased cost for the cofactors, reliance on the redox partner, and minimum catalytic efficiency (Bernhardt & Urlacher, 2014). Generally, four components accomplish the catalytic activity of CYP450 system involving substrate, CYP450 enzymes, redox partner, and the cofactor that lead toward the successful accomplishment of a particular task (Schmitz, Rosenthal, & Lütz, 2019; Urlacher & Girhard, 2019). Various CYP450 enzymes can directly utilize the H2O2 for the electron as well as a proton donor. The continuity of CYP450 catalytic cycle is dependent on persistent electron transfer via redox partner to heme group. CYP450 enzymes are categorized into various classes and subclasses based on the sequence homology of amino acids. CYP450 enzymes are narrowed to subclasses, i.e., CYP1, CYP2, CYP3, and CYP4. These subclasses are then further categorized into different isoforms (Prakash & Vaz, 2009). The most expressing forms of CYP450 are CYP: 1A2, 3A4, 2C8, 2C9, and 2E1 found in liver, whereas 2A6, 2D6, 2B6, 2C19, and 3A5 are comparatively less abundant isoforms. On the other hand, CYP: 1A1 and 1B1 are the forms that are expressed outside the liver (Wei, Ang, & Zhao, 2018; Xu & Du, 2018). The mechanism of action of CYP450 includes three possible ways: (i) insertion of an oxygen atom to sulfur or nitrogen electron pairs, leading toward S-oxide or N-oxide formation, (ii) addition of oxygen into N glyph_sbnd N or C glyph_sbnd H bond and ultimately the formation of hydroxyl derivatives, and (iii) catalyzing the epoxidation of aromatic-bonds (de Montellano, 2015). Correspondingly, the mechanism of action of CYP450 suggests that alcohol is the terminal product if oxidation occurs within the hydrocarbon chain. Hydroxylation at C glyph_sbnd H bond adjacent to heteroatom leads toward the formation of chemically unstable product, which further undergoes the elimination and forms two fragments; one with hydroxyl or thiol group according to the nature of heteroatom and the other with a carbonyl group (Ortiz de Montellano, 2013).

Functions

Many diagnostics, clinical, and epidemiological research has suggested that CYP450 enzymes have a crucial role in the development as well as prevention of cancer. Hence, the inhibition of CYP450 enzymes is linked with the reduction of cancer that depends upon the type of cancer, its etiology, and treatment. For this reason, many strategies should be developed for a particular understanding of DMEs in cancer therapy as well as chemoprevention (Alzahrani & Rajendran, 2020). Different isoforms of CYPs and subsequent drugs as their substrate for metabolism are summarized in Table 1. The members of CYP1 family are closely related to metabolic activation of many mutagens and pro-carcinogens. Likewise, CYP1B1 may impart a significant part in susceptibility to ovarian and mammary cancer by its participation in the metabolism of estrogen, along with many cancers related to the activation of polycyclic aromatic hydrocarbons present in cigarette smoke (Tsuchiya, Nakajima, & Yokoi, 2005). CYP450 enzyme system plays a critical role in the metabolism of many important endogenous compounds involved in the biosynthesis of cholesterol and its conversion into bile acids, steroid hormones production, estrogens and androgens, omega-hydroxylation of fatty acids metabolism of vitamin D3 to active metabolite, i.e., 1,25-dihydroxyvitamin D3, and also includes the biotransformation of various exogenous xenobiotics. The potential toxicity and pharmacological effectiveness of various drugs are greatly influenced by their process of metabolism, most of which is achieved by CYP450 systems (Cederbaum, 2015). The microsomal P450 enzymes present particularly in lung, skin and liver are crucial in converting the lipophilic xenobiotics such as insecticides, drugs, carcinogens, environmental pollutants,` and food additives to more comparatively polar compounds to make these more easier to eliminate from the body. CYPs found in intestine, particularly CYP3A4, might be very helpful in increasing the first-pass metabolism of various drugs. CYP450 systems are known to be very important in the recognition of their cellular detoxification, as a number of substances lost their potency or activity after being converted to more polar forms and metabolites that are excreted from the body. (McKinnon, Sorich, & Ward, 2008).

Table 1

Finally, summarizing the above discussion, we can conclude that these are the key elements that have numerous important tasks in the body and can be considered as novel targets for prevention along with treatment of many diseases, especially cancer. Understanding of these CYP450 enzymes leads toward the development of many economical compounds for multiple therapeutic targets.

2.1.2: Flavin-containing monooxygenases

Flavin-containing monooxygenases (FMOs) are also broadly distributed in the living organisms involving plants, bacteria, fungi, vertebrates, and invertebrates. These are present in the endoplasmic reticulum membrane in eukaryotes and metabolize the xenobiotics via phenomenon of oxidation (Rendic & Guengerich, 2015; Veeravalli et al., 2014).

Biochemical activation of flavin-containing monooxygenases

Biochemical activation of FMOs is dependent on the oxygen and NADPH. The biochemical activation and mechanism of action of FMOs involve various successive steps, i.e., first, NADPH donates an electron to FAD, causing reduction of FAD and converts it into FADH2. Second, this FADH2 is oxidized by oxygen and changes into reactive complex FADHOOH (Fig. 2). Finally, the mechanism of action involves the reaction of a reactive complex with substrate and changes it into the product, and enzyme comes back into initial stage via loss of water molecule (Coleman, 2019).

Fig. 2

Fig. 2 Biochemical activation/catalytic cycle of FMOs. Numerous enzymes are involved in the activation of FMO; first, NADPH cofactor bind to the enzymes and FAD is reduced by two electrons. Reduced FAD (FADH 2 ) reacts rapidly with O 2 and formation of FADOOH. In the next step, one water molecule is lost from FADOOH + NADP + and formation of FADOH, the reaction of FADOH is reversible; either to activate FAD or electron-transferring-flavoprotein (ETF). In the presence of ETF-oxidoreductase enzyme, ETF activates an electron transport chain. ETF , Electron-transferring flavoprotein; FMO , Flavin-containing monooxygenase; NADP , Nicotinamide adenine dinucleotide phosphate; FAD , Flavin adenine dinucleotide.

Additionally, FMOs oxygenate soft nucleophile-containing xenobiotics and drugs having heteroatoms like phosphorus, nitrogen, oxygen, and sulfur in liver and extrahepatic tissues. This FMOs gene family includes five distinct enzymes designated as FMOs1–5 (Carzaniga et al., 2017). FMOs are mainly present in fetal liver and kidney while not present in the adult liver. Similarly, FMO2 is present in kidney as well as lungs. While FMO3 is the major enzyme mediating metabolism in the adult liver. Yet, it is not responsible for all drugs metabolism in the liver, as many drugs are a substrate for CYP450 and others. Trimethylamine N-oxide is an end product of FMO3-mediated reactions; its increased plasma level is implicated in cardiovascular diseases (Mügge et al., 2020; Phillips & Shephard, 2020; Risk et al., 2016). Recently, preclinical studies have suggested that trimethylamine N-oxide is an independent risk factor of cardiovascular disease, and its increased level is not just a biomarker but also a causative factor of the disease (Heianza, Ma, Manson, Rexrode, & Qi, 2017; Phillips & Shephard, 2020; Qi et al., 2018; Schiattarella et al., 2017).

Functions

FMOs are the most significant non-CYP450 enzymes that play a crucial role in the phase I metabolism of drugs. FMOs were involved in about 6% of all phase I reactions and hence contributing nearly 2.5% for all metabolic reactions. These enzymes’ system performs versatile detoxification of xenobiotics, via conversion of lipophilic xenobiotics into the metabolites that are immediately excreted (Cruciani et al., 2014; Testa, Pedretti, & Vistoli, 2012). The substrates for FMOs are crucial drugs that may include nicotine, fluoxetine, chlorpromazine, itopride, imipramine, clozapine, tamoxifen, cocaine, and methimazole. While the endogenous substrate involves methionine, trimethylamine, cysteamine, triamine, and catecholamines (Başaran & Benay, 2017; Hayes'', 2010).

2.1.3: Monoamine oxidases

Monoamine oxidases (MAOs) are flavoproteins or flavin-containing amine oxidoreductases that are present in the outer membrane of mitochondria and responsible for catalyzing the deamination as well as dehydrogenation of amines, including neurotransmitters and xenobiotics (Maggiorani et al., 2017; Tzvetkov, Stammler, Hristova, Atanasov, & Antonov, 2019) (Table 2).

Table 2

Biochemical activation of monoamine oxidases

The mechanism of action of these MAOs in the catabolism of noradrenaline, as well as amine neurotransmitters, involves the oxidative removal of the amine group. There are two isozymes of MAOs (MAO-A and MOA-B) that mainly differ in their substrate affinities and organ distribution. This loss of amine group leads toward the inactivation of transmitter by preventing its binding to a prosthetic receptor that results in the formation of vanillylmandelic acid, which is a urinary metabolite (Penner et al., 2012b). Hence, the treatment approach for neurodegenerative disorders involves the development of selective MAO inhibitors. Correspondingly, a number of MAO inhibitors have been developed and approved in the world for combating neurological disorders (Fowler et al., 2015; Tzvetkov et al., 2019). A classification of MAO inhibitors on the basis of their differential selectivity for subtypes of MAO and reversibility with MAO is demonstrated in Table 4.

Alternatively, it is also well established that irreversible inhibition of MAO is interlinked with safety complications as well as adverse pharmacological effects (Kumar, Mantha, & Kumar, 2016). So, the biochemical activation and function of MAOs should be carefully understood.

Functions

Various biological functions performed by MAOs have been chiefly represented via their enzymatic reactions on biogenic-amines and the subsequent H2O2 production. Hence, the potential of MAOs has been summarized dominantly in reference to the metabolism of amine along with the formation of H2O2. MAOs play a crucial role in the center and peripheral nervous system via altering monoamine neurotransmitters (Yeung, Georgieva, Atanasov, & Tzvetkov, 2019). Hence, these MAOs are the important regulators of neurotransmitters levels, and subsequently, disturbance of MAOs is interlinked with various metabolic disorders such as inappropriate MAO-A genotype is engaged with autism (Cohen et al., 2011). Both isoforms seem to play their specific roles and functions in an organism as referred by their different expression profiles. Likewise, increased MAO-A levels are also associated with many psychiatric and neurological diseases like social anxiety and depression. In the same manner, there is an association between elevated MAO-B levels with neurodegenerative diseases like Parkinson’s and Alzheimer’s that is expected to be present (Kasture et al., 2009; Tong et al., 2013).

2.1.4: Molybdenum hydroxylases

Molybdenum hydroxylases are enzymes consisting of two equal size subunits, one subunit comprises of one FAD molecule while the other subunit contains one molybdenum atom in the form of a cofactor. This enzymes family is composed of two isoforms such as aldehyde oxidase (AO) and xanthine oxidoreductase (XO) (Fowler et al., 2015). These enzymes are generally present in many tissues such as kidney, lung epithelial cells, heart, liver, and placenta (Prakash & Vaz, 2009). However, tissues distribution has shown differences between XO and AO, having increased expression of XO in lactating mammary glands as well as proximal intestine, while AO is highly expressed in brain, lung, liver, and kidney (Garattini, Fratelli, & Terao, 2008; Prakash & Vaz, 2009).

Biochemical activation of molybdenum hydroxylase

Molybdenum hydroxylase is involved in both kinds of reaction involving oxidation as well as reduction, utilizes molecular oxygen as cosubstrate, and is then inserted into a substrate (Prakash & Vaz, 2009). Generally, oxidation reaction catalyzed by molybdenum hydroxylase involves the nucleophilic attack on electron-deficient carbon atom that leads toward the formation of either carboxylic acid or cyclic lactam from aldehydes and aromatic N-heterocyclic, respectively. Additionally, the mechanism of action of molybdenum hydroxylases also involves the catalytic reduction of S- and N-functional groups such as sulfoxides and N-oxides (Waller & Sampson, 2017). Both AO and XO differ in some specifications such as AO contributes to oxidizing aldehydes into carboxylic acids and also involved in the metabolism of various clinically important drugs, which may include ziprasidone, famciclovir, zonisamide, and zaleplon. Whereas XO has relatively narrow substrate specificity in comparison to AO. XO has also contributed to the oxidation of various chemotherapeutic agents (Garattini et al., 2008; Prakash & Vaz, 2009). Many factors affect these drug-metabolizing enzymes, as shown in Fig. 3.

Fig. 3

Fig. 3 Factors affecting the drug-metabolizing enzymes. Here is a representation of multiple factors affecting drug-metabolizing enzymes such as age, gender, polymorphism, diseased state, and various exogenous substances. These alter the normal metabolism of drugs and leads to undesirable effects such as subtherapeutic effect or toxic effect.

Functions

Molybdenum hydroxylase is essential for the maintenance of normal levels of XO in liver. XO and AO have been associated with many diseases such as ischemia and neurodegenerative disorders, respectively. These enzymes play a vital role in detoxification. AO oxidation may alter the production of ROS and may also lead to the development of neurological disorders such as schizophrenia, Parkinson's and Alzheimer’s disease. Additionally, AO and XO have been involved in the pathophysiology of many clinical syndromes such as alcohol-induced liver injury, tissue injury, and ischemia reperfusion injury. XO and AO both play a significant part in the metabolism of many exogenous compounds (Kitamura, Sugihara, & Ohta, 2006; Schwarz & Belaidi, 2013).

2.2: Reductive enzymes

Reductive enzymes include aldo-keto reductases, quinone reductase, and azo-reductases, which catalyze the reductive reactions. These enzymes are known to found along the periplasmic side of the membrane of cytoplasm and play a significant role in various energy conserving respiratory phenomena.

2.2.1: Aldo-keto reductases

The aldo-keto reductase (AKR) protein superfamily constitutes more than 190 members that are categorized into 16 families and generally present in all phyla. Such enzymes also reduce the carbonyl substrates such as sugar aldehyde, keto-prostaglandins, lipid peroxidation by-products, retinal, and keto-steroids (Penning, 2015). Furthermore, the carbonyl group has relatively increased chemical reactivity and ultimately leads toward a more rapid reaction with nucleophilic center, which may be a protein side chain constituting primary amino acid substituents. Therefore, alcoholic conversion from the aldehydes leads toward the diminished chemical reactivity (yet biological activity is not mandatory to decrease). Subsequently, this ends up with a mode of inactivation as well as detoxification (Barski, Tipparaju, & Bhatnagar, 2008). Actually, these aldo-keto reductases are nicotinamide adenine dinucleotide phosphate-dependent oxidoreductases and catalyze various alpha- and beta-unsaturated ketones or carbonyl groups on endogenous as well as exogenous substances (Chen, Jin, & Penning, 2015).

Bioactivation and function of aldo-keto reductase

Human AKR enzymes may cause the bioactivation of various endogenous substances (Jin & Penning, 2007). The biochemical activation and function of AKRs involve the binding of substrate and cofactor at different sites of enzymes. Hydride transfer from NADPH to a substrate is also a stereospecific (Kang & Kim, 2008). The members of AKR1 family constitute the maximum of human AKRs, these include human homologues of hydroxysteroid dehydrogenases, steroid 5β-reductase, and aldo-reductases.

Functions

AKRs of human beings are engaged in the metabolism of various synthetic hormones, CNS drugs, and chemotherapeutic agents. Along with this, AKRs are also involved in the detoxification of various substances such as nicotine-derived carcinogens (Penner et al., 2012a) and electrophilic substances such as 4-hydroxy-trans-2-nonenal compounds, produced mainly in oxidative stress situations. Additionally, they may be associated with the regulation of pre-receptors hormones, hence, to participate in transcriptional gene management/control (Petrash, 2004).

2.2.2: Quinones reductases

The quinones reductases (QRs) are the enzymes having two isoforms (QR1 and QR2), these are responsible for undergoing the alteration of quinones into hydroquinone’s, and these pathways are termed as a crucial detoxification pathway catalyzed by QRs. Subsequently, their physiological contribution in the eukaryotes dictates their function other than the detoxification and imparts in oxygen stress response. Similarly, QRs in mammalian are considered as a significant molecular switch that participates by regulating the life span of transcription factors like p53 and so contributing to cell transformation as well as apoptosis (Cuendet, Oteham, Moon, & Pezzuto, 2006; Deller et al., 2008). Many drugs involve the quinone pharmacophore, and this is a crucial part of antitumor drugs. Mostly, quinones are the prodrugs and mediate their activity upon reduction.

Biochemical activation of quinones reductase

QRs are the enzymes that induce the reduction of quinones and convert them into hydroquinone or semi-quinones. Hence, quinones are designed in such a way that upon bioactivation, these lose a leaving group and generates a reactive electrophile that mediates higher pharmacological activity in comparison to parent quinone (Siegel, Yan, & Ross, 2012).

NADPH mediates a primary role in the biochemical activation of QRs via donation of electrons. The biochemical activation and mechanism of action of QRs involve the following steps. First, NADPH binds with QRs and causes the reduction of FAD cofactor and released, causing the quinone substrate to be reduced upon binding with these enzymes. This further leads toward the conversion of the prodrug into the active form (Penner et al., 2012b).

Functions

Quinone reduction via QRs is a vital detoxification pathway and is now considered as a novel strategy for chemoprevention, i.e., minimizes the risk of cancer. The best way for detoxification is to minimize the levels of such metabolic enzymes that participate in the formation of reactive species, while the elevation of such enzymes generally phase II that are involved in deactivating the electrophile and radicals (Cuendet et al., 2006).

2.3: Hydrolytic enzymes

Hydrolytic enzymes are involved in catalyzing the hydrolysis and also known as hydrolases and cause splitting of various molecules such as ester, glycoside, and peptidase. Hydrolytic enzymes act as a biochemical catalyst that causes the splitting of high molecular weight biomolecules, such as fat, carbohydrates, proteins, and lipids, into their simple units (McKinney & Cravatt, 2005). Subsequently, lysosomal enzymes constituting β-galactosidase, acid proteinase, phosphatases, ribonucleases, and carboxylesterases are considered to participate crucial role in brain tumors (Prabha, Ravi, & Swamy, 2013). Many studies have indicated the contribution of hydrolytic enzymes in various pathological conditions like brain tumors (Wielgat et al., 2006) and also in several inherited metabolic disorders as well as neurodegenerative diseases like Alzheimer’s disease. Alkaline and acid phosphatases are two phosphomonoesterases that are highly studied in tumors in comparison to all other hydrolases (Prabha et al., 2013; Wielgat et al., 2006).

2.3.1: Esterases

Esterase is an enzyme that breaks down the ester-containing compounds into alcohol and acid during a chemical reaction along with the production of water. A large number of different esterases can exist in their different structure, specificity, and their biochemical and biological functions (Krisch, 1971; Wong, 2006).

Biochemical activation of esterases

Esterases are responsible for prodrug activation as well as detoxification of many drugs via hydrolyzing the compounds that contain ester, thioester bond, and amide in their structure. Among esterases, carboxylesterases (containing carboxyl group) are mainly involved in the hydrolysis of various drugs (Oakeshott, Claudianos, Campbell, Newcomb, & Russell, 2010). It is also observed that indiplon, flutamide, rifamycins (rifapentine, rifampicin, and rifabutin), and phenacetin are hydrolyzed via arylacetamide deacetylase, expressed promptly in gastrointestinal as well as human liver. Phenacetin is a prodrug and has been withdrawn from market owing to associated renal failure along with methemoglobinemia. Esterases are considered to participate in the metabolism of 10% of clinically marketed drugs (Fukami, 2015).

Functions

Currently, esterases have attained more importance owing to their pharmacological as well as toxicological functions. Many esterases are still unknown that contribute to hydrolyzing the acyl-glucuronides of the drugs (Fukami & Yokoi, 2012). For this reason, further studies should be conducted and carefully evaluated for understanding the pharmacological as well as toxicological importance of esterases in drug therapy (Fukami, 2015; Fukami & Yokoi, 2012).

2.3.2: Epoxide hydrolases

Epoxide hydrolases (EHs) are among the class of enzymes that are involved in epoxides biotransformation. There are five isoforms of EHs, two EHs isoforms are of great importance, including soluble EH, and microsomal EH. The remaining three isoforms of EHs, are known for limited substrate specificity and are merely aligned toward the endogenous epoxides formation, namely the cholesterol, hepoxilin, and leukotriene A4 EH (El-Sherbeni & El-Kadi, 2014).

Biochemical activation and mechanism of action of epoxide hydrolases

The basic molecular mechanism of hydration of epoxides by soluble EH and microsomal EH is almost identical (Morisseau & Hammock, 2005); it comprises of two important chemical steps. In the first step, an ester is formed in between the enzyme and the epoxide substrate and the second step is to release the dihydrodiol substrate. The soluble EH catalytic site containing the essential amino acid, i.e., Asp334 & Asp495 and His523 residues, along with two more residues, i.e., Tyr382 and Tyr465 that provide the required support to catalytic triad (Gomez, Morisseau, Hammock, & Christianson, 2004; Morisseau & Hammock, 2005).

Functions

EHs play a multifactorial role in the body including genotoxic epoxides detoxification (Decker, Arand, & Cronin, 2009). Soluble EHs plays a major role in the metabolism of arachidonic acid, which is crucial for the modulation of renal, cardiac, and vascular functions (Morisseau & Hammock, 2005). Hence, EHs impart a very critical role in an in vivo reactive metabolites detoxification by enhancing the epoxide hydrolysis to comparatively more soluble and rapidly excretable metabolites. Furthermore, EHs are responsible for regulating various pathological and physiological functions via controlling pharmacologically active mediators of epoxide at tissue levels.(Yu et al., 2000).

3: Phase II drug-metabolizing enzymes

Phase II DMEs are also known as conjugative DMEs including UDP-glucuronosyl transferases (UGTs), glutathione transferases (GSTs), sulfotransferases (SULTs), methyl-transferases, and N-acetyl transferases (NATs). Phase II DMEs carry out biotransformation reactions namely glucuronidation, glutathione conjugation, sulfonation, methylation, and acetylation. All the cofactors of Phase-II reactions react with suitable functional groups present on parent compounds (xenobiotics) or with conjugative products introduced by phase I biotransformation reactions, as shown in Fig. 4.

Fig. 4

Fig. 4 Schematic representation of phase II DMEs involved in the biotransformation of drugs.

3.1: UDP glucuronosyl transferases

Uridine diphospho-glucuronosyltransferases (UGTs) come under the phase II enzyme classification and are responsible for catalyzing all glucuronidation-related reactions located in endoplasmic reticulum of the kidney, liver, intestine, spleen, skin, nasal mucosa, and mammary glands (Riches & Collier, 2015). Based on sequence homology, the UGT enzymes family is further divided into two subclasses, first one is UGT1 (including 1A1, 1A3, 1A4, 1A5 1A6, 1A7, 1A8, 1A9, and 1A10) and second one is UGT2 (including 2A1, 2B4, 2B7, 2B10, 2B11, 2B15, 2B17, and 2B28). All the drugs comprising of a vast range of functional groups such as alcohols, phenols, thiols, carboxylic acids, aliphatic amines, and aromatic amines, all these groups act as substrates for UGTs. The glucuronidation reaction mainly occurs at the sites of electron-rich nucleophiles like N, S, or O heteroatoms (Rowland, Miners, & Mackenzie, 2013).

3.1.1: Biochemical activation of UGTs

In glucuronidation, the active site of this enzyme transfers the glucuronosyl group from the cofactor uridine-5′-diphospho-glucuronic acid (UDPGA) to the suitable compounds containing oxygen, nitrogen, carboxyl, or sulfur functional groups (Kaur et al., 2020). Substrates that are nonpolar in nature can easily diffuse from the endoplasmic reticulum membrane and then can be conjugated in the lumen of endoplasmic reticulum. Even so, UDPGA should be transported to the endoplasmic reticulum, and the resultant product formed should be carried out of the endoplasmic reticulum (Kobayashi, Sleeman, Coughtrie, & Burchell, 2006). It is reported for various transporters that are involved in the transportation of glucuronides from the endoplasmic reticulum lumen to cytoplasm, predominantly located in the membrane of the endoplasmic reticulum (Csala et al., 2004).

3.1.2: Functions

UGTs are predominantly among the important conjugating enzymes of liver, which are responsible for causing the rapid elimination of various endobiotics (Sheehan, Meade, Foley, & Dowd, 2001) along with xenobiotics (drugs, environmental chemicals, and pollutants) (Collier, Yamauchi, Sato, Rougée, & Ward, 2014). These compounds become easily soluble in the blood due to glucuronidation and are thereby excreted out via kidney. The glucuronidation process is accomplished by UGTs, and the glucuronidated products formed are secreted in urine and bile. UGTs are predominantly expressed in liver, but these are also present in abundance in the GIT, kidneys, brain, reproductive tissues, and lungs (Riches & Collier, 2015). Glucuronoconjugation by UGTs also occurs in many endogenous compounds such as steroids, thyroxine, and bilirubin. This particular detoxification pathway catalyzed by UGTs is present in many tissues in case of bilirubin that is the most significant.

3.2: Glutathione S-transferases

Glutathione S-transferase (GST) is a major enzyme among phase II detoxification enzymes mainly present in the cytosol. GSTs protect various cellular macromolecules from reactive electrophiles attack. Conclusively, GSTs catalyze all the reactions during which endogenous and exogenous xenobiotics are being conjugated with glutathione (GSH) (Sheehan et al., 2001). There are 13 different forms of GST subunits that have been recognized in human beings such as GSTA1, GSTA4, GSTM1, GSTM5, GSTP1, GSTT1, and GSTT2, and GSTZ1 belonging to different classes.

3.2.1: Biochemical activation of GST

GST is a dimeric enzyme having two subunits, each subunit of that has an active region along with two distinct functional sites: (1) GSH hydrophilic binding site (G-site) and (2) hydrophobic binding site of electrophilic substrates having different structures (H-site). The hydrophilic G-site is considered as a conservative site, whereas the hydrophobic H-site has an affinity for different substrates that regulate GST isozymes specificity (Deponte, 2013). The connection between these two GST subunits constructs an intrasubunit binding site for the ligands, which promotes the conjugate formation of GSH through one subunit and then insulated from the next subunit and therefore restrict the inhibition of product (Wu & Dong, 2012). In GSTs catalyzed biotransformation reactions, after carrying the electrophilic substrates to the vicinity of GSH on the active site of GST, this enzyme attacks the substrate as a nucleophile. GSTs distribution is also very diverse among different organs and mainly depends on sex, species, and age. GSTM1, GSTA1, and GSTA2 are expressed highly during the entire life, whereas GSTP1 expressed itself in fetal and embryonic organs that slowly diminished in the prenatal period (Singh, 2015).

3.2.2: Functions

In the last few years, it was observed that GSTs also have some nonenzymatic properties along with the catalytic activity as possessed by the conjugating GSH. GSTs regulate various signaling pathways associated with cell differentiation, cell division, and cell death (Cho et al., 2001). Among several isozymes of GST, GSTP1-1 was found to be greatly expressed in cancerous cells, where GST provides resistance to these cells against anticancer drugs (Ricci et al., 2005). Hence, the GST-inhibiting agents, particularly for GSTP1-1, may provide effective chemotherapy for diseases associated with anomalous cell proliferation and cancer. Moreover, GSTs enzymes owing to remarkable genetic polymorphisms in human beings are categorized into distinct classes, which contributes to about 30% sequence identification, and these are marked using Greek letters: alpha (α), mu (μ), omega (ω), pi (π), sigma (σ), theta (θ), and zeta (ζ). Although these classes having some genetic similarities, the affinities of these enzymes for their subsequent substrates vary owing to genetic mutation and also have discrete substrate specificity (Kaur et al., 2020). These classes of GSTs, their substrates, and functions are given in Table 3.

Table 3

3.3: Sulfotransferase

Sulfonation is a crucial biotransformation reaction responsible for metabolizing numerous xenobiotics, endogenous compounds, and drugs, which is catalyzed by a large supergene family namely sulfotransferases (SULTs). Two classes of SULTs are known to be found in mammals:

(1)Membrane-bound SULTs present in Golgi apparatus, which are involved in several biological processes including cell adhesion, axon function, T-cell response, cell proliferation, modulation of bacterial and viral infections (Grunwell & Bertozzi, 2002; Grunwell, Rath, Rasmussen, Cabrilo, & Bertozzi, 2002).

(2)Soluble SULTs present in cytoplasm responsible for sulfonation of many drugs (Gamage et al., 2006).

3.3.1: Mechanism and function of SULTs

SULTs are found predominantly in kidney, liver, intestine, platelets, brain, and platelets (Gamage et al., 2006). Sulfate conjugation by SULTs is generally a multistep process, which involves the inorganic sulfate activation, first by converting it from ATP to adenosine-5′-phosphosulfate (APS) and further converting it to an activated form which is termed as PAPS, 3′-phosphoadenosine-5′-phosphosulfate, as presented in the equations. Every step is catalyzed by a particular enzyme in the cytosol.

si2_e

All the members belonging to the SULTs family are involved in transferring a sulfuryl group (O2SX2) from the cofactor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to the most suitable hydroxyl group (OH) of different categories of substrates. The general mechanism by which SULTs transfer a sulfuryl group from PAPS substrate to an acceptor group is given in the following equation:

si3_e

The structure of SULTs enzymes is chiefly recognized via a histidine amino acid residue present in its active site (Hirschmann, Krause, & Papenbrock, 2014). Until now, knowledge or data on SULTs at the structural, molecular, and functional levels have lagged behind many other DMEs systems. On the other hand, the advancement over the past few decades has clearly indicated that SULTs enzyme system is consisting of a diverse family of enzymes that can be distinguished remarkably in their regulation, localization, and metabolic status (Gamage et al., 2006).

3.4: Methyltransferases

Methyltransferases (MTs) are also a part of phase II DMEs, responsible for catalyzing the S-adenosylmethionine (SAM)-dependent methylation of a vast range of xenobiotics and endogenous substrated (DNA substrates and proteins), which contains the functional groups such as C, N, O, or S (Gonzalo et al., 2006). There are several types of MTs, but the major ones involved in catalyzing the methylation of xenobiotics are thiopurine methyltransferase (TPMT), nicotinamide N-methyltransferase (NNMT), catechol-O-methyltransferase (COMT), histamine N-methyltransferase (HNMT), and thiol methyltransferase (TMT) (Penner et al., 2012a).

3.4.1: Functions of methyltransferases

Indolethylamine-N-methyltransferases (NNMTs) will mainly catalyze the N-methylation of a wide range of primary as well as secondary amines such as endogenous amines (such as tyramine, serotonin, and dopamine) and several drugs (such as normorphine, amphetamine, and desmethylimipramine). Among MTs, DNAMTs have a diverse mechanism of action and are responsible for performing an essential role in genomic disruption and integrity. It is also well documented that DNA-MTs are needed for carrying out transcriptional silencing of different sequence classes, such as transposable elements, imprinted genes, and genes present on inactive X-chromosome (Robertson, 2005), and transcriptional silencing of such sequences is necessary for maintaining the stability of chromosomes. The O-methylation of various xenobiotic and endogenous-catechol compounds is catalyzed by COMT. The substrates for COMT are basically neurotransmitters (including epinephrine, norepinephrine, and dopamine) and catechol drugs (antihypertensive-methyldopa and the anti-Parkinson’s disease agent L-dopa). S-methylation is considered very important for the metabolism of drugs, which are excreted in the forms of sulfoxides and sulfones, including captopril, 6-mercap topurine (6MP), D-penicillamine, and azathioprine.

3.5: N-acetyl transferases

N-acetyl transferases (NATs) are enzymes that are known to be present in the liver and other tissues of mammals. Two distinct isozymes of NATs express in human beings, including NAT-1 and NAT-2, showing 75%–90% of sequence homology. NAT-1 is highly expressed itself in several tissues; however, NAT-2 is primarily expressed in liver and gut tissues (Sim, Abuhammad, & Ryan, 2014).

3.5.1: Mechanism and function of NATs

NATs are among the very important carcinogens and DMEs, which catalyze the shift of acetyl group from a suitable donor-like acetyl coenzyme A (CoA) to a heterocyclic amine, aromatic, hydrazide, or N-hydroxylamine arylamine and hydrazine acceptor substrate. The basic mechanism of the acetyl group catalyzed by NATs undergoes double displacement, a presumed ping-pong mechanism as given in the following equation:

si4_e

This reaction generally progressed in 2 steps: formation of an intermediate, i.e., acetyl-CoA by the transfer of acetyl group from acetyl-CoA and the regeneration of enzyme along with the subsequent acetylation of arylamine (Gibson & Skett, 2013). Owing to their structural resemblance with the substrates, some substances may act as reversible inhibitors toward NATs, while other substances, such as p-chloromercurybenzoate and iodoacetate, are irreversible inhibitors .

NATs are known to be present in prokaryotes and eukaryotes, and these might also have some endogenous role along with metabolism of drugs, such as NAT present in Mycobacterium tuberculosis is reported to have a critical role in lipid biosynthesis of cell wall, and hence considered as a potential target of various drugs (Westwood & Sim, 2007).

3.6: Amino acid conjugation

Conjugation of amino acids is primarily seen in substrates having aromatic hydroxylamines or carboxylic acid. Conjugation of amino acid with carboxylic acids comprises of three basic steps:

(a)Reaction of CoA with acyl adenylate producing a very reactive acyl-CoA;

(b)Carboxylic acid activation via ATP to produce a pyrophosphate and acyl adenylate;

(c)Linkage between the activated acyl group and the amino group of amino acids.

The carboxylic acid group of the amino acid conjugates with aromatic hydroxylamines like serine and proline. Aminoacyl-tRNA caused the activation of the amino acid, which then reacts with aromatic hydroxylamine to produce reactive N-ester (Kato & Yamazoe, 1994). Such a type of conjugation of xenobiotics chiefly depends on groups of steric hindrance that may be present around carboxylic acid or found around the aromatic ring system. However, conjugation of carboxylic groups to amino acid is considered a detoxification pathway and conjugation of carboxylic acid to glucuronide may result in toxicity. Furthermore, hydroxylamines conjugation generates N-esters that can produce carbonium ions and electrophilic nitrenium upon degradation (Parkinson & Ogilvie, 2008). All the phase II DMEs along with the type of conjugation reaction involved, their enzymatic isoforms, cofactor used, cellular localization, and type of substrates are summarized in Table 4.

Table 4

4: Phase III biotransformation reactions

Although the significance and function of phase III biotransformation is not completely elucidated yet. Recently, it has been proposed that phase III biotransformation serves a dual function as membrane transporters involved in the biliary drug excretion and its metabolite along with the efflux of such drug or its metabolites across the membrane of hepatocytes (Phang-Lyn & Llerena, 2020). All the reactions in phase III occur post-phase II to make a chemical substance to carry out further metabolism and subsequent elimination. In phase III, drugs may be either transported through ATP binding cassette (ABC) transporters, such transporters actively uptake or outflow a substance from one part of cell membrane to another part requiring the consumption of energy in the form of ATP. Contrarily, a drug can also be transported through an solute carrier (SLC) transporters that facilitates the flow of particular solute particles across the membrane. These can actively transport solute particles against the electrochemical gradients by combining this work with another ion or solute (Nigam, 2015; Rosenthal, Bush, & Nigam, 2019). Phase III reactions are also known to carry out modification with the intake of various medications. A vital drug transporter that is known as P-glycoprotein is located in the small intestine that determines the efflux of several drugs from cytoplasm into the lumen of intestinal for the excretion and elimination in phase III reactions (Phang-Lyn & Llerena, 2020).

5: Conclusion

The aforementioned evidence suggests that DMEs mediate a key role in drug metabolism. Undoubtedly, most of the marketed drugs are proactive and dependent upon the DMEs for their therapeutic action. These DMEs perform their crucial function and convert prodrugs into their active metabolite, which possesses elevated pharmacological activity. These DMEs mediate their action via specific mechanisms and are grouped according to their mechanistic approach such as oxidative and reductive enzymes. Additionally, these DMEs not only participate in the conversion of prodrugs into their active forms but also converts biologically active metabolites into pharmacologically inactive, nontoxic, and easily excretable forms. Therefore, summarizing the above discussion, we can conclude that knowledge of DMEs is highly important during multiple prescription to avoid undesirable attributes like subtherapeutic or toxic effects.

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