Non-P450
Carrie M. Mosher and Allan E. Rettie
Department of Medicinal Chemistry, University of Washington, Seattle, Washington, U.S.A.
GENERAL INTRODUCTION
As noted in earlier chapters, P450 monooxygenases dominate oxidative enzymatic reactions that metabolize drugs and other xenobiotics to facilitate their excretion. Indeed, it is estimated that P450 pathways contribute significantly to the clearance of about two-thirds of all drugs that depend on metabolism (1). However, complete reliance on P450 clearance pathways can precipitate adverse drug reactions if plasma concentrations are elevated either because of competition of multiple coadministered drugs for the same P450 isoforms or because the P450 clearance pathway is polymorphic. Consequently, during drug discovery and development, distribution of a drug’s clearance pathways over multiple P450 isoforms and/or non-P450 enzymes is often considered beneficial.
Many non-P450 oxidative enzymes, including aldehyde dehydrogenase (ALDH), monoamine oxidase, and semicarbazide-sensitive amine oxidase, are capable of carrying out xenobiotic oxidation, but generally their quantitative contribution to drug metabolism is too low, or our present knowledge of their basic biochemistry is insufficiently comprehensive, to warrant detailed discussion here. However, interested readers are directed toward some recent excellent reviews on the dehydrogenases (2) and amine oxidases (3). This chapter will focus on three mammalian nonheme oxygenases, which can conveniently be categorized by the nature of their active centers as either flavin-containing or molybdenum-containing enzymes. The flavin-containing monooxygenase (FMO) isoforms are located predominantly in the microsomal cell fraction, whereas the molybdenum-containing enzymes, aldehyde oxidase (AO) and xanthine oxidoreductase (XOR), are both located in the soluble fraction. Each of the three enzymes will be discussed in terms of their catalytic mechanism, multiplicity, tissue distribution, enzyme regulation, biotransformation reactions, and relevance to human drug metabolism.
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MICROSOMAL FLAVIN-CONTAINING MONOOXYGENASE
Microsomal FMOs have been categorized as class B flavoprotein monooxygenases, along with the sequence-related Baeyer Villiger monooxygenases and microbial N-hydroxylating monooxygenases (4). All three enzymes exhibit common characteristics of a tightly bound FAD cofactor, two glycine-rich dinucleotide-binding domains (Rossman folds) for FAD and NADPH, and maintenance of bound NADPH/NADPþ throughout catalysis. Microsomal FMOs activate molecular oxygen in order to function as sulfur, nitrogen, phosphorus, and selenium oxygenases (5), and in this regard, as well as in their dependence on NADPH, they are indistinguishable from P450s, which can carry out many of the same catalytic reactions.
Moreover, both FMOs and P450s are localized in the endoplasmic reticulum of the cell, and so it is of interest to be able to distinguish between catalysis by these two monooxygenases in microsomal membranes. To accomplish this, investigators can take advantage of the increased thermolability of FMO relative to P450, human liver microsomes that have been phenotyped for FMO-selective substrate activity and/or immunochemical content, as well as commercially available recombinant preparations of FMO (and P450) isoforms. Examples of these various approaches can be found in several publications (6 10).
Catalytic Mechanism
The simplest catalytic cycle consistent with available experimental data involves the initial transfer of the pro-R hydrogen from NADPH to reduce FAD, followed by dioxygen binding to the reduced flavin to generate a C-4a-hydroperoxide (FAD-OOH in Fig. 1A) (11,12). Substrate nucleophiles (e.g., trimethylamine) attack the distal oxygen of this hydroperoxide with resultant oxygen transfer to the substrate and the generation of a hydroxyflavin (FAD-OH) species. The rate-limiting step in FMO catalysis is considered to be decomposition of the hydroxyflavin and/or release of NADPþ(5). Because the rate-limiting step follows transfer of oxygen to the substrate, it provides a rationalization for the observation that the Vmaxfor FMO-dependent reactions is relatively constant (13).
Recently, the crystal structures of Saccharomyces pombe FMO bound with NADPH and the substrate methimazole (Fig. 1B) have been solved (14). However, the quaternary complex of enzyme-FAD-NADP(H)-methimazole was not obtained, and in the two ternary complexes, methimazole bound in the same location as NADPH. These observations prompted the authors to suggest that substrate competes with NADPH and replaces the cofactor during the catalytic cycle of the yeast enzymes; however, no kinetic studies have yet been reported in support of this alternate mechanism. Yeast and mammalian FMOs share only 21% to 25% sequence similarity, but contain a highly conserved asparagine residue (Asn91 yeast; Asn61 FMO3), which is implicated in catalysis by yeast FMO, where it is proposed to be involved in oxygen binding (14). A homology model of human FMO3 based on the yeast FMO template also locates Asn61 at the catalytic center (15).
Moreover, site-directed mutagenesis of this residue, while maintaining enzyme expression and FAD binding, greatly diminished enzymatic activity for all substrates examined, consistent with a critical role for this residue in catalysis by human FMO3 (15,16).
Multiplicity, Tissue Distribution, and Species Differences Multiplicity
Molecular biology techniques have revealed the existence of 11 human FMO genes located on chromosome 1, 5 of which encode functional enzymes that are expressed in all mammalian species examined to date. These functional enzymes are FMO1, FMO2,
Figure 1 (A) Catalytic cycle for FMO dependent oxygenation. Step 1 illustrates reaction of the prototypical substrate trimethylamine with the enzyme’s activated oxygen species resulting in the formation of trimethylamine N oxide. Step 2 involves decomposition of the pseudobase FAD OH, with loss of water and release of NADPþ. Steps 3 and 4 are sequential reductions of the oxidized enzyme by NADPH and reaction of FADH2 with molecular oxygen to once again generate the reactive oxygen species FAD OOH. (B) Crystal structure of the yeast FMO (pdb 2GVC) active site.
The flavin cofactor, a residue involved in the catalytic mechanism (Asn91), and the inhibitor methimazole are highlighted. (C) Compounds used to elucidate FMO metabolic involvement via inhibition of FMO (methimazole) or P450s (1 aminobenzotriazole) as discussed within. Abbrevia tion: FMO, flavin containing monooxygenase.
FMO3, FMO4, and FMO5, which share 50% to 55% sequence identity (17). Human FMO6 mRNA is subject to alternative splicing that results in nonfunctional protein (18).
Three additional, possibly functional, FMO genes (FMO9, FMO12, and FMO13) exist in mice and rats, but apparently not in humans (19).
Tissue Distribution
On the basis of mRNA expression, the major forms of FMO expressed are FMO3 and FMO5 in human liver, FMO1 in human kidney, FMO2 in human lung, and FMO5 in human intestine (20,21). However, it should be noted that mRNA expression and protein expression are not always well correlated. Moreover, the human FMO enzymes differ substantially in their catalytic capabilities, and so this must also be factored into consideration of their contribution to drug or xenobiotic clearance in any given tissue.
In terms of human drug metabolism, FMO3 is clearly the most important isoform because it is highly expressed in the liver (22) at protein levels 2 to 10 times greater than FMO5 (23), which contrasts with the mRNA data for these two enzymes (20,21).
Human liver FMO1 protein expression is prominent in fetal liver, but is silenced within a few days after birth (24). In human kidney, FMO1 appears to be the functionally dominant form, present at protein levels that are 2 to 3 times higher than reported for fetal liver (25). As in nearly all animal species, human FMO2 is a pulmonary enzyme, but it is inactive in most human populations because of a polymorphism that results in a premature stop codon (26). FMO4 is the least well studied of the human FMO enzymes, perhaps because expression of the recombinant protein has proved challenging (27).
FMO5 is a quantitatively important hepatic FMO enzyme in humans and many experimental animals, but has attracted little attention because of its narrow substrate specificity (28).
Species Differences
As a general rule, FMO1 and FMO3 are expressed in the liver of most preclinical animal species, with the FMO1 isoform usually dominating FMO-dependent activity, a situation that contrasts strongly with that found in humans. While no gender differences in hepatic FMO activity have been reported in humans, a sex difference does exist in mice, as female mice exhibit higher liver FMO activity than males. The basis for this appears to be a combination of increased FMO1 levels in female mice and ablated FMO3 expression in male mice (29). Whereas FMO-mediated benzydamine clearance in cryopreserved rat hepatocytes was a good predictor of in vivo clearance, this was not true for cryopreserved human hepatocytes (30). Therefore, because tissue expression profiles and the substrate specificities of the individual FMO isoforms can differ across species, it is important to exercise caution when attempting to extrapolate liver microsomal data for FMO catalysis from experimental animals to humans.
Enzyme Regulation
Unlike the cytochrome P450s, the FMO system is not induced by exogenously administered xenobiotics, such as the barbiturates or polycyclic hydrocarbons. However, as noted above, the FMOs are subject to developmental, hormonal, and genetic regulation, all of which can influence FMO3 and/or FMO1 activity in humans.
Developmental Expression
The most striking example of developmental control concerns the selective expression of FMO1 in fetal human liver and FMO3 in adult human liver. Temporal expression patterns of human FMO1 and FMO3 protein have been investigated in human liver microsomal preparations representing ages from 8-week gestation to 18 years, using antibodies selective for each enzyme (24). FMO1 expression was highest in the embryo, whereas suppression occurred within three days postpartum in a process tightly coupled to birth. The onset of FMO3 expression was variable; most individuals failed to express this isoform during the neonatal period, but significant levels of FMO3 were generally detectable by one to two years of age. The authors concluded that birth is necessary, but not sufficient, for the onset of FMO3 expression. Hines and coworkers have undertaken additional studies to probe the trigger mechanism(s) for these events and recently identified that Pbx2 and Hox transcription factors are important to FMO3 developmental regulation (31).
Hormonal Regulation
Sex steroids have long been recognized to influence FMO activity, with induction of rabbit lung microsomal N,N-dimethylaniline N-oxygenase during pregnancy an early example of this phenomenon (32). Subsequently, upregulation of rabbit lung FMO2 was shown to correlate with plasma levels of progesterone and cortisol in pregnant animals (33).
Human FMO3 may also be induced in pregnancy (34). In male mice, sex steroids play an important role in hepatic FMO1 and FMO3 expression, with testosterone acting as a negative regulator of FMO3 in male mice (29). Finally, menstruation can be associated with a transient trimethylaminuria (35) (see following section), possibly because of changes in hormone levels affecting FMO3 expression and/or the presence of common genetic polymorphisms with independently modest effects on activity.
Genetic Polymorphism
Rare polymorphisms in the FMO3 gene that abolish functional activity cause the metabolic disorder trimethylaminuria, also known as fish-odor syndrome. Affected individuals are unable to metabolize trimethylamine for urinary excretion and resort, in part, to eliminating the compound unchanged in the sweat and exhaled breath (36,37). This is a consequence of the fact that FMO3 is the most efficient of all the human FMO enzymes for the conversion of dietary-derived trimethylamine to its non-odoriferous N-oxide metabolite (38).
Whereas the human FMO3 gene contains over 40 common single nucleotide polymorphisms (SNPs), common polymorphic variation in human FMO1 appears relatively minimal (39), suggestive perhaps of a critical endogenous role for this enzyme.
In FMO3, the coding region polymorphisms, E158K, V257M, and E308G, are each expressed at an allele frequency of greater than 5% in a variety of racial populations (16).
These common coding region variants maintain variable degrees of FMO3 activity, which may impact human drug metabolism to some degree, depending on the nature of the substrate (15,40,41).
Transformation Reactions
The wide panoply of metabolic transformations of which mammalian FMOs are capable is exemplified by the substrate specificity of pig liver FMO1, the first FMO enzyme to be described (42). This enzyme catalyzes the oxidative metabolism of a huge array of
amines, hydrazines, thiols, sulfides, thioamides, thiocarbamides, as well as numerous organic compounds bearing nucleophilic selenium and phosphorus atoms (43). Extensive structure-function studies with this enzyme have shown that, in principle, any soft nucleophile that gains access to the active site of FMO will be oxygenated (13,44);
however, access can be modulated by substrate charge and size (45). Presumably, differences in the dimensions and charge neutralization capabilities of FMO enzyme active sites and/or their access channels underlie the general view that mammalian FMO substrate specificity decreases in the order FMO1 > FMO3 > FMO2 > FMO5 (5).
A few illustrative examples of FMO-catalyzed oxidation at nitrogen and sulfur centers the most commonly encountered nucleophilic sites in drug molecules are presented below, together with discussion of methodology that can be used to discriminate between FMO and P450-dependent catalysis.
Oxidation at Nitrogen Centers
A very large number of nitrogen-containing functionalities are found in drugs and other xenobiotics. Given the nucleophilic mechanism of FMO catalysis, it might be expected that substrates for the enzyme would exhibit a minimum basicity requirement. Indeed, FMO catalyzes N-oxide formation on the pyrrolidine ring of nicotine (pKaof protonated nitrogen¼ 8), but not on the pyridine ring (pKa¼ 3). However, predictions of substrate specificity based on pKaalone do not accommodate all of the experimental observations (10), and it is clear that steric and electronic effects of the substrates themselves and the relative expression levels of specific FMO and P450 isoforms in a given species will determine the nature of the enzyme system involved in vivo. Regardless, xenobiotics that contain a basic sp3-hybridized nitrogen are generally good substrates for FMO.
A major group of substrates for the FMOs are the tertiary acyclic and cyclic amines.
Substrates such as trimethylamine (Fig. 1), clozapine (Fig. 2), benzydamine, and guanethidine are converted to stable N-oxides. However, the levels of N-oxide metabolite formed may often be underestimated owing to relatively facile reduction back to the parent amine. Although cytochrome P450s tend to preferentially N-dealkylate tertiary amines, P450-dependent formation of N-oxides is well documented (46,47). FMOs also catalyze the metabolism of secondary and primary amines (13), but are usually not involved in the oxidation of amides, heteroaromatic amines, benzamidines, guanidines, or diamines.
Oxidation at Sulfur Centers
FMOs readily catalyze the oxidation of thioethers to sulfoxides, often with a high degree of stereoselectivity, and the further metabolism of sulfoxides to sulfones. Thioethers, such as tazarotenic acid (Fig. 2), are generally better substrates for FMO than the tertiary amines discussed above, owing to the enhanced nucleophilicity of the sulfur atom. Relative nucleophilicity also explains why thioamides are excellent substrates and sulfoxides are usually poorer substrates for FMO. However, oxidation of sulfur-containing compounds is also carried out readily by cytochrome P450s. The relative participation of these two monooxygenase systems in sulfur oxidation will depend on their levels of expression in the metabolizing tissue and the substrate specificity of the FMO isoforms involved.
Diagnostic Substrates and Inhibitors
Approaches to the differentiation of P450-mediated and FMO-dependent catalysis may capitalize on the availability of (partially) selective substrates and inhibitors for both
enzyme systems. For example, the conversion of N,N-dimethylaniline to its N-oxide is one of the most widely used indicators of FMO catalysis in microsomal preparations, and as long as reactions are carried out at pH 8.5 to 9, only FMO(s) are likely to be involved.
However, at physiological pH, even this most diagnostic of substrates may be turned over, in part, by cytochrome P450s. A useful FMO substrate is the anti-inflammatory drug benzydamine (48) because several FMO enzymes, including human FMO3, form the Figure 2 Typical N and S oxygenation reactions catalyzed by FMO. Redox interconversions of sulindac illustrate the metabolic interplay of FMO and AO enzymes discussed in this review.
Abbreviations: FMO, flavin containing monooxygenase; AO, aldehyde oxidase.
highly fluorescent N-oxide metabolite. This has permitted the development of a simple, sensitive, LC-based metabolite assay for FMO activity (49,50).
Chemical inhibitors suffer from the disadvantage that they are rarely, if ever, specific for FMO. Unfortunately, no mechanism-based inhibitors of FMO have been identified, and antibodies raised against purified enzymes do not significantly inhibit the activity of microsomal FMOs. However, 1-aminobenzotriazole (Fig. 1C), a suicide inhibitor of most P450s at high substrate concentrations, is a useful indirect indicator of FMO catalysis if preincubation with this compound does not decrease the reaction rate that is being monitored. Methimazole (Fig. 1C) is a widely used inhibitor of FMOs, but it also competitively inhibits human CYP2B6, CYP2C9, and CYP3A4 at the relatively low substrate concentrations of 40 to 100mM (51). n-Octylamine, an activator of FMO1, is an inhibitor of human FMO3 (52). Therefore, it is prudent to employ a battery of “selective”
inhibition methods (6) when evaluating the in vitro role of FMO in a given oxidative pathway.
Relevance to Human Drug Metabolism
The conversion of lipophilic tertiary amines to polar N-oxides can be considered the prototypic FMO xenobiotic reaction pathway, and so it is not surprising that FMO has been implicated in the metabolism of a variety of tertiary amine-containing drugs.
However, although FMOs are clearly responsible for the N-oxygenation of widely used agents like nicotine and tamoxifen (53,54), this pathway does not dictate their metabolic clearance. Nonetheless, there are a number of examples where FMO-dependent metabolism is a significant contributor to drug clearance in vivo.
N-oxygenation is the major metabolic pathway for the gastroprokinetic agent itopride (Fig. 2), and FMO3 is the main catalyst of this pathway in human liver microsomes (55). More recently, an investigational Src kinase inhibitor TG100435 (Fig. 2) was shown to rely on FMO3 for metabolism to its major, active N-oxide metabolite (56).
Ranitidine, benzydamine, and olanzapine provide additional examples of marketed drugs where FMO plays a significant role in their metabolic elimination through formation of N-oxide metabolites (7,49,57).
Sulfoxides are quantitatively significant human metabolites of therapeutic agents such as the anti-inflammatory sulindac sulfide (Fig. 2), and tazarotenic acid, the major circulating metabolite of the topical antipsoriatic agent tazarotene. The former drug is administered as a sulfoxide prodrug and relies on metabolic reduction for its pharmacological activity. In vitro microsomal studies indicate that human liver FMO(s) S-oxygenate both sulindac sulfide (58) and tazarotenic acid (59).
An interesting variation on FMO-dependent S-oxygenation processes can occur following S-methylation of thiol precursors. In this regard, FMO catalysis has been documented for S-methyl metabolites of the alcohol aversion drug disulfiram (60), as well as MK0767, an investigational peroxisome proliferator-activated receptor (PPAR) dual agonist that initially undergoes thiazolidine ring scission to unmask the thiol (61). Figure 3 provides an example of this reductive scission reaction involving the antipsychotic drug ziprasidone, which can be catalyzed by molybdenum-containing hydroxylases.
XANTHINE OXIDASE/ALDEHYDE OXIDASE
The molybdenum hydroxylases are a family of homodimeric, 300-kDa enzymes that include AO, xanthine dehydrogenase (XDH), and xanthine oxidase (XOD). XDH and XOD are two forms of the same enzyme and are often collectively referred to as XOR.
Figure 3 Typical oxidative and reductive reactions catalyzed by XOR and AO. Abbreviations:
XOR, xanthine oxidoreductase; AO, aldehyde oxidase.
While there is no “alternative” form of AO, several homologs have been identified in certain species (see below). More information is available for XOR than AO, although extensive characterization of the human enzyme is still lacking for both. The physiological role of the mammalian molybdenum hydroxylases has yet to be elucidated;
however, these enzymes are involved in human health, and AO has an increasingly important role in human xenobiotic metabolism (62,63).
Catalytic Mechanism
Each 150-kDa subunit of a mammalian molybdozyme has a tripartite structure consisting of a 20-kDa N-terminal domain containing two nonidentical Fe2S2redox centers, a central 40-kDa flavin-containing region, and an 85-kDa C-terminal domain containing the molybdenum cofactor and substrate-binding sites (62). The molybdenum atom is anchored to its active site via a covalent bond to an organic pyranopterin dithiolene
Each 150-kDa subunit of a mammalian molybdozyme has a tripartite structure consisting of a 20-kDa N-terminal domain containing two nonidentical Fe2S2redox centers, a central 40-kDa flavin-containing region, and an 85-kDa C-terminal domain containing the molybdenum cofactor and substrate-binding sites (62). The molybdenum atom is anchored to its active site via a covalent bond to an organic pyranopterin dithiolene