Mechanism-Based Inactivation of Human Cytochromes P450s

Chem. Res. Toxicol. 2008, 21, 189–205

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Mechanism-Based Inactivation of Human Cytochromes P450s: Experimental Characterization, Reactive Intermediates, and Clinical Implications Paul F. Hollenberg,* Ute M. Kent, and Namandjé N. Bumpus Department of Pharmacology, UniVersity of Michigan, Medical Science Research Building III, 1150 West Medical Center DriVe, Ann Arbor, Michigan 48109 ReceiVed July 10, 2007

The P450 type cytochromes are responsible for the metabolism of a wide variety of xenobiotics and endogenous compounds. Although P450-catalyzed reactions are generally thought to lead to detoxication of xenobiotics, the reactions can also produce reactive intermediates that can react with cellular macromolecules leading to toxicity or that can react with the P450s that form them leading to irreversible (i.e., mechanism-based) inactivation. This perspective describes the fundamentals of mechanism-based inactivation as it pertains to P450 enzymes. The experimental approaches used to characterize mechanism-based inactivators are discussed, and the criteria required for a compound to be classified as a mechanism-based inactivator are outlined. The kinetic scheme for mechanismbased inactivation and the calculation of the relevant kinetic constants that describe a particular inactivation event are presented. The structural aspects and important functional groups of several classes of molecules that have been found to impart mechanism-based inactivation upon metabolism by P450s such as acetylenes, thiol-containing compounds that include isothiocyanates, thiazolidinediones, and thiophenes, arylamines, quinones, furanocoumarins, and cyclic tertiary amines are described. Emphasis throughout this perspective is placed on more recent findings with human P450s where the site of modification, whether it be the apoprotein or the heme moiety, and, at least in part, the identity of the reactive intermediate responsible for the loss in P450 activity are known or inferred. Recent advances in trapping procedures as well as new methods for identification of reactive intermediates are presented. A variety of clinically important drugs that act as mechanism-based inactivators of P450s are discussed. The irreversible inactivation of human P450s by these drugs has the potential for causing serious drug–drug interactions that may have severe toxicological effects. The clinical significance of inactivating human P450s for improving drug efficacy as well as drug safety is discussed along with the potential for exploiting mechanism-based inactivators of P450s for therapeutic benefits.

1. 2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

16. 17.

Contents Introduction Mechanistic Criteria Kinetic Aspects Mechanism-Based Inactivators: Structural Aspects, Functional Groups, and Formation of Reactive Intermediates Acetylenes Organosulfur Compunds Arylamines Cyclic Tertiary Amines Furanocoumarins Other Mechanism-Based Inactivators Types of Modifications Encountered with Reactive Intermediates Trapping and Identification of Reactive Intermediates Future Directions in the Identification of Reactive Intermediates and Their Targets Mechanism-Based Inactivators for Determining the Role of Specific P450s in Catalysis Role of Mechanism-Based Inactivators in the Identification of Critical Amino Acids in the Active Sites of P450s Clinical Implications for Mechanism-Based Inactivation of P450s Therapeutic Benefits of Mechanism-Based

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Inactivation 18. Future Directions

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1. Introduction The cytochromes P4501 are a superfamily of heme proteins that catalyze the metabolism of a wide variety of xenobiotics and endogenous compounds to products that generally are more * To whom correspondence should be addressed. Tel: 734-764-8166. Fax: 734-763-5387. E-mail: [email protected]. 1 ABT, 1-aminobenzotriazole; ADR, adverse drug reaction; BBT, N-benzyl1-aminobenzotriazole; BG, bergamottin; BITC, benzyl isothiocyanate; DAS, dially sulfide; DASO, diallyl sulfoxide; DASO2, diallyl sulfone; DASO3, monoallyl sulfone epoxide; DCE, 1,1-dichloroethylene; DCMB, N-(3,5dichloro-4-pyridyl)-3-(cyclopentyloxy)-4-methoxybenzamide; DMPB, N-(3,5dichloro-4-pyridyl)-4-methoxy-3-(prop-2-ynyloxy)benzamide; EC, 7-ethynylcoumarin; 17EE, 17-R-ethynylestradiol; 2EN, 2-ethynylnaphthalene; 9EPh, 9-ethynylphenathrene; ESI-LC-MS, electrospray ionization-liquid chromatography–mass spectrometry; GSH, reduced glutathione; ITC, isothiocyanate; LCMS/MS, liquid chromatography–tandem mass spectrometry; MALDI-MS, matrix-assisted laser desorption–ionization mass spectrometry; MALDI-TOF, matrix-assisted laser desorption–ionization mass spectrometry–time of flight; MBI, mechanism-based inactivator; RMBT, N-R-methylbenzyl-1-amino-benzotriazole; MDMA, 3,4-methylenedioxymethamphetamine; MS, mass spectrometry; 8-MOP, 8-methoxypsoralen; P450, cytochrome P450; 5P1P, 5-phenyl1-pentyne; NAC, N-acetylcysteine; NDMA, N-nitrosodimethylamine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; parathion, O,O-diethyl-O-pnitrophenylthiophosphate; PCP, phencyclidine; PEITC, phenethyl isothiocyanate; QA-GSH, quaternary ammonium GSH analogue; SRS5, substrate recognition site 5; TA, tienilic acid; tBA, tert-butyl acetylene; tBMP, tert-butyl 1-methyl-2-propynyl ether; tTEPA, N,N′,N′′-triethylenethiophosphoramide; TGZ, troglitazone; TZD, thiazolidinedione.

10.1021/tx7002504 CCC: $40.75  2008 American Chemical Society Published on Web 12/04/2007

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hydrophilic and contain an additional functional group, which may be used by conjugating enzymes to enhance the rate of clearance and excretion of the products. Although most of the reactions catalyzed by the cytochrome P450s are generally thought to lead to detoxication of xenobiotics by forming more hydrophilic metabolites that can be readily excreted from the body, P450s can also produce reactive intermediates that may react with macromolecules in the cell such as DNA, RNA, and proteins, ultimately leading to toxicity (1). It is not surprising that the cytochromes P450 that can bioactivate xenobiotics as well as endogenous compounds to form reactive intermediates may often be targets for modification by the reactive intermediates before they leave the enzyme active site. Those compounds that may be transformed by the P450s into reactive species, which can then react with moieties in the active site leading to inactivation of the P450, are oftentimes referred to as “mechanism-based”, “suicide”, “time-dependent”, or “catalysis-dependent” inactivators. The specific binding of these reactive intermediates to the active sites of the P450s that generate them prior to their release from the active site can be exploited in the design of very specific irreversible inhibitors (1–4).

2. Mechanistic Criteria The broad definition of mechanism-based or suicide inactivator includes any compound that is converted during the course of enzymatic catalysis to a form that then inactivates the enzyme carrying out the enzymatic conversion (5, 6). However, for this perspective, we will restrict the definition of mechanism-based inactivator to any compound that is catalytically transformed by a cytochrome P450 to give a reactive intermediate that inactivates the enzyme without leaving the P450 (5). A key to this concept is the requirement that the inactivation involves formation of a covalent adduct within the active site, which occurs without the release of the reactive intermediate from the active site. As a consequence, this definition rules out affinity labels, transition state analogues, and slow, tight-binding inhibitors. Although enzymologists generally consider that mechanismbased inactivation is relatively unusual in enzyme-catalyzed reactions, it is observed in P450-catalyzed reactions with somewhat higher frequency than most other reactions; presumably, this is because of the reactivity of the oxygenated intermediate. The utility of mechanism-based inactivators has recently become apparent in the design of new drugs since it appears to be possible that they could be designed such that they only affect the target enzyme. Mechanism-based inactivators are also of great interest to enzymologists because of their usefulness in elucidating enzyme mechanisms. Interest in mechanism-based inactivators has also arisen because of the potential for inactivating the P450s that form reactive intermediates that may ultimately lead to toxicity, carcinogenesis, or mutagenesis. Cytochrome P450-mediated oxidation reactions can in many instances generate highly reactive intermediates. In situations where these reactive intermediates do not readily react with water to form a stable metabolite, the subsequent fate of the reactive intermediate can lead to potentially adverse reactions. The reactive intermediates may form free radicals, oxidize thiols, and lower intracellular glutathione concentrations. Additional toxicological problems can be encountered when these reactive intermediates bind to endogenous macromolecules and interfere with normal cellular functions by a variety of different mechanisms. In particular, the inactivation of certain P450s that are required for drug metabolism and the clearance of metabolites from the body can lead to the build up of toxic

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Figure 1. Reaction pathway for mechanism-based inactivation.

metabolites resulting in adverse drug reactions (ADR). Covalent modification of cellular macromolecules by reactive intermediates may also elicit undesirable immune responses that can result in ADRs due to the formation of hapten–carrier conjugates that lead to autoimmune reactions. Reactive metabolites of halothane, dihydralazine, and tienilic acid, which become covalently bound to hepatic proteins, are thought to exert their toxic effects in this manner. Although studies with tienilic acid in cultured hepatocytes indicate that P450 2C is the primary target of the reactive intermediate, a significant fraction of the reactive intermediates also escapes to either be trapped by cellular GSH, thereby lowering the intracellular GSH concentrations, or modify other proteins (7). Finally, significant efforts have recently been put into the development of mechanism-based inactivators as diagnostic inhibitors that may be used to determine which P450(s) may be responsible for catalyzing the metabolism of a given substrate to give a specific product either in vivo or in vitro in cell homogenates or microsomal preparations.

3. Kinetic Aspects A kinetic scheme for mechanism-based inactivation is shown in Figure 1 (1). MBI stands for mechanism-based inactivator, E·MBI is the Michaelis–Menten enzyme–substrate complex, E·MBI* is the reactive intermediate reversibly bound in the active site of the protein, P is the product that leaves the enzyme, and EI** is the inactivated enzyme. The various kinetic constants, their derivation, and the kinetic implications of the various steps have been described by Silverman (6). The two principal kinetic constants routinely used to characterize mechanism-based inactivation are KI and kinact. As shown in Figure 1 and eq 1, KI is a complex mixture of all of the relevant kinetic constants. As shown in eq 2, kinact is a combination of k2, k3, and k4. The KI represents the concentration of the mechanismbased inactivator, which gives the half-maximal rate of inactivation, whereas the kinact represents the inactivation rate constant at an infinite concentration of the inactivator.

KI ) [( k-1 + k2)/k1] [(k3 + k4)/(k2 + k3 + k4 )]

(1)

kinact ) k2k4 ⁄ (k2 + k3 + k4)

(2)

The concept of the partition ratio was originally introduced by Walsh (8). It is determined by the ratio of k3/k4 and can be thought of as the number of substrate molecules metabolized and released from the enzyme as product for each molecule that is metabolized and forms a covalent adduct with the enzyme, thereby inactivating the protein. The partition ratio can also be thought of as the average number of cycles of metabolism that the enzyme traverses before it is inactivated. Thus, the partition ratio is, in fact, a measure of the efficiency of the mechanism-based inactivator. It is dependent on a number of different factors including the reactivity of the reactive intermediate, the proximity of the site of formation of the reactive intermediate to the appropriate target molecule(s) in the active site with which it reacts to form a covalent adduct, the presence of other molecules in the active site that may react with the reactive intermediate to convert it to a nonreactive form,

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and the rate of diffusion of the reactive intermediate out of the enzyme. In theory, the most efficient mechanism-based inactivator would have a partition ratio of zero. Thus, in a standard assay where metabolite formation is measured, it would be considered that this compound would not be a substrate for the enzyme since there would be no product formation. However, detection of a covalent adduct to the P450 or heme prosthetic group leading to inactivation would demonstrate that the compound is a mechanism-based inactivator. Mechanism-based inactivators having partition ratios ranging from slightly greater than zero to several thousand have been reported. Ortiz de Montellano and Correia (4) have pointed out that mechanism-based inactivators may prove to be much more enzyme-specific than reversible inhibitors. The reasons are as follows: (i) The initial reversible binding of the inhibitor to the enzyme must satisfy the constraints imposed on reversible inhibitors; (ii) the mechanism-based inactivator must also be acceptable as a substrate since catalytic activation to a reactive species is required for inactivation; and (iii) the reactive species formed as a consequence of metabolism must find an appropriate target within the active site and react with it leading to an irreversible modification of the enzyme, which then removes it permanently from the pool of active enzymes. The criteria that are routinely used to determine whether or not a substrate is a mechanism-based inactivator are as follows (1): (i) The loss of enzyme activity must be time-dependent (although loss of activity should exhibit pseudo first-order kinetics with respect to time, this is not absolutely necessary). A plot of the natural log of enzyme activity remaining after preincubation vs preincubation time, corrected for any loss of activity in the absence of inhibitor, should give a straight line. (ii) The rate of inactivation should exhibit saturation kinetics with respect to the concentration of the inactivator. (iii) The inactivation requires that all of the typical cofactors (in this case NADPH, O2, and P450 reductase) are present and that metabolism is occurring. It should be possible to demonstrate under these conditions that metabolism of the inactivator leads to the formation of a reactive intermediate, which ultimately results in the formation of product or inactivation. (iv) The presence of a substrate or an inhibitor of the enzyme should slow the rate of inactivation. (v) The inactivation should not be prevented by the inclusion of catalase and/or superoxide dismutase in the incubation mixture. P450s have been shown to be partially uncoupled, leading to the production of superoxide and/or hydrogen peroxide that can result in autoinactivation. The inclusion of catalase and/or superoxide dismutase protects against such inactivations. Inactivations due to reactive oxygen can seriously complicate the interpretation of studies with some mechanism-based inactivators. (vi) Generally, the inactivation should be irreversible and activity should not return upon dialysis or gel filtration because the mechanism-based inactivator should be covalently bound to the enzyme. (vii) The inactivation should result in a 1:1 stochiometry of labeling of the enzyme by the inactivator. In addition, the presence of added “scavenger” nucleophiles such as glutathione (GSH) should not affect either the rate of the inactivation or the stoichiometry. (viii) The kinetics for the inactivation should exhibit no lag time. Because P450s have been shown to convert a significant number of their substrates to reactive intermediates that ultimately form covalent adducts with DNA, RNA, or proteins, it is necessary to differentiate between specific inactivation, as already described, and nonspecific labeling by reactive intermediates, which may be much more extensive and ultimately lead to inactivation. Oftentimes, a careful analysis of the time course of the reaction

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can be very helpful in differentiating between the two possibilities since in the latter case there is a lag in the inactivation because the level of modification of the enzyme must increase to a certain point before any inactivation may be observed. Under ideal situations, it would be expected that all of these criteria should be satisfied for a mechanism-based inactivator. However, several instances have been reported where a mechanism-based inactivator does not satisfy all of these criteria. For example, mechanism-based inactivators that inactivate cytochrome P450s 2E1 and 2B4 have been reported where the inactivation is reversible under some conditions (9–11). In these cases, the reversibility is highly dependent on the size and chemical nature of the functional group as well as the identity of the reactive intermediate. To obtain KI and kinact, the natural log of the enzymatic activity remaining is plotted against the preincubation time. The apparent inactivation rate constant (kobs) is then determined from the slope of the initial linear phase of the inactivation reaction. The value of kobs is then plotted against the concentration of the inactivator, and the parameters (KI and kinact) are determined using a nonlinear least-squares method using eq 3 (6, 12). [I] represents the concentration of the inhibitor, and kobs is the apparent inactivation rate constant at that concentration of inhibitor [I]. The inactivation half-life (t1/2) can be calculated from eq 4.

kobs ) (kinact × [I]) ⁄ (KI + [I])

(3)

t1/2 ) 0.693 ⁄ kinact

(4)

The experimental determination of all of the parameters used in the previous equations requires measurement of enzyme activity after various preincubation times using various concentrations of the inactivators. At specific time points, aliquots are removed from the primary reaction mixture, diluted anywhere from 10- to 50-fold in a secondary reaction mixture, and assayed for remaining activity. The reactions must be carried out at several different concentrations of the mechanism-based inactivator to determine saturation. Although recent studies on mechanism-based inactivation of P450s have often used purified P450s in the reconstituted system, these studies do not need to be restricted to purified enzymes. Studies may be performed with microsomes using substrates that have been demonstrated to be specific for the individual isozymes under investigation to monitor their inactivation as long as the appropriate caveats are observed. Studies using radiolabeled mechanism-based inactivators can also be performed with microsomal preparations followed by HPLC or SDS-PAGE separation of the components of the microsomes to examine the specificity of the labeling reaction. Mechanism-based inactivators of the P450s can inactivate by three different pathways. They are (i) covalent binding to an amino acid residue in the apoprotein; (ii) alkylation or arylation of the prosthetic heme group; and (iii) destruction of the prosthetic heme group, which often leads to covalent modification of the apoprotein by the heme-derived products. Many of the mechanism-based inactivators may inactivate by more than one mechanism, and the predominant mechanism by which a given inactivator may inactivate may be determined by a variety of different factors including the identity of the P450 under study. Different inactivators having the exact same functional group may also inactivate via different pathways. So far, those factors that determine the pathways (apoprotein, heme, or both) operative for the modification of a particular form of P450 by a specific mechanism-based inactivator are not well-understood.


Complementary DNA (cDNA)-expressed P450s in the reconstituted system are now routinely used to investigate mechanism-based inhibition of specific P450s. These in vitro systems can be used to characterize time-, NADPH-, and concentration-dependent inactivation as well as to determine the kinetic parameters (KI, kinact, and the partition ratio). These systems can also be used in efforts to identify the reactive metabolite responsible for inactivation by trapping using glutathione or N-acetylcysteine. The modified protein can be isolated, and the covalent adduct and the site of attachment may be investigated using SDS-PAGE and LC-MS/MS approaches. Liver microsomes can also be used for these types of studies, although it is essential with microsomes that a probe substrate be used in the secondary reaction, which is specific for the P450 of interest. Studies with radiolabeled inactivators followed by SDS-PAGE or HPLC can be used to isolate the labeled proteins and may allow for determination of the isozyme that is labeled. However, the technology is not yet at the point where it would be possible to determine the site of adduct formation on the protein from microsomes. Finally, hepatocytes can also be used for these types of studies. Once again, it is essential that a very selective probe substrate be available for the isozyme or isozymes under investigation. Over the past 20–30 years, numerous compounds have been identified that function as mechanism-based inactivators for P450 enzymes. Several extensive reviews have been published that elaborate on many of these inactivators that have been studied with mammalian P450 enzymes (1, 13, 14). To describe all of the naturally occurring compounds or xenobiotics that have been identified as mechanism-based inactivators of P450 enzymes would be beyond the scope and readability of this perspective. For the purposes of this perspective, we will limit this extensive list to only some of those xenobiotics that have been studied more recently and primarily with human P450s, where the critical functional group is known or suspected and where reactive intermediates or covalent modification of human P450s has been demonstrated.

4. Mechanism-Based Inactivators: Structural Aspects, Functional Groups, and Formation of Reactive Intermediates Certain functional groups either occur naturally or have been specifically engineered into drugs to afford increased stability, solubility, or bioavailability to certain compounds. Some of these functional moieties have been shown to predispose the molecule to the metabolism by particular P450 enzymes in such a way as to generate potentially toxic reactive intermediates. These molecules can be grouped according to one of their structural aspects and fall into major categories such as the acetylenes, a large group of thiol-containing compounds that includes isothiocyanates, thiazolidinediones, and thiophenes, arylamines, quinones, furanocoumarins, cyclic tertiary amines, and numerous others. The fate of the reactive intermediate that is generated depends in part on the reactivity and longevity of the intermediate itself as well as the structure of the active site of the particular P450 where it was generated. The reactive intermediate may react with water and leave the active site as a non- or less toxic product. In other instances, the reactive intermediate may alkylate the heme moiety, which can lead to heme destruction, or it can react with a nucleophilic amino acid residue within the vicinity of the active site where the intermediate was formed.

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Figure 2. 17-R-Ethynylestradiol (17EE). The circled area indicates the site of metabolism resulting in the formation of reactive intermediates. The proposed ketene intermediate is thought to lead to protein adduction, whereas the R-ketocarbene intermediate results in heme adduction.

5. Acetylenes Acetylenes have been investigated as mechanism-based inactivators with bacterial and mammalian P450s (1, 15–18). Through these early studies, as well as subsequent investigations with 2-ethynylnaphatalene (2EN) (19), 9-ethynylphenathrene (9EPh) (20), 7-ethynylcoumarin (EC) (1), and 5-phenyl-1pentyne (5P1P) (21), we have obtained a better understanding of the mechanism that is involved when the acetylenic moiety is metabolized by various P450 enzymes. Initial oxidation at either the terminal carbon or the penultimate carbon of the acetylenic function may determine the site of attack on the P450 by the reactive acetylenic intermediate. The general trend appears to be that alkylation of the heme occurs if the initial site of transfer of oxygen from the activated oxygen intermediate of P450 is at the penultimate carbon (21). Protein adduction by the reactive intermediates of 2EN or 9EPh has been observed if the initial site of oxidation is on the terminal carbon of the acetylene (19, 20, 22). Transfer of the oxygen to the terminal carbon results in a 1,2-shift of the terminal hydrogen to the vicinal carbon, producing a reactive ketene intermediate (15–17). This intermediate then has two possible fates, either hydrolysis to the carboxylic acid product or reaction with a nucleophilic residue in the active site of the P450. Mechanism-based inactivation by the acetylenic compounds 2EN, 9EPh, EC, and 5P1P of rat and rabbit P450 enzymes and the identification of the site of adduct formation to T302 on the protein or to the P450 heme have been reviewed in detail (1). The inactivation of human P450s by acetylenes has been reported for the oral contraceptive 17-R-ethynylestradiol (17EE) (Figure 2) (23–25). 17EE was developed in 1938 and continues to be the primary estrogenic component in many oral contraceptives (26, 27). The acetylene functional moiety was introduced into the estradiol molecule to increase the oral bioavailability of the steroid. Recently, it was shown that in addition to inhibiting human P450 3A4 activity (28), 17EE is also a potent mechanism-based inactivator of rat P450 2B1 as well as the human P450s 2B6 and 3A5 (24, 25, 28, 29). Whereas the primary loss in activity of P450 3A4 is due to heme modification, P450s 2B1 and 2B6 are inactivated because of the formation of a covalent adduct to the apoprotein (25). Metabolism of 17EE by these enzymes results in the formation of reactive 17EE intermediates that inactivate these P450s in a mechanism-based manner by a combination of heme alkylation

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and protein modification in the case of P450 3A4 (23, 29) and by modification of the apo-protein with P450 2B6 (24). With each enzyme, the loss in activity was 17EE concentration-, time-, and NADPH-dependent and exhibited pseudo first-order kinetics. Inactivation of P450s 2B1 and 2B6 by [3H]-17EE resulted in exclusive labeling of the inactivated P450 apoprotein. A combination of approaches has been utilized to locate (i) the site of adduction and (ii) the mass of the reactive intermediate of 17EE that was responsible for the loss in enzymatic activity (24). Initial studies with four members of the P450 2B family revealed that only P450s 2B1 and 2B6 were inactivated by 17EE when incubated with NADPH in the reconstituted system. Mass spectral analysis of 17EE-inactivated P450 2B1 showed an increase in the mass of the apoprotein of 313 Da, which was consistent with the addition of the mass of 17EE plus one oxygen atom. N-Terminal amino acid sequencing of CNBr peptides obtained from P450 samples that had been inactivated by 17EE yielded the N-terminal sequences PYTDAVIHEI (for P450 2B1) and PYTEAV (for P450 2B6) that correspond to the peptides P347–N376 and P347–N365 in P450s 2B1 and 2B6, respectively. Electrospray ionization-liquid chromatography– mass spectrometry (ESI-LC-MS) and matrix-assisted laser desorption–ionization mass spectrometry (MALDI-MS) analysis of the P450 2B1-derived peptide resulted in a mass of 3654 Da for the modified peptide, which again corresponded to the theoretical mass of 17EE adducted to the P347–N376 peptide. Reactive intermediates of 17EE that were generated during metabolism by P450s 2B1 and 2B6 were trapped with GSH. ESI-LC-MS/MS analysis of the 17EE–GSH conjugates indicated that P450s 2B1 and 2B6 generated different reactive 17EE intermediates that were responsible for the inactivation and protein modification and for the formation of GSH conjugates by these two enzymes. ESI-MS/MS analysis of the 17EEinactivated 2B1 following digestion with trypsin demonstrated that the reactive intermediate formed from 17EE had formed an ester adduct with the S360.2 ESI-LC-MS/MS analysis of 17EE-inactivated P450 2B6 following digestion with trypsin showed that it also had been inactivated by formation of an ester adduct with S360. Interestingly, P450s 2B2 and 2B4 are completely refractory to inactivation by 17EE and they do not have a Ser at position 360. S360 is in the C-terminal end of the K helix and forms part of SRS5. The residues at positions 363 and 367 have been shown to be within 5 Å of the bound ligand in 2B4, and residue 363 plays a functional role in steroid metabolism (30, 31). Inactivation studies with a S360A mutant indicated that S360 was not critical for catalysis. Furthermore, examination of the crystal structure of P450 2B4 indicated that S360 is not located within 5 Å of the heme and may occupy a position in the access channel to the heme. Therefore, the loss in function of the P450s by the covalent modification of S360 does not appear to be a consequence of a catalytic role of this residue but rather due to steric hindrance by blocking access of substrates to the active site (32). N-(3,5-Dichloro-4-pyridyl)-4-methoxy-3-(prop-2-ynyloxy)benzamide (DMPB) (Figure 3) was synthesized based on the template structure of the P450 2B6 marker substrate N-(3,5dichloro-4-pyridyl)-3-(cyclopentyloxy)-4-methoxybenzamide (DCMB) (33). Incubations of expressed P450 2B6 in the reconstituted system or human liver microsomes with this terminal acetylene resulted in the time-, concentration-, and NADPH-dependent loss in DCMB hydroxylation activity with 2 Identification of S360 as the Residue Modified in CYPs 2B1 and 2B6 by a Reactive Intermediate of 17-R-Ethynylestradiol. Kent, U. M., Sridar, C., Spahlinger, G., and Hollenberg, P. F. Unpublished results.


Figure 3. N-(3,3-Dichloro-4-pyridyl)-4-methoxy-3-(prop-2-ynyloxy) benzamide (DMPB). The circled area indicates the site of metabolism resulting in the formation of a reactive intermediate. Epoxidation by P450 results in the proposed ketene intermediate.

Figure 4. 1,1-Dichloroethylene (DCE). The circled area indicates the site of metabolism resulting in the formation of a reactive intermediate. Two reactive intermediates are proposed, the DCE epoxide and the 2-chloroacetyl chloride.

a KI of approximately 5 µM and a kinact of 0.09 min-1 and formation of the DMPB carboxylic acid product. The loss in P450 2B6 activity correlated with the loss in the reduced P450 CO spectrum and the authors suggest that metabolism of DMPB to a reactive ketene intermediate may have resulted in heme alkylation. However, the possibility that the heme dissociated following protein modification and oxidation of the cys-thiolate axial ligand to the heme iron and/or conversion to P420 can not be excluded. Analysis of marker substrate metabolism in human liver microsomes indicated that DMPB did not affect P450s 3A4, 2D6, and 2E1, but reduced activity was also seen with P450s 2C9 and 2C19. 1,1-Dichloroethylene (DCE) (Figure 4) metabolism is mediated by P450 enzymes. DCE is a mechanism-based inactivator, and metabolism of DCE has been shown to elicit toxicity in lung tissue of mice exposed to this compound. Recent studies using recombinant rat and human enzymes indicated that metabolism of DCE resulted in the formation of several DCE metabolites such as 2,2-dichloroacetaldehyde, 2-chloroacetyl chloride, and a DCE epoxide (34). The DCE epoxide that was generated by P450s 2E1 and 2F2 in murine lung may be the reactive intermediate responsible for loss in activity and the observed toxicity.

6. Organosulfur Compunds Thiol functional groups are common in many food constituents (diallyl sulfide and isothiocyanates) (35–42), pesticides (parathion) (43), and drugs such as disulfiram, cimetidine (44),


Figure 5. Diallyl sulfide (DAS) is initially metabolized (1) to diallyl sulfoxide (DASO), which is metabolized (2) to DASO2 and then to a reactive epoxide intermediate (DASO3) responsible for mechanismbased inactivation of P450 2E1. The circled areas indicate the sites of metabolism resulting in the formation of a reactive intermediate.

tienilic acid (7), ticlopidine (45), and thiazolidinediones (46–50). The primary metabolism of many of these organosulfur compounds has been attributed to P450 enzymes, and metabolism by P450s has resulted in some cases in the mechanism-based inactivation of these enzymes. Beneficial health effects have been ascribed to the consumption of diallyl sulfide (DAS) (Figure 5) found as a component in garlic oil. The protective function of DAS against certain types of cancers in several tissues by chemical carcinogens such as nitrosamines including the potent lung carcinogens N-nitrosodimethylamine (NDMA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), benzo[R]pyrene, and ethylbenz[R]anthracene is thought to occur due to the initial conversion of DAS to diallyl sulfoxide (DASO) and then to diallyl sulfone (DASO2), a mechanism-based inactivator of P450 2E1 (35, 51). Thus, inactivation of P450 2E1 is believed to be responsible for decreased bioactivation of these carcinogens. P450 2E1 preferentially oxidizes the diallyl sulfide sulfur to form the sulfoxide and the sulfone. DASO2 has been shown to be converted further by P450 2E1 to a shortlived intermediate, the reactive monoallyl epoxide (DASO3), which inactivated the P450 2E1 activity in a time- and concentration-dependent manner and could be trapped with GSH (37). The time-, concentration-, and NADPH-dependent formation of DASO3 and its GSH conjugate paralleled the significant decrease in the P450 heme content, as measured by pyridine hemochrome, in liver microsomes incubated with DASO2. This decrease was greater than the corresponding loss in P450 2E1 activity and suggests that other P450s may also be sensitive to DASO2. Other protective effects of DAS metabolism by P450 2E1 and P450 2E1 inhibition have been ascribed to an increase in cellular glutathione levels. Isothiocyanates (ITCs) are found as glucosinolate complexes in relatively large quantities in cruciferous vegetables such as broccoli, cabbage, and watercress (52). The reactivity of the electrophilic carbon center of ITCs with sulfur, nitrogen-, or oxygen-containing nucleophiles contributes mainly to their effectiveness (53). As with diallyl sulfide, consumption of these compounds is thought to be beneficial for the prevention of certain cancers (1, 41). The anticarcinogenic properties ascribed to ITCs include induction of phase I and phase II enzymes, induction of apoptosis, and their antiproliferative activities (41, 42, 54). Many naturally occurring and synthetic ITCs have been shown to inhibit the activities of several P450 enzymes such as P450s 2B1/6, 2A6/13, and 2E1 in vitro and in vivo (1 and references therein, 40) and are thought to inhibit P450catalyzed bioactivation of the carcinogen via direct interaction of the ITC with one or more nucleophilic residues involved in P450 activity or by metabolic activation of the ITC to a reactive

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Figure 6. (A) Benzyl isothiocyanate (BITC); (B) phenethyl isothiocyanate (PITC). The circled area indicates the site of metabolism resulting in the formation of a reactive R-NCO intermediate.

intermediate that inactivates the P450. In particular, the inhibition of P450 2E1 by the naturally occurring benzyl isothiocyanate (BITC, Figure 6A) and phenethyl isothiocyanate (PEITC, Figure 6B) has received considerable attention (54). Although structurally very similar, these two ITCs may inhibit or inactivate members of different P450 families. Both compounds are mechanism-based inactivators for rat P450s 2B1 and rabbit 2E1, and they have been reviewed extensively (1 and references therein) and in part exert their protective effect by preventing the metabolism of pro-carcinogens such as NNK and Nnitrosomethylbenzylamine. P450s 2A6 and 2A13 are thought to be the primary enzymes involved in the metabolism of the tobacco-specific pro-carcinogen NNK. Recently, the effects of BITC and PEITC on the metabolism of coumarin by P450s 2A6 and 2A13, the oxidation of nicotine by P450 2A6, and the metabolism of NNK by P450 2A13 were investigated (40). Noncompetitive inhibition of coumarin metabolism was seen with P450s 2A6 and 2A13 and BITC, whereas partial noncompetitive and uncompetitive inhibition of coumarin metabolism was seen with PEITC and P450s 2A6 and 2A13, respectively. In contrast, nicotine (2A6) and NNK (2A13) metabolism was inactivated in a time-, concentration-, and NADPH-dependent manner characteristic of mechanism-based inactivation (40). Little loss in heme was observed with the inactivation of either enzyme by BITC or PEITC, suggesting that the primary means of activity loss was adduction of a reactive BITC or PEITC intermediate to the apo-protein. In support of this hypothesis, mass spectral analysis of apo-P450 2A13 showed an increase in the mass of the inactivated enzyme by 159 or 187 mass units corresponding to the mass of one molecule of BITC or PEITC and 1–2 oxygen atoms. The mechanism-based inactivation and metabolism of BITC or PEITC by P450s 2B1 and rabbit 2E1 in the reconstituted system and in microsomes has been studied extensively and is summarized in ref 1. Recently, it has also been shown that expressed and purified human P450 2E1 is inactivated by both BITC and PEITC in a reconstituted system in a mechanismbased manner.3 The KI values for human P450 2E1 with BITC and PEITC were approximately 1 and 13 µM, respectively. No loss in spectrophotometrically or HPLC-detectable heme was observed upon inactivation, again suggesting that the loss in enzymatic activity is due to adduction of a functionally critical residue in the P450 active site. The exact nature of the reactive intermediate responsible for the loss in P450 activity is not known. Studies using [14C]-labeled BITC or PEITC showed that radiolabel became covalently attached to the inactivated protein. Because no inactivation of human P450 2E1 was observed with phenethylisocyanate, the possibility that a protein modification 3

Yoshigae, Y., and Hollenberg, P. F. Unpublished results.

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Figure 8. (A) Tienilic acid (TA); (B) ticlopidine. The circled areas indicate the sites of metabolism resulting in the formation of the reactive intermediates, tienilic acid S-oxide and ticlopidine S-oxide. Figure 7. Parathion. The circled area indicates the site of metabolism resulting in the formation of the putative reactive phosphooxythiran intermediate responsible for protein modification.

by the isocyanate may not lead to inactivation has to be considered. In light of studies with other sulfur-containing compounds such as parathion (discussed below), where metabolism results in the release of a reactive sulfur molecule that may bind to a functionally critical site, such a reaction cannot be excluded for the isothiocyanates. With the exception of one metabolite, the metabolic profile observed for the metabolism of BITC and PEITC by human P450 2E1 was similar to that seen with the rabbit enzyme. Although the sequence identities between rat, rabbit, and human P450 2E1 are greater than 85%, there appear to be significant structural changes within the active site of the human enzyme that result in a complete change in the specificity for the formation of one of the metabolites.3 Metabolic activation of the pesticide parathion (O,O-diethylO-p-nitrophenylthiophosphate) (Figure 7) has been shown to be responsible for hepatotoxicity and immunotoxicity. Parathion has been shown to be a mechanism-based inactivator of P450 3A4 and is oxidized primarily by human P450s 3A4 and 3A5 and to some extent by P450 2D6 to form a reactive phosphooxythiran intermediate (55, 56). This intermediate may then spontaneously dearylate to the nontoxic products p-nitrophenol and diethyl phosphate/diethyl thiophosphate. In contrast, oxidative desulfuration of the intermediate results in the formation of the toxic paraoxon metabolite. Microsomes incubated with parathion in the presence of NADPH showed covalent binding of sulfur to macromolecules, which was thought to arise from the conversion of parathion to paraoxon by P450s. Recent studies using rat liver microsomes indicated that the 2B family may be involved in the metabolism of parathion to a greater extent than previously thought. Liver microsomes from mice treated with phenobarbital generated the highest levels of the p-nitrophenol metabolite together with decreased hepatic GSH levels, suggesting that parathion was metabolized to a greater extent by 2B enzymes (55–57). Tienilic acid (TA, Figure 8A) is a potent inducer of immunemediated hepatotoxicity characterized by the presence of antiLKM2 autoantibodies in the serum of affected individuals. TA is metabolized by human liver P450 2C9 to 5-hydroxy tienilic acid. During the metabolism of TA, a highly reactive electrophilic intermediate is generated that covalently binds to the P450 2C protein and results in the inactivation of the enzyme (58–60). Some of the reactive intermediates that are generated also diffuse

Figure 9. (A) Troglitazone (TGZ). The circled area indicates the site of metabolism resulting in the formation of a reactive intermediate. (B) Proposed reactive thiazolidinedione intermediate.

out of the active site and then are able to react with other microsomal proteins and GSH (7). Serum from patients containing anti-LKM antibodies was found to recognize native and alkylated P450s 2C9 and 2C11 but not members of other P450 families (61). Ticlopidine (Figure 8B) is a selective mechanism-based inhibitor of P450 2C19 (45). The selectivity of ticlopidine for P450 2C19 has been ascribed to the presence of two aryl rings where one of the rings has to be a thiophene ring. An additional requirement appears to be the presence of an amine function. Oxidation of ticlopidine resulted in a ticlopidine-, time-, concentration-, and NADPH-dependent inactivation of P450 2C19. The kinetics of inactivation were described by a KI of 87 µM and a kinact of 0.003 min-1. The major metabolites observed were 2-hydroxyticlopidine and a dimer of the ticlopidine S-oxide. The primary cause for the loss in activity of P450 2C19 was the binding of a reactive intermediate of ticlopidine, which may be the thiophene S-oxide or possibly a thiophene ring epoxide to the P450 apo-protein. Thiazolidinedione (TZD) derivatives such as troglitazone (TGZ, Rezulin) (Figure 9) or MK-0767 (46–50) undergo P450dependent metabolism via activation of the TZD ring and ring scission to generate several reactive intermediates. With TGZ, the initial S-oxidation of the TZD ring was followed by a spontaneous ring opening, reduction, and sulfation (48). TGZ was the first clinically beneficial glitazone used for the treatment of type II diabetes (62). It was voluntarily removed from the market in 2000 due to its sometimes severe hepatotoxicity, which has resulted in approximately 90 cases of hepatic failure


Hollenberg et al.

Figure 11. (A) Phencyclidine (PCP). The circled area indicates the site of metabolism resulting in the formation of a reactive intermediate. (B) Formation of the proposed PCP reactive intermediates.

Figure 10. (A) N-Benzyl-1-aminobenzotriazole (BBT); (B) N-R-methylbenzyl-1-aminobenzotriazole (aMBT). The circled area indicates the site of metabolism resulting in the formation of a reactive intermediate.

requiring liver transplantations and approximately 26 deaths. TGZ is a potent inducer of P450 3A4 and is metabolized primarily by P450 3A4 and to some extent by P450 2C8. The primary metabolism in humans involves sulfation to the TGZsulfate (TGZS), oxidative chroman ring opening to a TGZquinone (TGZQ) metabolite, and glucuronidation to form the TGZG metabolite. Covalent binding of [14C]TGZ to macromolecules was seen primarily in dexamethasone-induced rat liver microsomal preparations, indicative of a role for P450 3A4. This binding was only observed in samples that were incubated with TGZ in the presence of NADPH and required active P450. Ketoconazole completely inhibited the binding of radiolabeled TGZ, presumably by blocking the metabolic formation of the reactive intermediate. Although the hepatotoxic properties of TGZ are thought to come about through a variety of mechanisms, reactive TGZ intermediates such as the quinone or epoxide metabolite may play an important role in many pathological consequences (49, 50).

7. Arylamines 1-Aminobenzotriazole (ABT) is a relatively nonselective mechanism-based inactivator for several P450 enzymes. The reactive benzyne intermediate formed upon incubation in the presence of NADPH leads to a loss in enzymatic activity by alkylating the heme moiety of the P450s. N-Aralkylated derivatives of ABT such as N-benzyl-1-aminobenzotriazole (BBT) and R-methylbenzyl-1-amino-benzotriazole (RMBT) (Figure 10A,B) that structurally mimic the 2B selective substrate benzphetamine were synthesized in an effort to generate a specific inhibitor for the 2B family. The initial studies with these derivatives showed that BBT and RMBT were indeed more specific for the P450 2B family and have been previously reviewed (1 and references therein). BBT was found to be a mechanism-based inactivator of P450 2B1 with a KI ) 2.1 µM. BBT bound to the P450 apo-protein with a stoichiometry of 0.4:1. Radiolabeling of the P450 2B1 apo-protein was observed when BBT, labeled at either the 2,3-aminotriazole or the 7-benzyl position, was incubated in the reconstituted system in the presence of NADPH. Metabolite studies showed that BBT metabolism results in the formation of aminobenzotriazole, benzotriazole, benzaldehyde, and a BBT dimer. RMBT appears to be a more potent inactivator of P450 2B1 and P450 2B6 than BBT and also labels the 2B1 apo-protein (63–65). Recent studies from our laboratory have narrowed down the possible

site of adduction and have shown a distinct difference in the identities of the reactive intermediates generated by the rat and the human P450s.4 The site of adduction to the apo-protein was studied by a combination of enzymatic and chemical digestions of the labeled proteins together with mass spectrometric analyses of the adducted peptides. The digestion experiments suggested that the site of adduction was located in a peptide spanning amino acid residues 359–370. The reactive intermediate(s) generated by the metabolism of BBT or RMBT by P450s 2B1 and 2B6 were trapped with GSH and analyzed using electrospray ionization mass spectrometry. Metabolism of BBT and RMBT by P450 2B1 results in three different reactive intermediates that were trapped by GSH, a benzotriazole intermediate from BBT or RMBT, an aminobenzyl, and a methylaminobenzyl intermediate from BBT and RMBT, respectively. Metabolism of BBT and RMBT by P450 2B6 resulted in trapping of two different reactive intermediates with GSH, an aminobenzyl and a methylaminobenzyl intermediate from BBT and RMBT, respectively. No adduct corresponding to the benzotriazole moiety could be isolated from incubation mixtures with P450 2B6. The adducted peptide in P450 2B6 spanned the residues 359–378. Preliminary data from digestions with trypsin of P450s 2B1 or 2B6 and analysis of the peptide digests using ESI-LCMS/MS suggest that the adducts are near to or located on R378 (R370 in 2B6). This observation correlates well with the findings that V367 plays a role in substrate specificity (30).

8. Cyclic Tertiary Amines Phencyclidine (PCP, Figure 11) is a mechanism-based inactivator of P450s 2B1 and 2B6 (66, 67). With each enzyme, the inactivation was PCP concentration-, time-, and NADPHdependent and exhibited pseudo first-order kinetics. Virtually no loss in the spectrally detectable heme was observed; therefore, the inactivation was thought to occur through binding of a reactive intermediate of PCP to the apoprotein. The stoichiometry of [3H]PCP binding was approximately 1:1. This stochiometry and protein adduction of a reactive PCP intermediate were supported by ESI-LC-MS analysis of PCP-inactivated P450s 2B6 and 2B1 and 2B4. The mass difference between the PCP-inactivated and the noninactivated P450s was 244 Da, which corresponded to the binding of one PCP molecule per P450. Five major metabolites of PCP were detected and identified as the PCP iminium ion, cis/trans-1-(1-phenyl-4hydroxy-cyclohexyl)piperidine, cis/trans-1-(1-phenylcyclohexyl)4-hydroxypiperidine, and 5-[N-(1′-phenylcyclohexyl)amino] 4 Identification of the Reactive Intermediates and Amino Acid Modifications Formed During the Metabolism of N-benzyl-aminobenzotriazole or R-Methylbenzyl-aminobenzotriazole by CYP 2B1 and CYP 2B6. Kent, U. M., and Hollenberg, P. F. Unpublished results.

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Figure 12. Furanocoumarins, bergamottin (BG), 8-geranyloxypsoralen, and 8-methoxypsoralen (8-MOP). The circled area indicates the site of metabolism resulting in the formation of the reactive furan epoxide intermediate leading to protein adduction.

pentanoic acid (66). The primary site of metabolism to form the reactive intermediate responsible for inactivating members of the P450 2B family appears to be on the piperidine ring. P450s 2B1 and 2B4 formed a novel metabolite with an m/z of 240 corresponding to the mass of the 2,3-dihydropyridinium species of PCP. Reduction with NaBH4 resulted in the disappearance of the signal at m/z 240, consistent with reduction of the 2,3-dihydropyridinium species. Trapping studies using GSH and NAC resulted in conjugates that were consistent with the mass of the captured 2,3-dihydropyridinium ion. These data suggested that the reactive intermediate responsible for inactivating P450s 2B1 and 2B4 was the enamine, which was formed following oxidation of the R-carbon of the piperidine ring to generate the iminium ion. Previous studies had proposed that the iminium ion might be the reactive indermediate. However, this finding together with the observation that NADPH was required for the iminium ion to inactivate the P450 ruled out the iminium ion as the inactivating species (66, 67, and references therein). P450 2B6 favors oxidation pathways for the metabolism of PCP that differ from those utilized by P450s 2B1 or 2B4. The human enzyme formed a completely different reactive intermediate corresponding to a dioxygenated species that could be trapped as a GSH or NAC adduct. P450 2B6 may have generated this reactive intermediate from the enamine intermediate by oxidation at the 4-position of the piperidine ring followed by a second hydroxylation at the 3-position or possibly formation of a 3,4-epoxide (66).

9. Furanocoumarins Furanocoumarins (Figure 12) such as bergamottin (BG), 8-geranyloxypsoralen, and 8-methoxypsoralen (8-MOP) are found as components of many foods and have been shown to inhibit the metabolism of several xenobiotics. BG, one of the components responsible for the “grapefruit juice effect”, is a mechanism-based inactivator of cytochrome P450s 2B1, 2B4, 2B6, 3A4, and 3A5 (68–70). The inactivation of both P450s 2B6 and 3A5 was NADPH-dependent and irreversible (a KI of 5 µM for 2B6 and a KI of 20 µM for 3A5). Covalent binding of a reactive BG intermediate to the apoprotein has been observed. ESI-LC-MS analysis of the BG inactivated P450s 2B1, 2B4, 2B6, and 3A5 resulted in an increase in mass of the apoprotein by 388 Da. It has been suggested that BG is first metabolized to 6,7-dihydroxy BG followed by the addition of one oxygen to the furanocoumarin moiety. The reactive epoxide intermediate could then bind to a nucleophilic residue in the P450. The catalytic pathway leading to the production of the

reactive intermediate of BG responsible for inactivating P450s was investigated by studying the metabolism of BG by P450s 2B6 and 3A5 (70). P450 2B6 metabolized BG primarily to two major metabolites, 5′-OH-BG and a mixture of 6′- and 7′-OHBG, and one minor metabolite, bergaptol. Because the 6′- and 7′-OH-BG were the primary metabolites, it suggested that P450 2B6 preferentially oxidized the geranyl-oxy chain of BG. The metabolism of BG by P450 3A5 resulted in three major metabolites: bergaptol, 5′-OH-BG, and 2′-OH-BG, and two minor metabolites, 6′,7′-dihydroxy-BG and the mixture of 6′and 7′-OH-BG. Because bergaptol was the most abundant metabolite formed, it suggested that P450 3A5 metabolized BG mainly by cleaving the geranyl-oxy chain. Molecular modeling studies confirmed that the docking of BG in the P450 2B6 active site favors oxidation in the terminal region of the geranyl-oxy chain, whereas an orientation positioning the 2′-carbon of BG nearest the heme iron is preferred by P450 3A5. The predominant GSH–BG conjugates formed by both P450s exhibited m/z values of 662. MS/MS analysis of the GSH conjugates indicated that the oxidation leading to the formation of a reactive intermediate occurred on the furan moiety of the BG, presumably through the initial formation of an epoxide at the furan double bond. These data showed that the oxidation of the geranyl-oxy chain resulted in the formation of stable metabolites of BG, whereas oxidation of the furan ring produced reactive intermediates that may be responsible for inactivating P450s 2B6 and 3A5. 8-Geranyloxypsoralen is a naturally occurring inhibitor of P450 3A4 (71). This compound is closely related to BG and contains the same furanocoumarin core structure. The only difference between the two molecules is that the geranyloxy chain of BG is located at the 5-position. Studies on P450 3A4 activity in human liver microsomes with different synthetic analogues of 8-geranyloxypsoralen confirm earlier findings that the furan ring is required for inactivation (72). The most likely candidate for the reactive intermediate was the 2′,3′-epoxide. 8-MOP is a potent mechanism-based inactivator of P450s 2A6 (KI of 0.8 µM) (72), 2B1 (KI of 2.9 µM) (73), and 2A13 (KI of 0.11 µM) (74) and also contains the same furanocoumarin core structure as BG and 8-geranyloxypsoralen. The mechanism of inactivation was ascribed to the covalent modification of the P450 apo-protein by a reactive intermediate of 8-MOP. The steps in generating this reactive intermediate were found to require an initial epoxidation reaction followed by hydrolysis or attack by a nucleophile to form dihydrofuranocoumarin products. As with BG and 8-geranyloxypsoralen, the furan epoxide is believed to be the key reactive intermediate responsible for the modification and inactivation of the P450 apoprotein.

10. Other Mechanism-Based Inactivators Zafirlukast (Figure 13) is used clinically as a leukotriene receptor antagonist in the treatment of asthma (75). Isolated cases of idiosyncratic hepatotoxicity in individuals have been associated with the use of this drug (76). Zafirlukast is metabolized by P450 3A4 in an NADPH-dependent manner to yield reactive electrophilic intermediates that arise from the oxygenation of the indole moiety of the molecule. P450 3A4 was inactivated in a mechanism-based manner with a KI of 13.4 µM and a kinact of 0.026 min-1. The reactive intermediate was trapped with GSH and was found to be a R,β-unsaturated iminium intermediate (77). It is not clear at this time if the hepatotoxic consequences associated with Zafirkulast are brought about by covalent modification of P450s.


Hollenberg et al.

P450s 2B6 and 2B1 (83). The loss in P450 2B1 activity could primarily be ascribed to modification of the apoprotein by a reactive tTEPA intermediate. In contrast, the inactivation of P450 2B6 was due primarily to modification of the heme, suggesting that the inactivation of these two P450s occurs by distinctly different mechanisms. tTEPA is initially oxidized by P450s to the primary active metabolite TEPA. The structure of the reactive intermediates responsible for protein or heme alkylation has not been elucidated.

11. Types of Modifications Encountered with Reactive Intermediates

Figure 13. Zafirlukast. The circled area indicates the site of metabolism resulting in the formation of a reactive iminium intermediate.

Tamoxifen is a nonsteroidal antiestrogen that is used to treat hormone-dependent breast cancer (Figure 14A). Nonstereoidal antiestrogens are also used in hormone replacement therapy, and some links between long-term therapy and increased risks for endometrial and breast cancers have been observed. Tamoxifen was found to be a potent mechanism-based inactivator of several human P450s such as P450s 2B6, 2D6, and 2C9 (78, 79). The principal loss in activity occurs as a result of apoprotein modification. The reactive intermediate involved may be formed after oxidative metabolism of tamoxifen to 4-OH-tamoxifen, which could be further oxidized to form the reactive quinone methide. Raloxifene (Figure 14B), another nonsteroidal antiestrogen related in structure to tamoxifen, is used in the treatment of osteoporosis. Raloxifene was found to inactivate P450 3A4 in human liver microsomes in a mechanism-based manner with an approximate KI of 9.8 µM and no loss in spectrally detectable P450 (80). Raloxifene was bioactivated by P450 3A4 (and 2D6 to some extent) primarily on the 7-position of the benzothiaphene ring and to some lesser extent on the 5-position of the benzothiaphene and the 3-position of the phenol ring. The mechanism that was postulated involves the initial epoxidation of the phenol to form the reactive arene oxide intermediate. However, the possibility that the initial step of activation involves a quinone intermediate could not be ruled out. Recent detailed studies of the P450 3A4 peptide that was adducted by the raloxifene reactive intermediate were able to identify Cys239 as the site of modification and determine that the raloxifene diquinone methide was the reactive intermediate responsible for protein modification (81). Glabridin (Figure 15), an isoflavan isolated from alcoholic extracts of licorice root, was shown to be a mechanism-based inactivator of P450s 3A4 and 2B6 (82). Studies with P460 2B6 in a reconstituted system demonstrated that the inactivation was due to modification of the P450 apoprotein. The structure of the reactive intermediate is not known. The inability of the 2,4dimethyl derivative of glabridine to inactivate P450 3A4 raises the possibility that the dihydroxyphenyl ring may be the ultimate site of metabolism leading to the formation of a reactive intermediate. A mechanism similar to that seen with tamoxifen may be involved, but that has not yet been studied in detail. The anticancer drug N,N′,N′′-triethylenethiophosphoramide (tTEPA) (Figure 16) was studied in the reconstituted enzyme system and was found to be a mechanism-based inactivator of

Modification of the P450 apoprotein by reactive intermediates generally involves covalent binding to a nucleophilic amino acid residue such as lysine, serine, threonine, tyrosine, or cysteine. In contrast to modifications of the apoprotein, the fate of the heme moiety resulting from attack by a reactive intermediate can be more complex. Studies with the mechanism-based inactivators allylisopropylacetamide and 5-phenyl-1-pentyne (5PIP) suggest that the reactive intermediate formed by the P450s during the metabolism of each compound alkylates the heme moiety (84, 21). Earlier studies on the metabolism of 1-aminobenzotriazole by P450 enzymes showed that the reactive benzyne intermediate bound to the iron-depleted P450 heme by bridging two adjacent heme nitrogens (85). Heme modification that involves the complete breakdown of the tetrapyrole ring at the meso carbons has been described for the mechanismbased inactivation of P450 3A4 by 3,3-dicarbethoxy-2,6dimethyl-4-ethyl-1,4-dihydropyridine, spironolactone, and cumene hydroperoxide (86–88). A role for a peroxyl radical was invoked in generating the resulting monopyrolic (hematinic acid, methylvinylmaleamide) and dipyrolic fragments. The dipyrolic fragments may interact with the P450 apo-protein either directly or after further breakdown. Mechanism-based inactivation of P450s 3A4 and 3A5 by 17EE and BG has resulted in both protein modification and the loss of spectrally detectable heme with the appearance of heme-related products that eluted at a later time following HPLC separation (23, 69). HPLC analysis of the 17EE-inactivated P450 3A4 demonstrated that the inactivation resulted in the disappearance of approximately onehalf the native heme with the concomitant generation of modified- and 17EE-labeled heme. The two structurally related compounds tert-butyl acetylene (tBA) and tert-butyl 1-methyl-2-propynyl ether (tBMP) inactivated P450 2E1 and a T303A mutant of 2E1 (89). The decrease in enzyme activity was ascribed primarily to a loss in the P450 heme. LC-MS analysis revealed that inactivation resulted in the appearance of modified heme products with masses consistent with the mass of an iron-depleted heme conjugated to a tBAor tBMP-reactive intermediate containing one oxygen atom. In addition, the formation of a new spectral intermediate absorbing maximally at 485 nm was discovered during the course of the inactivation (90). The formation of this adducted heme in the T303A mutant was reversible, and it appears to have been generated from an intermediate that was formed in a reversible fashion and could slowly decompose to regenerate the active enzyme. Subsequent detailed studies revealed that exogenously supplied protons were required (similar to what is believed to occur in the native proton transfer pathway involving T303 in the wild-type P450 2E1) for the intermediate to covalently N-alkylate the P450 heme. The reactive intermediate of tBA capable of inactivating P450 2E1 may be formed by the concerted attack of OH+ from the electrophilic hydroperoxoiron species on one of the nitrogens of the heme (90). tBA and

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Figure 14. (A) Tamoxifen; (B) raloxifene. The circled areas indicate the sites of metabolism resulting in the formation of the reactive tamoxifen quinine methide and the raloxifene diquiononemethide intermediates.

Figure 15. Glabridin. The circled area indicates a possible site of metabolism resulting in the formation of a reactive intermediate.

Figure 16. N,N′,N′′-Triethylenethiophosphoramide (tTEPA). The circled area indicates the site of oxidation resulting in the formation of TEPA.

tBMP also inactivate P450 2B4 with concurrent losses in the reduced CO spectrum and the native heme, as measured by HPLC (91). LC-MS analysis demonstrated that the losses in native heme were accompanied by the appearance of two modified hemes with m/z values of 705 Da, consistent with formation of a heme adduct by tBMP containing one oxygen atom and attached to an iron-depleted heme. 1H NMR analysis of the two modified heme products suggested that they are N-alkylated on pyrrole rings A and D (91).

12. Trapping and Identification of Reactive Intermediates Because of the potential for P450-catalyzed reactions to form reactive intermediates that can lead to detrimental clinical effects down the line, several approaches have been developed to identify compounds that undergo bioactivation by P450s early in the drug discovery and development process. Small sulfhy-

dryl-containing compounds such as reduced GSH, GSH esters, and NAC have been employed successfully to trap electrophilic intermediates generated by P450s as S-linked conjugates for subsequent structural analysis using mass spectrometry (13, 35, 66, 92–95). Reactions of GSH with electrophiles via (i) nucleophilic substitution at oxirane rings and saturated and aromatic carbons, (ii) Michael addition, and (iii) addition to nitroso and carbonium ions have been described. One of the advantages of using GSH as a trapping reagent is that the conjugates are more polar, amphoteric, and nonvolatile, making the isolation, extraction, and structural identification of a reactive intermediate more feasible. Because the reactive intermediates are generally present in low abundance, identification of these trace amounts has been difficult and requires sensitive assay methods. However, recent advances in liquid chromatography coupled with electrospray ionization mass spectrometry techniques have resulted in the identification of many low abundant reactive intermediates. A further advantage to using GSH to trap reactive intermediates of unknown mass and structure is the ability to utilize tandem mass spectrometry under neutral loss scanning conditions. GSH conjugates will undergo a neutral loss of 129 for the pyroglutamate, which is lost from the glutamate residue of the [M + H]+ ion of the GSH conjugate. This means that the instrument will detect this neutral ion loss when it occurs and can then be interrogated to give information as to the mass of the parent conjugate. The associated MS/MS spectrum can then be used to elucidate the structure of the conjugate and draw conclusions as to the structure of the reactive intermediate. Such analyses can then aid in the modification of the structure of parent compound so as to avoid bioactivation by P450 enzymes and potentially overcome their toxicological properties. Because not all GSH conjugates, such as aliphatic or benzylic thioether conjugates, can be successfully analyzed using neutral loss scanning, negative ion MS/MS spectra with precursor ion scanning for the parent conjugates at m/z of 272, which arise from the deprotonated glutamyl-dehydroalanyl-glycine species, have been employed (96). In many instances, it is not only desirable to identify the reactive intermediate but to quantify how much of the intermediate is generated. To that end, trapping reagents coupled to spectrophotometrically detectable and


quantifiable functional groups such as dansyl glutathione have been developed (97). Other electrophiles that can be generated as a result of bioactivation by P450 enzymes such as iminium ions, aldehydes, or acyl glucuronides may escape detection when GSH is used as the trapping agent. These hard electrophiles have been trapped using anions such as CN upon incubation in the presence of equal amounts of unlabeled and isotopically labeled CN salts. These CN conjugates can then be detected by LC-MS/MS using neutral loss scanning of 27 and 29 Da for the unlabeled and isotopically labeled CN, respectively (98).

13. Future Directions in the Identification of Reactive Intermediates and Their Targets ADRs resulting from the P450-catalyzed formation of reactive intermediates or toxic metabolites are more difficult to predict than some other ADRs because the molecule requires metabolic activation. Clinical manifestations of such toxic effects are therefore highly dependent on a variety of factors including the reactivity of the intermediate, the species or tissue in which the compound is metabolized, the genetic predisposition or polymorphic expression of certain P450 enzymes in an individual, and the relative expression of various phase I or phase II drugmetabolizing enzymes. It therefore becomes highly desirable to rapidly identify target compounds that may be bioactivated early in development and to determine which enzyme is responsible for the formation of the reactive intermediate. Proteomic analysis approaches where macromolecular targets of radiolabeled reactive intermediates have been separated using 2D gel electrophoresis coupled to MALDI-TOF mass spectral analysis have been utilized to identify numerous protein targets in tissues. For instance, treatment of rats with [14C]bromobenzene resulted in the identification of 33 different [14C]bromobenzene-labeled proteins in rat liver cytosol using such a proteomic approach (99). Development of new reagents that are capable of trapping the majority of reactive intermediates and subsequent analysis using high-throughput screening techniques would be most useful. Recently, a quaternary ammonium GSH analogue (QAGSH) with chemical reactivity similar to GSH has been developed (100). Trapping studies with this fixed charge compound would overcome high background issues and the requirement to separate and resolve samples using liquid chromatography making QA-GSH suitable for high-throughput analysis. In another approach, GSH has been mixed in a 1:1 ratio with stable isotope-labeled γ-glutamylcysteinylglycine13 C2-15N (GSX) and used to trap reactive intermediates (101). The presence of GSH conjugates was detected by LC-MS/MS as a neutral loss of 129 Da. Definitive identification of the trapped intermediate was achieved by searching for the unique signature of a doublet with 3 Da difference in mass that arose from the 1:1 mixture of the GSH:GSX. Two human P450 cell-based models have been developed to evaluate metabolism-dependent toxicity (102). The first cell system consisted of microsomes expressing P450 3A4 that were incubated together with human liver-derived HepG2 target cells. The second model contained HepG2 cells transiently transfected with P450 3A4. Toxicity was assessed by measuring ATP levels and the reduction of a mitochondrial activity marker substrate to monitor cell viability. Incubation of each of these systems with several P450 substrates such as troglitazone, tamoxifen, triazolam, and others demonstrated the utility of such systems in the early screening process. Because cellular proteins are a major target of reactive intermediates, it would be advantageous to be able to predict if

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certain proteins may be more susceptible than others to modification. In an innovative approach to addressing this challenge, two thiol-reactive biotin-tagged model electrophiles were developed to label cytosolic and nuclear proteins. Capture of the biotin-labeled peptides was accomplished following digestion of the labeled samples with trypsin using biotin–avidin affinity chromatography. This was followed by multidimensional LC-MS-MS analysis of the biotin-labeled peptides (103). In the majority of cases, the adduct was confined to a single cysteine residue, suggesting that most proteins were modified at only one or possibly two sites. Susceptible protein motifs were evaluated by analyzing the frequencies at which a particular amino acid was found flanking the adducted cysteine residue (-5 to +5 relative to the adduct). Lysine residues in positions -2,-3, and +5 were observed at significantly higher levels, but this result was somewhat dependent on which of the two biotin-tagged compounds was used.

14. Mechanism-Based Inactivators for Determining the Role of Specific P450s in Catalysis One of the inherent difficulties in investigating P450-catalyzed reactions, both in vitro and in vivo, is distinguishing between the catalytic activities of individual P450s. Strategies for addressing this issue have included the use of chemical inhibitors and inhibitory antibodies of individual P450s. However, one caveat of this approach is that many inhibitors of P450s and inhibitory antibodies are not completely selective for an individual form of P450. Because mechanism-based inactivators require metabolism by the targeted P450 to a reactive intermediate, which then leads to the inactivation of the enzyme that formed it, there is the potential for a higher level of specificity. The higher degree of selectivity and the irreversible nature of the inactivation make mechanism-based inactivators powerful tools for determining the relative contributions of individual P450s to a particular reaction. In addition, many mechanismbased inactivators are drugs that have already been approved for use in humans; therefore, they can be utilized both in vitro and in vivo as probes for P450 catalytic activity. For instance, paroxetine, a selective serotonin reuptake inhibitor, is also a mechanism-based inactivator of P450 2D6 and has been used both in human liver microsomes and in patients to phenotype the P450-mediated metabolism of 3,4-methylenedioxymethamphetamine, also known as MDMA or “ecstasy” (104, 105). These studies have established a role for P450 2D6 in the metabolism of MDMA, although it is not completely clear whether P450 2D6 is the principal enzyme involved in its metabolism (104, 106). tTEPA is a P450 2B6-specific mechanism-based inactivator and has been used to determine that P450 2B6 is the primary P450 involved in the metabolism of efavirenz, a non-nucleoside reverse transcriptase inhibitor used to treat HIV-1 (107). Subsequently, the role of P450 2B6 in the metabolism of efavirenz has been confirmed in vitro (108) and by clinical studies reporting differential effects of P450 2B6 genetic polymorphisms on efavirenz clearance (109–111). Erythromycin is commonly used to phenotype P450 3A4 reactions and has been used to demonstrate the role of P450 3A4 in a number of reactions including the N-demethylation of levo-R-acetylmethadol, a long-acting opioid agonist pro-drug used for preventing opiate withdrawal (112).

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15. Role of Mechanism-Based Inactivators in the Identification of Critical Amino Acids in the Active Sites of P450s The structures of a number of mammalian and bacterial P450s have been solved and reveal that P450s contain several common structural elements including a series of helices denoted by letters A–L (113). The A helix is closest to the N terminus of the catalytic domain (113). Site-directed mutagenesis studies have indicated that residues in helices B, F, and I are in contact with the substrates (114). There are also six well-conserved substrate recognition sequences (SRS 1–6) that are present in most P450s (115). The availability of P450 crystal structures has greatly increased our knowledge regarding the relationship between P450 structure and function (116). Because of the important role that P450s play in a number of biological processes such as drug metabolism, carcinogen activation, and metabolism of endogenous substrates, elucidation of the key structural elements, including amino acid residues, involved in substrate metabolism is critical. However, in order for this information to be most beneficial, the static structural information gained through X-ray crystallography studies must be coupled with experimental data obtained from dynamic systems. Mechanism-based inactivators offer a unique approach for identifying critical amino acid residues within the active sites or access channel regions of P450s. Combination studies involving several inactivators and P450s will help to generate a broader knowledge base regarding those residues that are important for the specific catalytic function of a particular P450. The information provided from these studies could then lead to more rational design of drugs and inhibitors that target specific P450s.

16. Clinical Implications for Mechanism-Based Inactivation of P450s In the clinical setting, P450 inhibition can result in elevated plasma levels of other drugs that are primarily metabolized by the particular P450 that is inhibited. As a result, inhibition of P450s has the potential to cause severe adverse events, particularly if the coadministered drug has a narrow therapeutic index. Because a P450 modified by a mechanism-based inactivator is irreversibly inactivated and has to be replaced by newly synthesized P450 in order for activity to be regained, mechanism-based inactivation has a greater potential to lead to drug–drug interactions when compared to reversible inhibition (117). A number of clinically relevant drugs and dietary components have been shown to be mechanism-based inactivators in vitro and subsequent clinical studies have confirmed that some of these mechanism-based inactivators affect the pharmacokinetics of coadministered drugs. A drug–drug interaction is said to have occurred when the efficacy or toxicity of one drug is altered by the coadministration of another drug. Clinically relevant pharmacokinetic interactions are often the result of changes in P450-mediated metabolism, which can be a consequence of P450 inactivation. Because the experiments used to characterize mechanism-based inactivators are performed in vitro, a major challenge is to determine whether these results will have any clinical relevance. However, there are several examples where drug–drug interactions have occurred involving compounds that have been demonstrated to be mechanism-based inactivators in vitro. As discussed earlier, 17EE is a mechanism-based inactivator of P450s 3A4 and 2B6 and can also reversibly inhibit 2C19 (118). 17EE has been reported to interact with coadministered drugs; however, the


most striking interaction described thus far comes from a study with selegiline. Selegiline is a selective, irreversible inhibitor of monoamine oxidase-B used in the treatment of Parkinson’s disease and is extensively metabolized by P450s 2B6 and 2C19 (119). Healthy female volunteers taking 17EE and selegiline together exhibited a 20-fold increase in the selegiline AUC and a concomitant decrease in the formation of selegiline metabolites (120). In some instances, the resulting drug–drug interaction can be severely toxic or even fatal. When terfenadine is coadministered with a mechanism-based inactivator of P450 3A4 such as erythromycin, metabolism of terfenadine is decreased. Unchanged terfenadine in the plasma can result in torsades de pointes, a form of ventricular arrhythmia that can lead to cardiac arrest and death in patients (121, 122). Prolonged use of inactivators of P450 3A4 along with simvastatin, which is primarily metabolized by P450 3A4 (123, 124), can cause severe skeletal muscle toxicity. In a clinical study, the incidence of myopathy was 10 times greater in participants who received verapamil, a mechanism-based inactivator of P450 3A4, along with 20–80 mg of simvastatin daily (125). Finally, P450 inactivation may also result in decreased metabolism of prodrugs, such as cyclophosphamide, that have to be metabolically activated (126). The effect of grapefruit juice on the pharmacokinetics of prescription drugs has received increasing attention over the past decade and a half, and several reviews have been published recently (127, 128). Patients who ingest grapefruit juice as part of their diets exhibit significantly greater mean oral bioavailability of drugs belonging to several different classes including lipid-lowering drugs, calcium channel blockers, and immunosuppressive agents. Studies have reported an effect of grapefruit juice on the pharmacokinetics and metabolism profiles of more than 40 drugs (129). The “grapefruit juice effect” has been largely attributed to mechanism-based inactivation of enteric P450 3A4 by furanocoumarins, such as bergamottin, present in grapefruit juice, although other P450s have been shown to become inactivated as well (69). Both acute and extended exposures to grapefruit juice result in P450 inhibition (130). Subsequently, several other juices and dietary components have been shown to be mechanism-based inactivators of P450s. Seville orange juice, which also contains bergamottin, was shown to increase the bioavailability of felodipine and alter its P450 3A4-mediated metabolism (131). Low concentrations of grape juice and tea were shown to inhibit approximately 90% of flurbiprofen hydroxylation, a marker of P450 2C9 activity, in human liver microsomes (132). However, in the same study, neither grape juice nor tea had an effect on flurbiprofen hydroxylation in humans when compared to placebo. Other dietary components identified as mechanism-based inactivators of P450s include glabridin, found in licorice root (82); oleuropein, a complex phenol found in olive oil (133); diallyl sulfone, which is formed during cooking or after the ingestion of garlic (134); and resveratrol, found in red wine (135, 136). A recent study isolated 19 alkamides from Piper nigrum (black pepper) and tested them for mechanism-based inactivation of P450 2D6 using human liver microsomes (137). Interestingly, P450 2D6 was inhibited by all of the isolated alkamides. Two of these compounds decreased the activity of P450 2D6 by greater than 50% and acted in a mechanism-based manner. Although reports of toxicity due to a drug–diet interaction are rare, the outcome of the interactions may also vary depending on the age of the patient, gender, genetic polymorphisms, and pathological conditions. Although a two-fold or greater increase in drug plasma concentration increases the risk of adverse events


(138), less dramatic changes in plasma concentrations may still be clinically relevant if the coadministered compound has a narrow therapeutic index.

17. Therapeutic Benefits of Mechanism-Based Inactivation The effects of mechanism-based inactivators on the pharmacokinetics of coadministered compounds can also be exploited to provide therapeutic benefits. The coformulation of ritonavir, a potent inhibitor of P450 3A4 (139), with lopinavir, a novel protease inhibitor with relatively low bioavailability (140), is proof of this concept. The combined drug formulation was shown to significantly improve the pharmacokinetic properties and hence the activity of lopinavir against HIV-1 protease (140). Along the same lines, inactivation of P450 2A6 has been proposed as a novel therapeutic approach for smoking cessation. P450 2A6 has been shown to be the primary P450 responsible for the metabolism of nicotine (141). Inhibition of P450 2A6 results in altered pharmacokinetics for nicotine, resulting in an increased plasma half-life (142). With this in mind, it is reasonable to postulate that inhibition of nicotine metabolism will lead to a decrease in the desire to smoke as well as a decrease in exposure to toxic chemicals and pro-carinogens associated with cigarette smoke. Increased bioavailability of nicotine could also aid individuals relying on nicotine replacement products as a means of quitting smoking, as lower nicotine blood levels may result in poorer clinical outcomes with regard to smoking cessation (143). Recently, a series of 3-heteroaromatic analogues of nicotine were synthesized to help elucidate the critical structural and mechanistic features necessary for selectively inhibiting human cytochrome P450 2A6 (144). A number of these compounds were found to be mechanism-based inactivators and exhibited highly selective inhibition of P450 2A6 as compared to other drug-metabolizing enzymes. Through further studies, selective inhibitors of P450 2A6 may be identified that can be delivered to patients as part of a smoking cessation strategy.

18. Future Directions Mechanism-based inactivators are powerful molecular probes for experimentally delineating the catalytic specificities of particular P450s and for obtaining important information regarding P450 structure and function. Although mechanismbased inactivators can be responsible for toxic events in vivo, the knowledge gained from these drug–interaction studies can assist in the development of strategies for using mechanismbased inactivators to favorably modulate the pharmacokinetics of coadministered drugs. In the future, coformulation of drugs such as protease inhibitors, which have low oral bioavailability, with mechanism-based inactivators may be a key aspect of their therapy. To achieve this, mechanism-based inactivators specific for each of the clinically relevant P450s are necessary. The design of these inactivators will also be of great value in vitro as they can be used to assist in the elucidation of critical structural elements of the individual P450s. This could also lead to targeted drug design, for use in instances where inactivation of a specific P450 is a potential therapeutic approach to treating a clinical condition. However, gender, age, and genetic polymorphisms are all very important factors that need to be taken into consideration as we move towards future studies and clinical applications of mechanism-based inactivation. These factors, particularly genetic polymorphisms, may result in differential inactivation of P450s. Clinical studies have demonstrated that certain P450 genetic polymorphisms play a role in adverse drug

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reactions. There is also significant evidence that mechanismbased inactivation of P450s is involved in some adverse drug reactions. So far, studies focusing on the potential overlap between the two are lacking. Thus, studies investigating whether common genetic polymorphisms affect P450 inactivation and may thereby affect the frequency of drug–drug interactions are needed.

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TX7002504

Mechanism-Based Inactivation of Human Cytochromes P450s

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