Quantitative Distinctions of Active Site Molecular Recognition by P

enzymes cytochrome P450 3A4 and P-glycoprotein. Although expressed in ... CYP3A4 inhibitors that do not affect P-gp active transport function. Remarka...
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Quantitative Distinctions of Active Site Molecular Recognition by P-Glycoprotein and Cytochrome P450 3A4 Er-jia Wang, Karen Lew, Mary Barecki, Christopher N. Casciano, Robert P. Clement, and William W. Johnson* Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, Lafayette, New Jersey 07848 Received July 30, 2001

The bulk of characterized xenobiotic defense and disposition is conferred by the abundant enzymes cytochrome P450 3A4 and P-glycoprotein. Although expressed in many tissues, these enzymes are most abundant in the liver and intestine and seem to share most substrates and inhibitors, with the apparent synergy between these two promiscuous enzymes asserted because of their extensive overlap of substrates and shared tissue location. Since the broad-spectrum tolerance to lipophilic compounds of various sizes naturally results in a similar pattern of substrate/inhibitor recognition, the cause or mechanism of many drug/drug and drug/herb interactions can be difficult to determine. These two seemingly indiscriminate enzymes, however, do not share some unique inhibitor selectivity. Particularly, we show various potent CYP3A4 inhibitors that do not affect P-gp active transport function. Remarkably, we have also identified several compoundssvalinomycin, norverapamil, reserpine, nobiletin, emetine, gallopamil, fluphenazinesthat uniquely inhibit P-gp function with affinities comparable to benchmark P-gp inhibitors despite a lack of effect on CYP3A4 function at physiologically relevant concentrations. Indeed, valinomycin inhibits P-gp with an IC50 similar to cyclosporin A yet apparently does not affect CYP3A4 function, and emetine and nobiletin are also specific for interaction with P-gp. Additionally, norverapamil and reserpine have, respectively, a 60and 40-fold preference for inhibition of P-gp over CYP3A4. Some striking structural analogies among these compounds are discussed. These distinguishing qualities of substrate recognition between CYP3A4 and P-gp should reveal nuances of active-site architecture unique to each and could serve as tools to probe for the specific discernment of P-gp-mediated drug/drug or drug/herb interactions. Learning more about binding distinctions and quantitative activity relationships of substrate/inhibitor interactions with these two enzymes and the differences between them may indicate how they recognize such a wide variety of molecules as substrates (and/or inhibitors). Moreover, identification of specific inhibitors will allow the determination of which enzyme is responsible for drug interactions and/or the extent of contribution in a multiple exposure situation.

Introduction Mammalian cells possess a natural battery of defense mechanisms against xenobiotic assault. A particular class of enzymes actively transports an extensive array of structurally unrelated large lipophilic compounds from the cell, hence providing what is often known as multiple drug resistance (MDR)1 (1, 2). Multidrug resistance is characterized by active efflux or pumping of xenobiotics, including pharmaceuticals, via transmembrane proteins. Of these transporters, the MDR1 protein is the best known and most extensively studied and thus far appears to have the largest substrate list (3). The MDR1 gene encodes a 170 kDa integral plasma membrane phosphorylated glycoprotein, also known as P-glycoprotein (Pgp), which is primarily expressed in the epithelial cells of the intestine, liver, and kidney, and in the endothelial * To whom correspondence should be addressed. Phone: (973) 9404336. Fax: (973) 940-4211. E-mail: [email protected]. 1 Abbreviations: P-gp, P-glycoprotein; ABC, ATP-binding cassette; DNR, daunorubicin; MDR, multidrug resistance; NBD, nucleotide binding domain; NBS, nucleotide binding site.

cells of the placenta and brain. The gross structural features of P-gp appear to be shared by a large family of membrane transporters known as ATP-binding cassette (ABC) transporters. The most abundant P450 enzyme in humans, P450 3A4 is present in both liver and small intestine, two major sites for the oxidation of xenobiotics as well as the active efflux conferred by P-gp. P450 3A4 can account for as much as 60% of the total P450 in a human liver (4-6) and sometimes as much as 70% in enterocytes (7). Most drugs as well as many other xenobiotics are preferentially oxidized by the P450 CYP3A4, with well over 100 substrates having been identified thus far. Notably, the definition of a substrate of both CYP3A4 and the active transporter P-gp is essentially the same and quite imprecise: generally large (MW > 180) and generally very lipophilic. The list of known CYP3A4 substrates varies in size from smaller molecules such as acetaminophen (MW 151) to cyclosporin A (MW 1201), with molecule types ranging from nonionic detergents to polycyclic aromatics to oligopeptides.

10.1021/tx010125x CCC: $20.00 © 2001 American Chemical Society Published on Web 11/28/2001

CYP3A4 and P-gp Substrate Binding Sites

The purported xenobiotic protection role of P-gp (8) mimics that of CYP3A4. The apparent significant overlap between qualities of P-gp and CYP3A4 has been noted (9), as they are sometimes coordinately upregulated (10), have similar tissue distribution (11) and cellular localization (12), and a remarkable and extensive overlap in substrate selectivity (13-17). Both enzymes provide a protective role to many of the same cells and defend against an extensive shared list of xenobiotic substrates. Naturally, this coincidental effect is synergistic, as CYP3A4 confers protection by oxidizing the xenobiotic to generally less toxic metabolite(s) and P-gp by restraining cellular access of the compound. Indeed, many drugdrug interactions of CYP3A4 substrates previously ascribed to CYP3A4 now appear to have been significantly mediated by P-gp as well (18-21). The overlap of CYP3A4 and P-gp substrate specificity, and therefore their combined contribution, is likely the most significant impediment to determining the contribution of CYP3A4 to a drug’s disposition when using CYP3A4 inhibitors. Conversely, the contribution of P-gp activity can be determined only when a probe inhibitor has no significant effect on CYP3A4. The extensive overlap between these two enzymes is probably fortuitous as opposed to concerted because of their great tolerance for and acceptance of large lipophilic substrates. Both enzymes appear to have large accommodating hydrophobic binding sites that do not discriminate among many lipophilic compounds. However, substrate recognition and preference are not this simple as both enzymes have shown cooperativity and a role for decisively oriented hydrogen bonding in the substrate binding sites. Learning about the binding selectivities and interactions of these enzymes may provide greater understanding of how both recognize and bind to their respective substrates and may also delineate the causes of many drug-drug interactions. Accordingly, the objective of this study was to investigate the extent or specificity of the relationship of CYP3A4 inhibitors and P-gp inhibitors using two previously reported methods of evaluation (22, 23). Hence, the rationale for compound selection was initial evaluation of suggested P-gp substrates/inhibitors not conspicuously known for interacting with CYP3A4 followed by comprehensive scrutiny of CYP3A4 substrate/ inhibitor tables and databases. Through screening these compounds experimentally with quantitative characterization of interaction, important divergent selectivities were revealed and described herein.

Materials and Methods Chemicals. The sources for these chemicals are followed in parentheses: reserpine (Lancaster Synthesis, Boston, MA), emetine dihydrochloride, cimetidine, norfloxacin (ICN Biomedicals, Aurora, OH), norverapamil (Alltech, State College, PA), azithromycin (US Pharmacopeia, Rockville, MD), ciprofloxacin (US Biological), gallopamil, nor-gallopamil (Knoll AG, Germany), fluphenazine hydrochloride (Alfa Aesar Ward Hill, MA), trimethoprim (Teknova Inc., Half Moon Bay, CA). Ketoconazole, valinomycin, daunorubicin (DNR), verapamil, colchicine, cyclosporin A, mannitol, dithiothreitol, ATP disodium, ammonium molybdate, ascorbic acid, sodium meta-arsenite, aprotinin, leupeptin, EGTA, EDTA, HEPES, ouabain, phenylmethyanesulfonyl fluoride, and TRIZMA base were purchased from Sigma Chemical Co. (St. Louis, MO). Hanks’ balanced salt solution, Alpha Minimum Essential Medium, DMEM, penicillin/strepto-

Chem. Res. Toxicol., Vol. 14, No. 12, 2001 1597 mycin, fetal bovine serum (FBS), and trypsin-EDTA were obtained from Life Technologies, Inc. (Rockville, MD). Microplates (Fisher 96-well), plastic tubes, and cell culture flasks (75 cm2) were purchased from Corning Inc. (Corning, NY). All other reagents were of the highest grade commercially available. Cell Lines. The NIH-3T3-G185 cell line presenting the gene product of human MDR1 was licensed from NIH and maintained in DMEM as described previously (22). CR1R12 cell line, provided by Dr. Alan Senior (University of Rochester, Rochester, NY), was maintained as described previously (22). Fluorescence-Activated Cell Sorter Flow Cytometry. Fluorescence measurements of individual cells (NIH-3T3-G185) were performed using a Becton-Dickinson FACScalibur fluorescence-activated cell sorter (San Jose, CA), equipped with an ultraviolet argon laser (excitation at 488 nm, emission at 530/ 30 and 570/30 nm band-pass filters). Analysis was gated to include single viable cells on the basis of forward and side lightscatter and was based on acquisition of data from 10 000 cells. Log fluorescence was collected and displayed as single-parameter histograms as previously described (22). Cell Viability Test. Cell viability was assessed using propidium iodide staining. Dead cells in which propidium iodide was bound to double strands of DNA or RNA were detected in certain regions of the cytometry dot plots and excluded from data acquisition. Calculation of Relative Fluorescence. The DNR (daunorubicin) fluorescence intensity of individual cells was recorded as histograms since DNR has been shown to be the most sensitive of all conventional P-gp marker substrates. The mean fluorescence intensity of 10 000 cells was used for comparison among different conditions. Verapamil was selected as a positive control and used to normalize the measurements. Relative fluorescence was used for quantitation and comparison among different compounds. The relative fluorescence (% maximal or reference inhibition) represents a ratio obtained through the following formula: the geometric mean fluorescence of a discrete sample divided by the geometric mean fluorescence in the presence of 100 µM verapamil, times 100 or expressed as

Relative fluorescence ) Fluorescence of sample geometric mean × 100 Fluorescence of reference std. geometric mean Membrane Microsome Preparations. CR1R12 cell membranes enriched with the MDR1 gene product transport enzyme were used for preparation of membrane microsomes. Colchicine (0.5 µg/mL) was added to the growth culture medium to prevent reversion. Cells were washed with complete Hanks’ buffer before being resuspended in 10 mL of lysis buffer (Tris-HCl, 50 mM; mannitol, 50 mM; EGTA, 2 mM; and dithiothreitol, 2 mM; pH 7.0 at 25 °C) containing protease inhibitors (phenylmethanesulfonyl fluoride, 1 mM; aprotinin, 10 µg/mL; leupeptin, 10 µg/ mL). All subsequent steps were performed at 4 °C. The cells were lysed by nitrogen cavitation (Kontes Glass Co., Vineland, NJ) at 500 psi for 15 min twice. Nuclei and mitochondria were sedimented by centrifugation at 4000g for 10 min. The microsomal membrane fraction was then sedimented by centrifugation at 100000g for 60 min. The pellet was resuspended in 0.25 M sucrose buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) and homogenized using a Potter-Elvehjem homogenizer. Aliquots of membrane microsomes were rapidly frozen and stored at -80 °C until analysis. Protein content was determined by a microassay adaptation of the Lowry method. ATP Hydrolysis and Phosphate Release. The consumption of ATP was quantified by determining the amount of liberated inorganic orthophosphate which forms a color complex with molybdate. The ATP hydrolysis assay is based on phosphaterelease determination using membrane microsome preparations; the assay was carried out in a 96-well microplate (23). The microsomes were thawed on ice prior to diluting to 3.5 µg of protein/well in ice-cold ATPase buffer (sodium ATP, 3 mM; KCl, 50 mM; MgSO4, 10 mM; dithiothreitol, 3 mM; Tris-HCl, 50 mM; pH 7.0) containing 0.5 mM EGTA (to inhibit Ca-

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Wang et al. Table 1. Concentration Parameters for Compound Interaction with P-Glycoprotein and CYP3A4

compds

P-gp IC50 (µM)

ATPase activity (Km, µM)

3A4 inhibition (I) or IC50 (µM)

examples of compounds that bind to both P-gp and CYP3A4 Carvedilol 4.6 ( 0.2 93 ( 48 6.8 ( 1.0 Cyclosporin A 1.4 ( 0.1 3 ( 1.7 4 (a,b) Itraconazole 1.7 ( 0.2 0.8 ( 0.2 1.2 Nicardipine 3.2 ( 0.1 24 ( 2 8 Simvastatin 8.9 ( 0.3 18 ( 11 I (a,b) Terfenadine 1.8 ( 0.3 nd 10 Verapamil 4.2 ( 1.0 2.6 ( 0.6 24

Figure 1. Intracellular retention of daunorubicin in NIH3T3G185 cells versus competing valinomycin (a) or cyclophosphamide (b) concentration. Fluorescence intensity is expressed as relative fluorescence. The average number of cells per assay was 10 000. The function for the line through the data is the Hill equation: v ) VmaxSn/(K′+Sn). The parameters IC50 and the Hill coefficient along with the standard deviation are shown in Table 1. Cyclophosphamide has no significant effect on retention of the marker substrate in the MDR1-transfected cell line as shown by the open symbols. ATPase), 0.5 mM ouabain (to inhibit the Na/K-ATPase), and 3 mM sodium azide (to inhibit the mitochondrial ATPase). The total incubation volume including the various inhibitors was 100 µL. The incubation reaction was initiated by transferring the plate from ice to 37 °C, the plate was incubated for 30 min and then the reaction was terminated by the addition of 50 µL of 12% SDS solution at room temperature, followed by the addition of 50 µL of a mixture solution (equal volumes) of 18% fresh ascorbic acid in 1 N HCl and 3% ammonium molybdate in 1 N HCl. After 4 min, 100 µL of a solution of 2% sodium citrate and 2% sodium meta-arsenite in 2% acetic acid was added to fix the color formation. After 30 min of incubation at room temperature, the fixed released phosphate was quantitated colorimetrically using a microplate reader (Bio-Tek FL600, VT) at 750 nm. By comparison to a standard curve, the amount of phosphate releasedsand hence ATP consumedswas quantified. Waterinsoluble drugs were dissolved in methanol or DMSO; the maximum methanol or DMSO concentration (2% v/v) was shown not to affect the ATPase activity. Cytochrome P450-3A4 Activity Assay. The 6β-hydroxylation of testosterone was determined by a modification of the method of Soderfan et al. (24). Pooled human liver microsomes (0.24 mg/mL) were incubated for 10 min at 37 °C with 50 mM potassium phosphate buffer, 3.3 mM MgCl2, 200 µM testosterone, and 1 mM NADPH. Ketoconazole (2 µM) served as the positive inhibitor control. The reaction was terminated with acetonitrile containing 11β-hydroxytestosterone (internal standard). The protein precipitate was removed by centrifugation. The supernatant was injected onto a Waters 2690 Alliance HPLC system equipped with a Waters 996 photodiode array detector (245 nm wavelength) and a Zorbax RX-C8 (4.6 × 150 mm) column maintained at 40 °C. The flow rate was 1 mL/min and the run time was 30 min. The mobile phase consisted of a 20 mM ammonium acetate and methanol gradient system. Initial conditions were 45% ammonium acetate for 15 min followed by ramping to 100% methanol at 30 min. The data (peak area ratio) was collected and analyzed using Waters Millennium software, version 3.20.

Results Using two methods of evaluation, several typical CYP3A4 substrates/inhibitors as well as various P-gp substrates/inhibitors were quantitatively evaluated for their interaction with P-gp. The IC50 (concentration at half-maximum inhibition) could be determined from a simple function, as shown in Figure 1, where the retained

compounds selective for CYP3A4 Acetaminophen ne nd Azithromycin ne nd Ciprofloxacin ne 28 ( 9 Cyclophosphamide ne nd Dextromethorphan ne nd Lidocaine ne 10 ( 2 Nifedipine 113.2 ( 11.4 17 ( 12 P-Nitrophenol ne nd Pravastatin ne nd Diclofenac ne nd

I (a,b) I (a,b) I (a,b) I (a,b) I (a,b) I (a,b) 10 I (a,b) I (a,b) I (a,b)

compounds selective for P-gp Clarithromycin 4.6 ( 0.3 27 ( 9 Clofazimine 0.6 ( 0.2 0.3 ( 0.1 Emetine 9.2 ( 0.3 1.0 ( 0.9 Fluphenazine 6.5 ( 0.4 5.7 ( 1.2 Gallopamil 3.2 ( 0.5 1.5 ( 0.3 N-Norverapamil 0.9 ( 0.1 2.7 ( 0.8 Nobiletin 11.5 ( 0.9 1.7 ( 0.4 Reserpine 0.5 ( 0.1 0.6 ( 0.2 Spironolactone 23.6 ( 1.9 8.9 ( 6.8 Valinomycin 3.2 ( 0.2 0.8 ( 0.3

49 (a,b) 4.1 ( 1.6 >500 (ne) 76 ( 32 67 ( 20 62 ( 21 ne 20.4 ( 5.5 >100 ne

a Determination of concentration parameters is described in the text. Some CYP3A4 inhibitors are noted by citation from previous reports. I ) inhibitor as reported in the literature. ne ) No effect; nd ) no data. b Refs 5, 6, and 59.

fluorescence was measured for samples of viable cells by a flow cytometer at varying concentrations of compound (25). The concentration dependency of inhibition displayed a sigmoidal response curve (Figure 1), a consequence of cooperativity (25), with the Hill equation for allosteric interaction enzymes therefore being the appropriate function for fitting to the data: v ) VmaxSn/ (K′+Sn). The IC50 for Valinomycin on DNR transport in the NIH3T3-G185 cell line (which overexpresses the gene product of human MDR1) was ≈3.2 µM, and Valinomycin achieved about 86% of maximal (reference) inhibition (Figure 1). Several other well-characterized substrates of CYP3A4 (references indicated) were shown to inhibit the P-gp-mediated transport of DNR efflux and are designated in Table 1 with the IC50. However, select CYP3A4 substrates had no effect on the transport of the marker substrate (DNR) over a concentration range of 0-250 µM (Figure 1, one example shown) and are noted in Table 1 (ne, no effect). Conversely, we have identified some significant inhibitors of P-gp that did not inhibit CYP3A4 at physiologically relevant concentrations. These compounds included valinomycin, norverapamil, reserpine, emetine, gallopamil, fluphenazine, and nobiletin and are noted by an IC50 under the P-gp inhibition column in Table 1. Remarkably, valinomycin, emetine, and nobiletin had no detectable effect on CYP3A4 function at concentrations up to 100 µM. ATP Hydrolysis. As ATP is consumed at a purported rate of about one or two per transport event, the rate of

CYP3A4 and P-gp Substrate Binding Sites

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Discussion

Figure 2. P-gp-mediated ATP hydrolysis rates in the presence of itraconazole. The data are fit to a hyperbola and the Vmax ) 2-fold above control with a Km ) 0.8 ( 0.2 µM. Enzyme activity from microsomes of CR1R12 cells overexpressing hamster P-gp.

Figure 3. Inhibition of cytochrome P450 3A4 mediated testosterone hydroxylation versus concentration and IC50 determination. The estimated IC50 values were determined with nonlinear regression analysis: y ) Imax + (V0 - Imax)S/(IC50 + S)) using GraphPad Prism 2.01 software (San Diego, CA). Where y ) activity (% control), Imax ) maximum inhibition, Vo ) control activity, S ) concentration of test compound, and IC50 ) concentration at half-maximal enzyme inhibition.

hydrolysis of ATP represents transport rate or activity assay of function (23, 26-31). In the absence of exogenous substrate, the enzyme is still able to hydrolyze ATP to produce a basal level of activity, which is probably due to the transport of endogenous substrates. Therefore activity data are presented as a percent of the basal or control activity, as any change in the rate of ATP hydrolysis represents the sum of the basal activity and the contribution of the exogenous substrate to ATP hydrolysis. The presence of itraconazole causes a concentration-dependent increase in the rate of ATP hydrolysis relative to the baseline rate, which indicates that it is a comparatively rapid substrate for P-gp (Figure 2). The Km is ∼0.8 µM, and the Vmax is ∼2-fold above baseline, as indicated in Figure 2 (Table 1). Similar quantitative results following evaluation of selected compounds investigated in this study are shown in Table 1. The Km results are in general compatible with the observed IC50s. Cytochrome P450 3A4 Inhibition. The IC50s of selected compounds for the inhibition of CYP3A4-mediated testosterone hydroxylation are displayed in Table 1. Figure 3 illustrates an example of the evaluation and IC50 determination of reserpine.

The number of substrates/inhibitors common to both CYP3A4 and P-gp appears due in large part to the coincidental tolerance or promiscuity of the hydrophobic binding sites of these two enzymes. Substrate recognition and preference, however, are apparently not this simple for compounds have been identified with specificity for one or the other enzyme’s substrate binding site. Moreover, much evidence indicates at least two distinct and unique substrate binding sites for P-gp. The role of the plasma membrane and its ability to concentrate hydrophobic compounds must also be considered when addressing substrate affinity for the CYP3A4 and P-gp enzymes. The substrate binding domains of P-gp are apparently located within the putative transmembrane segments, and the substrate binding site of CYP3A4 may be partly embedded in the membrane. Even lipid detergents such as Triton X-100 have been shown to associate with the substrate binding sites of CYP3A4 [also Triton N-101 and others (32)] and P-gp (33). Indeed, it appears that P-gp recognizes its substrates directly from the lipid phase (33-37), where they are expected to be much more concentrated due to partitioning of the lipophilic compounds. However, the lipophilicity factor logP (a partition coefficient phase preference) often is not correlated with P-gp binding affinitysand certainly not across compound classes or series (38, 39). Structure activity relationships have shown direct correlation of MDR inhibition to log P only for compounds within a closely related series (3). Litman et al. (40) showed that 34 inhibitors from different pharmacological classes have no significant correlation with calculated partition coefficients and that the size of the molecule (van der Waals surface area) was a better corollary. In fact, a P-gp inhibitor has been defined as a compound containing at least two aromatic rings separated by a basic chain with a secondary or tertiary amine (41-43), and even stereospecific effects have been observed for chiral compounds (44). Furthermore, the contribution of hydrogen bonding has been shown for P-gp substrates (45-47). Certain recognition elements formed by 2- or 3-electron donor groups with a fixed spatial separation have been noted for typical substrates along with a high percentage of amino acids with hydrogen bond donor side chains in the transmembrane (substrate interaction) sequence of P-gp (46). CYP 3A4 substrate recognition and binding are not entirely amorphous either; distinct selectivity is observed among the P450 superfamily (5, 6, 48), and a role for unique H-bond or polar interactions (49) have been described for CYP3A4. Various CYP3A4 oxidation reactions have shown both homotropic and heterotropic cooperativity and even allosteric alteration in product patterns (48). Additionally, many of the reactions exhibit regiospecific hydroxylation products indicating a somewhat precise orientation in the binding site (50, 51)sall clear indications of the complicated nature of CYP3A4 substrate recognition. It therefore appears that the extensive overlap of substrates recognized by P-gp and CYP3A4 is a fortuitous accommodation driven largely by tolerant hydrophobic contacts supplemented with H-bond interactions within the unique active sites. We have shown examples of inhibitors not shared by both P-gp and CYP3A4.

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Wang et al.

Figure 4. Molecular two-dimensional structural representations of compounds selective for P-glycoprotein over CYP3A4.

Surprisingly, valinomycin inhibits P-gp function in the NIH-G185 cell line with an potency (IC50 ≈ 3 µM) comparable to cyclosporin A, despite a lack of effect on CYP3A4 function. Because this cell line overexpresses the MDR1 transporter, the IC50 for an inhibitor would be higher in these evaluations than under in vivo conditions, where far fewer copies of the enzyme would be contained per cell. The hydrophobic cyclic decapeptide ionophore valinomycin is transported by P-gp (52) and evidently has a rapid diffusion rate cross the membrane (“flip-flop” rate, 10-4-10-5 s). Although effective rapid diffusion back into the inner leaflet is a component of effective P-gp inhibition (38, 53), valinomycin has been shown to have an affinity for P-gp of 0.78 µM which is similar to that of cyclosporin A (33). Furthermore, emetine and nobiletin are also specific for interaction with P-gp. Despite apparent absence of interaction with CYP3A4, emetine has an ATP hydrolysis activity Km of 1 µM and an IC50 in the highly resistant cell line employed here of 9 µM. Remarkably, norverapamil potently inhibited P-gp function (IC50 ≈ 0.9 µM) yet did not inhibit CYP3A4 at physiologically relevant concentrations (IC50 ∼ 62 µM). Norverapamil is the N-demethylated oxidative metabolite of the calcium channel antagonist verapamil. Gallopamil also favors inhibition of P-gp over CYP3A4, though by a more modest ratio of about 23-fold. Although metabolites of verapamil, gallo-

pamil, and carvedilol have higher IC50 values (lower apparent P-gp affinity) than the respective parent compounds, they still exhibit comparatively high affinity. Notably, some stereoselectivity is observed with verapamil and carvedilol (54). Furthermore, reserpine has at least a 40-fold affinity preference for inhibition of P-gp over CYP3A4. The antihypertensive alkaloid from Rauwolfia serpentina, reserpine, has been known for over a decade to inhibit P-gp (55, 56)sand does so with remarkably high affinity (IC50 ≈ 0.5 µM)syet should not inhibit CYP3A4 at clinical exposures (IC50 ≈ 20 µM). Reserpine has high affinity for P-gp with a Kd of 0.73 µM (33). Finally, fluphenazine has a ∼12-fold affinity preference for inhibiting P-gp over CYP3A4 and should not affect CYP3A4 function in vivo at even toxic concentrations. The structures of these P-gp selective inhibitors share some interesting qualities (Figure 4). Valinomycin, clarithromycin cyclosporin A, gramacidin, aureobasidin, and PSC833 are all hydrophobic cyclic peptides (gramacidin is a linear pentadecapeptide) that can occupy multivariate 3-D conformations. These compounds share a high affinity for P-gp (33) and are exemplified by cyclosporin A and aureobasidin A exhibiting such structural analogies as hydrophobic residues, a large antiparallel B sheet domain, and an arrowhead-like overall conformation (57, 58). Of particular interest are the similarities among reserpine, verapamil, and yohimbine.

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Figure 5. Molecular 2-dimesional structural representations of compounds selective for CYP3A4 over P-glycoprotein.

Despite the difference in stereochemistry between reserpine and yohimbine, P-gp interaction studies showed that the compounds with the benzoyl substituent next to the basic indolo-piperidine ring system are the most potent inhibitors of P-gp mediated vinblastine transport (43). Molecular dynamics analyses show that verapamil can achieve a thermodynamically probable conformation similar to that of reserpine (43). This preferred conformation of verapamil is also similar to that of vinblastine, the vinca alkaloid substrate/inhibitor of P-gp. Emetine, fluphenazine, and to some extent even nobiletin appear to have significant structural features in common with reserpine in 2-dimensional overlay (Figure 4). Remarkably, we have shown several examples of compounds that uniquely inhibit P-gp function with affinities comparable to benchmark P-gp inhibitors, despite a lack of effect on CYP3A4 function at even toxic concentrations. This preference is unusual though there are many examples of potent CYP3A4 inhibitors without effect on P-gp function (i.e., Figure 5). These unique compounds could prove useful as tools to probe for the specific discernment of P-gp-mediated drug/drug or drug/ herb interactions. Learning more about the binding interactions of these two enzymes and the differences between them may help identify how they recognize such a wide variety of molecules as substrates (or inhibitors). Moreover, identification of specific inhibitors will allow the determination of which enzyme is responsible for drug/drug interactions and/or the extent of contribution in a clinical situation.

Acknowledgment. The authors are very grateful Prof. Adriane L. Stewart for editorial assistance and Dr. Peter Wislocki for comments on the manuscript.

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