QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.106.012609v1 35/4/614    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Locuson, C. W. Articles by Tracy, T. S. Search for Related Content PubMed PubMed Citation Articles by Locuson, C. W. Articles by Tracy, T. S.

Visible Spectra of Type II Cytochrome P450-Drug Complexes: Evidence that "Incomplete" Heme Coordination Is Common

Pfizer Animal Health, Veterinary Medical Research and Development Metabolism and Safety, Kalamazoo, Michigan (C.W.L.); Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Pfizer, Inc., St. Louis, Missouri (J.M.H.); and University of Minnesota, College of Pharmacy, Department of Experimental and Clinical Pharmacology, Minneapolis, Minnesota, (T.S.T.)

(Received August 31, 2006; accepted January 23, 2007)

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
The visible spectrum of a ligand-bound cytochrome P450 is often used to determine the nature of the interaction between the ligand and the P450. One particularly characteristic form of spectra arises from the coordination of nitrogen-containing ligands to the P450 heme iron. These type II ligands tend to be inhibitors because they stabilize the low reduction potential P450 and prevent oxygen binding to the heme. Yet, several type II ligands containing aniline, imidazole, and triazole moieties are also known to be substrates of P450, although P450 binding spectra are not often scrutinized to make this distinction. Therefore, the three nitrogenous ligands aniline, imidazole, and triazole were used as binding spectra standards with purified human CYP3A4 and CYP2C9, because their small size should not present any steric limitations in their accessing the heme prosthetic group. Next, the spectra of P450 with drugs containing the three nitrogenous groups were collected for comparison. The absolute spectra demonstrated that the red-shift of the low-spin Soret band is mostly dependent on the electronic properties of the nitrogen ligand since they tended to match their respective standards, aniline, imidazole, and triazole. On the other hand, difference spectra seemed to be more sensitive to the steric properties of the ligand because they facilitated comparison of the spectral amplitudes achieved with the drugs versus those with the standard nitrogen ligands. Therefore, difference spectra may help reveal "weak" coordination to the heme that results from suboptimal orientation or ligand binding to more remote locations within the P450 active sites.

Generally, nitrogen-containing heme ligands, which bind more avidly than oxygen-containing ligands, are more often inhibitors than substrates (Gigon et al., 1968). Hence, many substrates (i.e., type I ligands) actually displace the heme water ligand, shifting the iron spin equilibrium toward the high-spin form, whereas nitrogen heterocycles and anilines (i.e., type II ligands) can replace the water ligand to stabilize the low-spin form. However, some type II ligands are also substrates (Kunze et al., 2006; Pearson et al., 2006), whereas others derive little, if any, affinity for P450 from this interaction (Ha-Duong et al., 2001), even though it is commonly described as being tight or high affinity in nature.

Thus, the question remains, if it is assumed that the above examples are not just exceptions to the rule, can the substrate-bound P450 spectrum tell us more about the type II binding interaction? Recently, we were intrigued by the use of absolute P450 spectra by Atkins and coworkers (Roberts et al., 2005) in which CYP3A4 was saturated with a type II ligand (aniline) to shift the majority of the CYP3A4 sample to the nitrogen-liganded low-spin form. Based on this work, the question was raised whether absolute spectra or difference spectra reveal more insight into the different degrees of type II binding. The amplitude of any difference spectrum induced by a type II substrate should depend partly on the natural abundance of high-spin P450 present for the isoform in question (Kumaki et al., 1978). However, within the same isoform, different type II substrates can give different A_max values, but it is not always clear whether this is due to a population of substrate binding in type I fashion, steric restraints that limit optimal nitrogen-iron interaction, electronic effects, or a combination of the aforementioned. It should be noted that A_max values also are not indicative of binding affinity as shown with some anilines (Hodek et al., 2004). Therefore, both absolute and difference spectra of two major drug-metabolizing enzymes, CYP3A4 and CYP2C9, were recorded with drugs that elicit type II difference spectra and compared with the type II ligands aniline, imidazole, and triazole, which are functionalities commonly found in approved drugs. It is postulated that _max in absolute spectra of type II binders is primarily determined by the electronic structure of the coordinating group. Alternatively, the ability of difference spectra to accentuate changes in the high-spin P450 makes them more apt to reveal multiple substrate binding modes and/or binding modes that are more peripheral to the P450 heme prosthetic group.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. Potassium phosphate, glycerol, acetonitrile, HCl, and imidazole were purchased from Fisher Scientific (Fairlawn, NJ). Sulfaphenazole, aniline, 1,2,4-triazole, DMSO, itraconazole (mixed isomers), lyophilized isocitrate dehydrogenase (containing sodium citrate and manganous sulfate), and D-(+)-threo-isocitric acid monopotassium salt were purchased from Sigma (St. Louis, MO). [1-methyl]-1,2,4-Triazole was purchased from Alfa Aesar (Ward Hill, MA). PH-302 [2-((2-(1H-imidazol-1-yl)-6-methylpyrimidin-4-yl)(3-((benzo-[d][1,3]dioxol-5-ylmethyl)(methyl)amino)propyl)amino)acetamide] was provided by Pfizer, Inc. (St. Louis, MO). Chemically competent E. coli F^'IQ cells were obtained from Invitrogen (Carlsbad, CA). Corning Costar 3904 96-well plates were purchased from Fisher Scientific (Hampton, NH).

Enzymes. Full-length human CYP2C9 containing the Barnes (1996) modified N-terminus was expressed in E. coli F^'IQ from the pCW plasmid provided by Dr. A. E. Rettie from the University of Washington. Exact methods for expression and purification were described previously (Locuson et al., 2006, and references therein). Full-length expressed and purified human CYP3A4 and human cytochrome P450 reductase were purchased from Invitrogen. Both P450s contain a C-terminal histidine tag used for purification via nickel-chelate chromatography.

Visible Spectroscopy. All spectra were carried out in split-beam mode with an Olis upgraded Aminco DW-2000 spectrophotometer (Olis, Inc., Bogart, GA) with the settings as described previously (Locuson et al., 2006). The wavelength range typically encompassed 340 to 500 nm and readings averaged over 10 scans were taken in 1-nm steps. Some absolute spectra were also recorded through 600 nm to obtain the (569-nm) and ß (535-nm) bands.

Substrate Preparation. PH-302 and sulfaphenazole were dissolved in DMSO and diluted in water to obtain the desired stock solutions. Aniline was used directly, or first dissolved in DMSO, and further diluted in water to achieve lower concentrations for stock solutions. Imidazole was prepared as a 2 M solution and the pH was adjusted to 6.0, 7.4, or 8.5 using 5 M HCl before being diluted further into buffer (0.1 M potassium phosphate, pH 7.4). Itraconazole (3.33 mM) was prepared in DMSO because it was found to be more soluble in this solvent than acetonitrile. Further dilution was carried out in DMSO, or in water, if the final concentration was <100 µM.

Substrate Titrations. All experiments were carried out at 30°C in matched micro quartz cuvettes (2 mm x 10 mm internal) containing 0.5 µM P450 and 20% glycerol (v/v) in 0.1 M potassium phosphate buffer, pH 7.4, unless otherwise noted. Substrates (Fig. 1) were then titrated into the cuvettes containing P450. To obtain difference spectra, a baseline was recorded with P450 in both the sample and reference cuvettes followed by substrate titration into the sample cuvette only and buffer titration of the same volume into the reference cuvette. In obtaining absolute spectra, a baseline was recorded with only the buffer and glycerol in the sample and reference cuvettes. Next, P450 was added to the sample cuvette followed by titration of the substrate into both cuvettes so that the majority of any interfering signal of substrate was subtracted except when high concentrations of ligand were needed to saturate the enzyme (e.g., aniline). All absolute spectra were normalized to the absorbance at 500 nm. Except for one case (itraconazole equilibration was not reached for 3 min), equilibration of samples after addition of substrate was reached immediately after mixing since no change in spectra could be detected upon repeated scans. Ranges of ligand concentrations used are presented in Table 1.

View larger version (24K): [in this window] [in a new window]   FIG. 1. Structures of type II P450 ligands. Arrows denote the primary site of metabolism by CYP3A4 and asterisks denote chiral centers.

Data Fitting. Ligand binding curves were fit by nonlinear regression to one of four equations with SigmaPlot 8.0 (SSI, Inc., Richmond, CA). Equation 1 was used to determine K_S from difference spectra where only one binding event was evident and the K_S was higher than P450 concentration

(1)

where Y is the spectroscopic signal, E is enzyme, and L is ligand. Equation 2 was used to determine K_S when the plot of substrate concentration versus change in absorbance appeared hyperbolic, but for which the Eadie-Hofstee transform revealed curvature

(2)

Equation 3 was used to determine K_S for the high-affinity binding mode when multiple binding events were evident, but the high K_S binding mode was not saturable

(3)

where m is the slope of the linear portion of the curve at high ligand concentrations. Equation 4 was used to determine K_S when its value was lower than the concentration of P450 in the experiment

(4)

where E is P450 concentration. Equation 5 was used to determine K_S when binding led to a sigmoidal response

(5)

where Y₀ is the signal at zero ligand concentration. The following term was multiplied by eqs. 1 to 3 when K_S was determined from absolute spectra because the signal at zero ligand concentration was not zero

(6)

where Y₀ and Y are the signals at zero ligand and saturating ligand, respectively.

P450 Reconstitution. Purified CYP3A4 or CYP2C9 was incubated with two molar equivalents of human P450 reductase on ice for 5 min, followed by addition of extruded 1,2-dilauroyl-sn-glycero-3-phosphocholine to 1 µg/pmol P450. After 5 min, the reconstituted enzyme mixture was used in reaction mixtures containing substrate, an NADPH-regenerating system, and NADPH as described immediately below.

NADPH Consumption. The consumption of NADPH was monitored by a coupled enzyme method. Cytochrome P450 reductase uses NADPH to reduce P450 at two points in the P450 catalytic cycle. The conversion of NADP back to NADPH by isocitrate dehydrogenase then generates -ketoglutarate, which is reacted with 2,4-dinitrophenylhydrazine to form a hydrazone chromophore (Friedemann, 1957). More recently, a similar method was used by Waskell and associates (Gruenke et al., 1995), but in the present case, the assay was adapted for use in 96-well plates. Reactions were carried out in 96-well plates in 50 mM potassium phosphate buffer, pH 7.4, using 20 pmol of reconstituted P450, the substrate concentrations shown in Table 1, 1 U of isocitrate dehydrogenase, and 10 mM D-(+)-threo-isocitrate in a total volume of 0.090 ml. Plates containing all the reaction components were preincubated in a water bath at 37°C for 3 min, and then 0.010 ml of NADPH (0.1 mM) was added to start the reactions. Trichloroacetic acid (70%, 10 µl) was used to quench the incubations after 30 min. Next, 0.05 ml of 1 mM 2,4-dinitrophenylhydrazine/1 M HCl solution was added and the plates were incubated at room temperature. Formation of the hydrazone of -ketoglutarate was monitored spectroscopically at 390 nm to determine that 1 h was sufficient to ensure completion of the reaction. Next, 0.075 ml of 20% NaOH was added and the samples were monitored immediately at 450 nm along with standard curve samples in a Molecular Devices (Sunnyvale, CA) Thermomax microplate reader. Standard curves (duplicate samples) were prepared using -ketoglutarate at 5 to 160 µM and processed at the same time as enzyme reaction samples.

Gaussian Curve Fitting of P450 Spectra. Analysis was carried out using OriginPro version 7.5 (OriginLab Corp., Northampton, MA). All spectra were fit to three components. Absolute spectra were fit to three Gaussian curves at 360, 390, and 417 nm representing the delta, high-spin, and low-spin peaks, respectively. Difference spectra were fit to three Gaussian curves at 390, 410, and 430 nm. The shoulder of the trough at 390 nm represents the decrease in inherent high-spin P450, whereas the trough at 410 to 412 nm and peak at 425 to 432 nm result from the red-shift of the low-spin peak due to coordination of a ligand to the heme iron. The line through the data points in each plot represents the fit that is achieved using all three components, even though only the high-spin component is shown in difference spectra for clarity.

	Results

Top Abstract Materials and Methods Results Discussion References

 
CYP3A4. The absolute spectrum of CYP3A4 displayed a low-spin Soret band with a _max of 417 nm (Fig. 2A). A slight shoulder on the blue side of this peak (385–390 nm) was presumed to be the small percentage that exists in the high-spin state (Jefcoate, 1978) (Fig. 2A). High-spin CYP3A4 became more evident when the type II ligand imidazole was added to saturating concentrations (>20 mM) because the decrease at 385 nm was greater than would be expected if the spectrum was only red-shifted by imidazole (Fig. 2A). Saturating aniline (>20 mM) and 1,2,4-triazole (>40 mM) shifted the band 5 nm to 422 (Fig. 2, B and C), whereas imidazole ligation to the heme iron red-shifted the low-spin Soret band 7 nm to 424 nm. In addition, aniline decreased absorbance of _max more than did imidazole (Table 1). No correction for dilution was made in this case since the total volume of the sample was changed less than 0.5%.

View larger version (10K): [in this window] [in a new window]   FIG. 2. Absolute spectra of 0.5 µM CYP3A4 in 0.1 M potassium phosphate buffer, pH 7.4, containing 20% glycerol (v/v) and type II ligands at 30°C. A, free () and with 25 mM imidazole (). B, 25 mM aniline () and 25 mM aniline after addition of 1% DMSO (—). C, 40 mM 1,2,4-triazole ().

Difference spectra were also obtained with CYP3A4 in the presence of the type II ligands aniline, imidazole, and 1,2,4-triazole (Fig. 3). These compounds acted as standards because there should be no steric restrictions in their ability to access the P450 heme. As noted with the absolute spectra, it was evident that imidazole coordination to the heme group differed from aniline and triazole since the A_max between the peak and trough was 24% lower for aniline and 59% lower for triazole. In addition, the peak induced by aniline (429 nm) was located at a lower wavelength than that observed with imidazole (434 nm) as expected from the absolute spectra. All type II CYP3A4 spectra contained a significant shoulder in the trough that was not as intense as the lowest point at 410 nm, indicating that some high-spin P450 exists in the absence of substrate (i.e., mixture of type IIa and type IIb spectrum). If very little high-spin P450 was present before addition of type II ligand, only a trough centered around 410 nm would be observed (type IIb). Conversely, if very little low-spin P450 was present, a type II ligand would create a trough centered closer to 390 nm, representing the decrease in the high-spin Soret (type IIa).

View larger version (18K): [in this window] [in a new window]   FIG. 3. Difference spectra of 0.5 µM CYP3A4 in 0.1 M potassium phosphate buffer, pH 7.4, containing 20% glycerol (v/v) and type II ligands at 30°C. Saturating concentrations of type II ligands (see Table 1) include imidazole (—), aniline (——), PH-302 (), itraconazole (), and fluconazole ().

CYP3A4 Substrates. The compounds PH-302 and itraconazole were tested with CYP3A4 because they are not only type II binders, but also substrates that are metabolized in regions other than their nitrogen-containing rings. PH-302 is oxidized on the benzodioxole group to give a catechol (Hutzler et al., 2006), and itraconazole is metabolized at the alkyl side chain of the triazolone ring (Kunze et al., 2006). In addition, itraconazole is known to be a more potent inhibitor of CYP3A4 than of CYP2C9, whereas fluconazole was tested because it is a weaker inhibitor of CYP3A4 than CYP2C9 (Niwa et al., 2005). Hence, these differences in relative inhibition potency of the compounds toward individual P450s allowed assessment of isoform differences as well. Differences in electronic features could also be evaluated since PH-302 heme ligation involves binding of an imidazole group, whereas the antifungals itraconazole and fluconazole bind via their triazole groups.

PH-302 red-shifted the low-spin Soret band in the absolute spectra as effectively as imidazole, but the absorbance at _max was decreased 7.6% (Fig. 4). In the difference spectra, the peak (431 nm) and trough (410 nm) induced by PH-302 were similar to the wavelengths given by imidazole, but A_max was only 72% of that achieved with imidazole. Low-spin Soret band intensity was not affected as much with itraconazole and fluconazole compared with PH-302, but the red-shift of the Soret band of itraconazole and fluconazole was notably weaker in both forms of spectra, as were the A_max values (Table 1). Titration of 1,2,4-triazole indicated that the degree of red-shift of the low-spin Soret is the same as that found for the triazole-containing drugs (Fig. 4B), as were the A_max values from difference spectra.

View larger version (12K): [in this window] [in a new window]   FIG. 4. Absolute spectra of CYP3A4 with type II coordinating drugs. A, titration of PH-302 from 1 to 50 µM. B, saturating concentrations of fluconazole (, 100 µM) and itraconazole (, 3 µM) compared with the free enzyme (). Conditions were identical to those in Fig. 1.

View larger version (13K): [in this window] [in a new window]   FIG. 5. Itraconazole titration (0.1–3 µM) with CYP3A4 used to calculate the spectral dissociation constant, KS. Both the difference spectra () peak minus trough absorbance values and the difference between the low- and high-spin Soret bands in absolute spectra () were fit to eq. 5.

The substrate itraconazole produced a sigmoidal binding response (Fig. 5). Although this type of binding behavior has not been reported previously, differences in metabolism of specific itraconazole isomers in other studies (Kunze et al., 2006) suggest that our results may have arisen from using a mixture of isomers.

CYP2C9. Absolute spectra of CYP2C9 with and without the type II ligands aniline, imidazole, and triazole followed the same rank order with respect to the _max of the low-spin Soret (imidazole > aniline triazole > no ligand) as observed with CYP3A4. However, slightly different values were obtained for the peak and trough in difference spectra compared with CYP3A4 (Table 1). In fact, the peaks for several type II ligands in CYP2C9 difference spectra were low in intensity and broad (Fig. 6). Also, the shoulder of the trough was less evident in difference spectra, suggesting that CYP2C9 in the absence of anything except buffer and glycerol exists with a smaller population of high-spin heme iron than does CYP3A4.

View larger version (11K): [in this window] [in a new window]   FIG. 6. Visible spectra of CYP2C9 with and without a saturating concentration of type II ligand (see Table 1). A, imidazole difference spectrum. B, fluconazole absolute spectra (). The inset shows the difference spectra at the concentration that is equal to KS1 (–) and the highest concentration tested (––). C, sulfaphenazole absolute spectra (). The inset shows the difference spectrum under saturating concentrations.

CYP2C9 Substrates. Similar to triazole and aniline, most of the drug substrates produced only minor red-shifts of the low-spin Soret band (Table 1; Fig. 6). In addition, the percentage change of the Soret band was greater than 5-fold more negative for CYP2C9 than for CYP3A4 on average. As a consequence, difference spectra typically possessed less intense peaks that were positioned at lower wavelengths. In several cases, this led to the difference spectra peaks being either very broad or not distinguishable from the baseline, as was the case with fluconazole (Fig. 6B). Unlike CYP3A4, fluconazole produced a much lower A value compared with triazole with CYP2C9.

Sulfaphenazole was studied because it is a potent inhibitor of CYP2C9 that was initially thought to derive its affinity through type II binding via its aniline ring (Mancy et al., 1996). In concurrence with the results of others (Mancy et al., 1996), titration of sulfaphenazole in the present study resulted in type II difference spectra, although A_max was only 4.8% of that obtained with imidazole (Fig. 6C). In addition, the _max value (426 nm) obtained for the peak was similar to that reported by others (425 nm) (Ha-Duong et al., 2001), but at least 8 nm lower than that observed with imidazole. Absolute CYP2C9 spectra with saturating concentrations of sulfaphenazole resulted in a decrease in the absorbance of the low-spin Soret band and a very small red-shift of 1 nm.

Overall, CYP2C9 displayed more complex binding behavior than CYP3A4. Multiple binding events were evident in difference spectra with fluconazole and PH-302, which produced small A_max values for the highest-affinity site (Fig. 7). The binding response for both drugs was best described as biphasic, containing a high-affinity/low-K_S component and an unsaturable low-affinity/high-K_S component. A biphasic response was also evident in absolute spectra for these compounds when the absorbance of the high-spin Soret band (385–390 nm) was subtracted from that of the low-spin Soret band (420–424 nm). The absorbance at 424 nm was used as the nitrogen-liganded low-spin Soret band because it represents the maximum red-shift observed (with imidazole), but use of the isosbestic point at 420 nm generally gave similar results.

View larger version (10K): [in this window] [in a new window]   FIG. 7. Type II ligand titrations with CYP2C9 used to calculate the spectral dissociation constant, KS.A, A obtained from difference spectra with fluconazole as the ligand. Eadie-Hofstee transform is shown in the inset. B, absolute spectra A obtained with PH-302. C, fit of sulfaphenazole binding data using absolute () and difference spectra () A values.

K_S Values. Equilibrium binding constants of the type II ligands were determined for both P450 isoforms using difference (A_peak-trough) and absolute spectra (A_424–390). The best model (i.e., r²) for describing the nonlinear fit and shape of the binding curve for a ligand using difference spectra was usually the same model giving the best fit of the corresponding absolute spectra. In the case of triazole, whose spectrum appeared hyperbolic, re-plots of A/[L] versus A (i.e., Eadie-Hofstee-like plot) were used to determine when a multiple-binding site model was required in nonlinear regression (e.g., eq. 2). The K_S values obtained from the two forms of spectra differed significantly for the CYP2C9 substrates. Difference and absolute spectra K_S values for fluconazole and sulfaphenazole binding to CYP2C9 differed by approximately 8.5- and 10-fold, respectively (Table 1; Fig. 7C). Also, K_S values for imidazole, aniline, and triazole were generally higher than those for the drug ligands indicating a role for hydrophobic interactions in binding for drug-like type II ligands.

In an attempt to discern factors that may be responsible for the multiple binding phases of the type II ligands, two possibilities were considered: 1) multiple types of populated heme coordination modes, and 2) simultaneous occupancy of a P450 active site by multiple ligands that may have a cooperative effect on binding or the binding signal. Since triazole has three nitrogens that may produce different spectra, 1-methyl-1,2,4-triazole was used to mimic the 1-N-substituted drugs fluconazole and itraconazole. The apparent affinity was not altered, nor was the detection of multiple binding events attenuated with the methyl analog as compared with triazole with CYP2C9 (C. L. Locuson, J. M. Hutzler, and T. S. Tracy, unpublished data). This observation suggests that the multiple binding phases result from a single nitrogen or that the methyl group does not sufficiently hinder the nitrogens at positions 1 and 2. Next, the ionization state of the ligand was considered. Although the protonation of a second nitrogen in triazole should not occur near physiological pH (pK_a = 2.45) (Satchell and Smith, 2002), pH produced noticeable changes in the spectra of imidazole-CYP2C9 complexes (Fig. 8). Under conditions where the majority of the imidazole should contain only one protonated nitrogen, a single binding event was observed (K_S = 279 ± 10). Conversely, under more acidic conditions, multiple binding events became more evident, although the affinity of the higher-affinity binding event changed only modestly (K_S = 274 ± 10).

View larger version (12K): [in this window] [in a new window]   FIG. 8. Effect of pH on the detection of multiple binding modes of imidazole with CYP2C9 as shown by Eadie-Hofstee plots. Imidazole concentrations ranged from 1 to 500 µM.

View larger version (11K): [in this window] [in a new window]   FIG. 9. Evaluation of Gaussian curve fitting of spectra to determine relative changes in high-spin P450 upon addition of type II ligands. A, CYP3A4 absolute spectrum displays a small high-spin component in the absence of ligand near 390 nm. B, difference spectrum of CYP2C9 with a saturating concentration of imidazole demonstrates a mixed type II response with a moderate amount of a type IIa shoulder near 390 nm. C, difference spectrum of CYP3A4 with a saturating concentration of imidazole demonstrates a mixed type II response with a higher amount of type IIa than CYP2C9 according to the shoulder near 390 nm. See Materials and Methods for a description of the fitting procedure.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Type II complexes result from P450 heme coordination of low-pK_a nitrogen-bearing ligands. This binding directly to the heme of P450s can inhibit metabolism of a coadministered drug and result in a drug-drug interaction. Furthermore, drugs designed to target P450s have been made more potent by adding imidazole groups to them to enable type II coordination (Hackett et al., 2005). Interestingly, some type II ligands may also be substrates and some aniline- or imidazole-containing drugs do not always contribute to binding energy even though they give type II spectra, suggesting the possibility of different degrees of type II binding. The present studies compared the difference and absolute spectra of purified P450s with type II ligands to determine the spectral characteristics of varying degrees of type II binding.

Type II difference spectra of nitrogen-containing drugs were characterized with CYP3A4 and CYP2C9 enzymes. All compounds tested produced smaller A_max values in difference spectra than imidazole alone (Table 1). Several compounds also decreased the amplitude of the low-spin Soret band. Interestingly, PH-302 and itraconazole are both metabolized by CYP3A4 at positions removed from the imidazole group, requiring binding of these compounds in multiple modes (Fig. 1). Conversely, fluconazole is oxidatively metabolized, to a small extent (Brammer et al., 1991), on the heme-coordinating triazole to give an N-oxide. If carried out by P450, fluconazole oxidation also argues for the existence of multiple binding modes, except that the motion of the substrate relative to the heme would be translational rather than rotational. These findings suggest that clues about multiple binding modes, steric factors (Chiba et al., 2001), and/or metabolism might be detected by comparing binding spectra to a standard spectrum, such as that of imidazole or triazole.

Although some of the spectral differences appear minor (Table 1), the case of CYP2C9 and sulfaphenazole provides evidence that difference spectra may indeed indicate which type II ligands have higher drug-drug interaction potential. Sulfaphenazole produces a weak type II signal with CYP2C9, but binds with equal affinity when the aniline group is replaced with a tolyl group (Rao et al., 2000). Given this information, compounds like sulfaphenazole can reveal the spectral characteristics of a weak type II ligand. In general, the A_max is weaker than changes induced by saturating amounts of the standard, in this case, aniline. Further studies are needed to confirm this hypothesis.

In several cases where very different binding modes must exist to achieve metabolism, the nitrogen-bearing drug produced a red-shift in absolute spectra similar to that of the respective imidazole or triazole. The red-shift induced by imidazole was most similar to that observed with the imidazole-containing PH-302, and likewise for triazole compared with fluconazole and itraconazole. Since the small imidazole and triazole standards should not be hindered in accessing the heme group, these results suggest that the P450 absolute spectrum may be useful in identifying the electronic structure of the ligand. This is in agreement with previous work (Sono and Dawson, 1982) characterizing the Soret red-shifting ability of P450 ligands containing different coordinating atoms following the order: sulfur > nitrogen > oxygen.

Alternatively, differences in steric properties or in binding that is, on average, distant from the heme, may be more detectable with difference spectra. In all but one case (CYP3A4 with itraconazole), the A amplitude for the type II drug ligands did not match that achieved with imidazole, aniline, or triazole, although this was sometimes complicated by multiple binding phases in CYP2C9. These A_max differences between ligand standard and drug may occur because distal heme ligands exhibit strong preferences for orientation (Roberts et al., 2001; Scott et al., 2004). The observed differences in A_max are much greater than the electronic effects reported earlier (Kulkarni et al., 1974; Mailman et al., 1974) supporting a possible relationship between low A_max values and suboptimal coordination orientation or hindered binding. Thus, the trough amplitude in difference spectra may be more important than peak intensity, which should be somewhat dependent on the degree of low-spin Soret red-shift (Fig. 6B).

Field strength of the ligand and the planarity of the heme (Roberts et al., 2001) may also affect the _max and A_max values in absolute and difference spectra, respectively, of type II P450 complexes. However, the most basic and probably the most nucleophilic compound, imidazole, produced the most significant changes in binding spectra, agreeing with earlier work by Dawson (Sono and Dawson, 1982). Since hydrophobic forces play a large role in P450 ligand binding (Regev-Shoshani et al., 2004), the hydrophilic nature of the nitrogen ligand standards may also contribute to the lack of correlation between the affinity of nitrogen ligands and spectral characteristics. Donation of charge from the proximal cysteine axial ligand to the iron may also disfavor coordination at the distal axial position regardless of resulting spectral properties (Sono and Dawson, 1982). Thus, the nucleophilicity that allows imidazole to stabilize low-spin P450 more effectively than triazole may also explain its lower affinity.

Reduction of P450, indirectly monitored as NADPH consumption, was also used to probe the ability of a type II ligand to stabilize the low-spin state. Again, imidazole was alone in its ability to stabilize low-spin P450 (Table 2; Fig. 9). Total NADPH consumption also includes any uncoupled reactions carried out by P450 or P450 reductase in which a metabolite is not produced from the type II ligand. However, since the rate with imidazole is actually lower than that obtained in the absence of any substrate, the net effect observed with imidazole is interpreted here as fewer P450 reduction events. It should be noted that type II complexes are still reducible, so that the measured rates will be dependent on multiple factors.

Human P450 isoform differences were also observed, as CYP2C9 displayed more complex binding behavior than CYP3A4. Except in the case of triazole, this was not a consequence of the type of spectra used for the nonlinear regression analysis of the binding constant, because both difference and absolute spectra usually fit best to the same binding model. By altering the pH of the titration experiment buffer and the ligand solution, the binding of imidazole, but not triazole, could be "normalized" to a single binding event. The multiple nitrogens in triazole may give rise to a population of different heme complexes. Alternatively, the multiple binding phases may result from a population of enzyme with triazole directly coordinated to the heme iron and another population bound to the heme water ligand. Recently, fluconazole was found to occupy two distinct binding modes in CYP121 crystals and in electron paramagnetic resonance experiments even though, spectrally, hyperbolic binding was observed (Seward et al., 2006).

Another intriguing observation was that the CYP2C9 low-spin Soret band in type II ligand complexes exhibited decreased intensities compared with CYP3A4. This finding suggests that the deconvolution of absolute spectra obtained with type II ligands into low- and high-spin components may not be straightforward with some P450 isoforms. For instance, even though the fraction of low-spin enzyme increases with type II ligands, the low-spin complexed enzyme apparently has a lower extinction coefficient, as shown for CYP2C9 with several ligands (Fig. 6B; Table 1). Moreover, the decrease in high-spin enzyme, which is already low before addition of ligand, makes accurate deconvolution of the high-spin component difficult (Fig. 9A). Both of these observations argue that absolute spectra are less than optimal for making quantitative comparisons. The peaks of difference spectra are also suspect in their representation of low-spin enzyme because ligand-specific extinction coefficients and _max values sometimes hinder peak detection altogether (Fig. 6B, inset). Conversely, the trough region of a difference spectrum does not appear to suffer as much from the abovementioned problems. In particular, it is hypothesized that A_500–390 would be useful when comparing a type II difference spectrum obtained with drug to that of a standard chemical with the same heme-coordinating group (Fig. 9, B and C). The intensity of the 390-nm region reflects the ability of a ligand to decrease the fraction of inherent high-spin enzyme present and, therefore, is not expected to produce ligand-specific effects on trough intensity or _max. Deconvolution of the negative high-spin component of CYP3A4 and CYP2C9 produced with imidazole was accomplished readily, but the use of A_500–390 would eliminate the error resulting from having two baselines due to substrate interference at lower wavelengths (Fig. 9). However, the degree of type IIa spectrum (i.e., inherent high spin) exhibited by a P450 ultimately determines the analytical sensitivity in this approach unless a type I ligand is first added to the sample.

In conclusion, type II ligand-P450 complexes display varying spectral fingerprints based on their physicochemical properties and the P450 isoform under study. Several drugs have been described that undergo P450 oxidation at regions distinct from moieties containing type II-coordinating nitrogens, and other nitrogen-containing heterocycles may be preferentially metabolized by P450s (Dowers et al., 2004). Hence, P450 heme coordination by a drug lead does not necessarily imply drug-drug interaction potential. Furthermore, recent studies demonstrating substrate-dependent inhibition by the type II ligands ketoconazole and miconazole (Kumar et al., 2006) suggest that inhibition studies involving type II ligands may not always be sufficient for assessing inhibition potential. Thus, further exploration of type II binding characteristics may result in better decisions during the drug development process.

	Acknowledgments

 
We thank Dr. Peter M. Gannett for thoughtful discussions.

	Footnotes

 
This work was supported in part by a grant from the National Institutes of Health to T.S.T. (GM063215).

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.012609.

ABBREVIATIONS: P450, cytochrome P450; DMSO, dimethyl sulfoxide; PH-302, 2-((2-(1H-imidazol-1-yl)-6-methylpyrimidin-4-yl)(3-((benzo[d]-[1,3]dioxol-5-ylmethyl)(methyl)amino)propyl)amino)acetamide.

Address correspondence to: Timothy S. Tracy, Department of Experimental and Clinical Pharmacology, University of Minnesota, College of Pharmacy, 7-115B Weaver-Densford Hall, 308 Harvard Street SE, Minneapolis, MN 55455. E-mail: tracy017{at}umn.edu

	References

Top Abstract Materials and Methods Results Discussion References

 


Backes WL and Canady WJ (1981) The interaction of hepatic cytochrome P-450 with organic solvents. The effect of organic solvents on apparent spectral binding constants for hydrocarbon substrates. J Biol Chem 256: 7213–7227.[Abstract/Free Full Text]

Barnes HJ (1996) Maximizing expression of eukaryotic cytochrome P450s in Escherichia coli. Methods Enzymol 272: 3–14.[CrossRef][Medline]

Brammer KW, Coakley AJ, Jezequel SG, and Tarbit MH (1991) The disposition and metabolism of [14C]fluconazole in humans. Drug Metab Dispos 19: 764–767.[Abstract]

Chiba M, Tang C, Neway WE, Williams TM, Desolms SJ, Dinsmore CJ, Wai JS, and Lin JH (2001) P450 interaction with farnesyl-protein transferase inhibitors metabolic stability, inhibitory potency, and P450 binding spectra in human liver microsomes. Biochem Pharmacol 62: 773–776.[CrossRef][Medline]

Dowers TS, Rock DA, Rock DA, Perkins BN, and Jones JP (2004) An analysis of the regioselectivity of aromatic hydroxylation and N-oxygenation by cytochrome P450 enzymes. Drug Metab Dispos 32: 328–332.[Abstract/Free Full Text]

Friedemann T (1957) Determination of alpha-keto acids. Methods Enzymol 3: 414–418.

Gigon PL, Gram TE, and Gillette JR (1968) Effect of drug substrates on the reduction of hepatic microsomal cytochrome P-450 by NADPH. Biochem Biophys Res Commun 31: 558–562.[CrossRef][Medline]

Gruenke LD, Konopka K, Cadieu M, and Waskell L (1995) The stoichiometry of the cytochrome P-450-catalyzed metabolism of methoxyflurane and benzphetamine in the presence and absence of cytochrome b(5). J Biol Chem 270: 24707–24718.[Abstract/Free Full Text]

Hackett JC, Kim YW, Su B, and Brueggemeier RW (2005) Synthesis and characterization of azole isoflavone inhibitors of aromatase. Bioorg Med Chem 13: 4063–4070.[Medline]

Ha-Duong NT, Marques-Soares C, Dijols S, Sari MA, Dansette PM, and Mansuy D (2001) Interaction of new sulfaphenazole derivatives with human liver cytochrome P450 2Cs: structural determinants required for selective recognition by CYP 2C9 and for inhibition of human CYP 2Cs. Arch Biochem Biophys 394: 189–200.[CrossRef][Medline]

Hodek P, Sopko B, Antonovic L, Sulc M, Novak P, and Strobel HW (2004) Evaluation of comparative cytochrome P450 2B4 model by photoaffinity labeling. Gen Physiol Biophys 23: 467–488.[Medline]

Hutzler JM, Melton RJ, Rumsey JM, Schnute ME, Locuson CW, and Wienkers LC (2006) Inhibition of cytochrome P450 3A4 by a pyrimidineimidazole: evidence for complex heme interactions. Chem Res Toxicol 19: 1650–1659.[CrossRef][Medline]

Jefcoate CR (1978) Measurement of substrate and inhibitor binding to microsomal cytochrome P-450 by optical-difference spectroscopy. Methods Enzymol 52: 258–279.[Medline]

Kulkarni AP, Mailman RB, Baker RC, and Hodgson E (1974) Cytochrome P-450 difference spectra. Type II interactions in insecticide-resistant and -susceptible houseflies. Drug Metab Dispos 2: 309–320.[Abstract]

Kumaki K, Sato M, Kon H, and Nebert DW (1978) Correlation of type I, type II, and reverse type I difference spectra with absolute changes in spin state of hepatic microsomal cytochrome P-450 iron from five mammalian species. J Biol Chem 253: 1048–1058.[Free Full Text]

Kumar V, Wahlstrom JL, Rock DA, Warren CJ, Gorman LA, and Tracy TS (2006) CYP2C9 inhibition: impact of probe selection and pharmacogenetics on in vitro inhibition profiles. Drug Metab Dispos 34: 1966–1975.[Abstract/Free Full Text]

Kunze KL, Nelson WL, Kharasch ED, Thummel KE, and Isoherranen N (2006) Stereochemical aspects of itraconazole metabolism in vitro and in vivo. Drug Metab Dispos 34: 583–590.[Abstract/Free Full Text]

Locuson CW, Gannett PM, and Tracy TS (2006) Heteroactivator effects on the coupling and spin state equilibrium of CYP2C9. Arch Biochem Biophys 449: 115–129.[CrossRef][Medline]

Mailman RB, Kulkarni AP, Baker RC, and Hodgson E (1974) Cytochrome P-450 difference spectra: effect of chemical structure on type II spectra in mouse hepatic microsomes. Drug Metab Dispos 2: 301–308.[Abstract]

Mancy A, Dijols S, Poli S, Guengerich P, and Mansuy D (1996) Interaction of sulfaphenazole derivatives with human liver cytochromes P450 2C: molecular origin of the specific inhibitory effects of sulfaphenazole on CYP 2C9 and consequences for the substrate binding site topology of CYP 2C9. Biochemistry 35: 16205–16212.[CrossRef][Medline]

Niwa T, Shiraga T, and Takagi A (2005) Effect of antifungal drugs on cytochrome P450 (CYP) 2C9, CYP2C19, and CYP3A4 activities in human liver microsomes. Biol Pharm Bull 28: 1805–1808.[CrossRef][Medline]

Ost TW, Clark J, Mowat CG, Miles CS, Walkinshaw MD, Reid GA, Chapman SK, and Daff S (2003) Oxygen activation and electron transfer in flavocytochrome P450 BM3. J Am Chem Soc 125: 15010–15020.[CrossRef][Medline]

Pearson J, Hill J, Swank J, Isoherranen I, Kunze K, and Atkins W (2006) Surface plasmon resonance analysis of antifungal azoles binding to CYP3A4 with kinetic resolution of multiple binding orientations. Biochemistry 45: 6341–6353.[CrossRef][Medline]

Rao S, Aoyama R, Schrag M, Trager WF, Rettie A, and Jones JP (2000) A refined 3-dimensional QSAR of cytochrome P450 2C9: computational predictions of drug interactions. J Med Chem 43: 2789–2796.[CrossRef][Medline]

Regev-Shoshani G, Shoseyov O, and Kerem Z (2004) Influence of lipophilicity on the interactions of hydroxy stilbenes with cytochrome P450 3A4. Biochem Biophys Res Commun 323: 668–673.[CrossRef][Medline]

Roberts AG, Campbell AP, and Atkins WM (2005) The thermodynamic landscape of testosterone binding to cytochrome P450 3A4: ligand binding and spin state equilibria. Biochemistry 44: 1353–1366.[CrossRef][Medline]

Roberts SA, Weichsel A, Qiu Y, Shelnutt JA, Walker FA, and Montfort WR (2001) Ligand-induced heme ruffling and bent no geometry in ultra-high-resolution structures of nitrophorin 4. Biochemistry 40: 11327–11337.[CrossRef][Medline]

Satchell JF and Smith BJ (2002) Calculation of aqueous dissociation constants of 1,2,4-triazole and tetrazole: a comparison of solvation models. Physiol Chem Chem Phys 4: 4314–4318.

Scott EE, White MA, He YA, Johnson EF, Stout CD, and Halpert JR (2004) Structure of mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl)imidazole at 1.9-A resolution: insight into the range of P450 conformations and the coordination of redox partner binding. J Biol Chem 279: 27294–27301.[Abstract/Free Full Text]

Seward HE, Roujeinikova A, McLean KJ, Munro AW, and Leys D (2006) Crystal structure of the Mycobacterium tuberculosis P450 CYP121-fluconazole complex reveals new azole drug-P450 binding mode. J Biol Chem 281: 39437–39443.[Abstract/Free Full Text]

Sono M and Dawson JH (1982) Formation of low spin complexes of ferric cytochrome P-450-CAM with anionic ligands. spin state and ligand affinity comparison to myoglobin. J Biol Chem 257: 5496–5502.[Free Full Text]

This article has been cited by other articles:

				L. Perrin, C. Aninat, V. Hamon, Y. Hayashi, C. Abadie, B. Heyd, F. Andre, and M. Delaforge Metabolism of Phenylahistin Enantiomers by Cytochromes P450: A Possible Explanation for Their Different Cytotoxicity Drug Metab. Dispos., November 1, 2008; 36(11): 2381 - 2392. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.106.012609v1 35/4/614    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Locuson, C. W. Articles by Tracy, T. S. Search for Related Content PubMed PubMed Citation Articles by Locuson, C. W. Articles by Tracy, T. S.