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Effect of Cholesterol on the Interaction of Cytochrome P450 Substrate Drug Chlorzoxazone with Phosphatidylcholine Bilayers Ayumi Yamada, Nobutaka Shimizu, Takaaki Hikima, Masaki Takata, Toshihide Kobayashi, and Hiroshi Takahashi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00286 • Publication Date (Web): 27 Jun 2016 Downloaded from http://pubs.acs.org on June 29, 2016
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Effect of Cholesterol on the Interaction of Cytochrome P450 Substrate Drug Chlorzoxazone with Phosphatidylcholine Bilayer Ayumi Yamada,† Nobutaka Shimizu,‡ Takaaki Hikima,§ Masaki Takata,§,# Toshihide Kobayashi,┴, ¶ and Hiroshi Takahashi*,†, §, ┴ †
Biophysics Laboratory, Division of Pure and Applied Science, Graduate School of Science and
Technology, Gunma University, 4-2 Aramaki, Maebashi, Gunma 371-8510, Japan ‡
Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research
Organization, 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan §
#
RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyougo 679-5148, Japan
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1
Katahira, Aoba-ku, Sendai, Miyagi 980-8577, Japan. ┴
Lipid Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
*Author to whom correspondence should be addressed. Funding Sources This research was support by JSPS KAKENHI Grant Number 20261860 and 16K05509 to H.T. 1
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ABSTRACT: Many drugs are oxidized by membrane protein cytochrome P450 (CYP) enzymes in their metabolism process. CYPs are located mainly in endoplasmic reticulum (ER) membranes. Recent studies have suggested that CYP-substrate drugs first bind the lipid bilayers of ER membranes and then, the drugs reach the active site of CYP by way of an access channel. The entrance of the channel is located in the hydrophobic regions of the lipid bilayers. One of the feature of ER membrane is lower cholesterol content as compared with other biomembranes. In the present study, cholesterol-concentration dependence of the interaction of a CYP substrate drug, chlorzoxazone (CZX) with model membranes composed of phosphatidylcholine (PC) and cholesterol was examined with differential scanning calorimetry (DSC), UV-visible spectroscopy, and X-ray diffraction. Experimental results indicated that CZX can bind to pure PC bilayers in the absence of cholesterol and that, by contrast, high cholesterol concentration (30-50 mol%) tends to prevent CZX from binding to PC bilayers. Interestingly, the effect of cholesterol on the binding and insertion of CZX was biphasic. In the case of palmitoyl-oleoylphosphatidylcholine (POPC) bilayers containing 5-10 mol% cholesterol, the CZX’s binding and penetration into the bilayer were found to be greater than pure POPC bilayers. The concentration of 5-10 mol% nearly corresponds to the cholesterol concentration of ER membranes. The low cholesterol contents (12-20 mol%) of ER membranes might be the most suitable for CYP-drug metabolism process in ER membranes.
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Introduction: Cytochrome P450 (CYP) enzymes are a superfamily of monooxygenases. Fifty-seven CYPs have already identified in the human genome.1 Microsomal CYPs play an important role in the metabolism of xenobiotic chemicals and drugs by oxidizing these compounds.1,2 It has been reported that nearly 75% of the top 200 prescribed drugs in USA are metabolized by CYP.3 The oxidation by CPYs induces to increases of the polarity of the drugs. Consequently, the oxidized drugs are easily excreted from a body with urine. The majority of CYPs is localized in endoplasmic reticulum (ER) membranes that are recognized to be a responsible main site for the metabolism of xenobiotic chemicals and some drugs. Recently, some papers have reported that CYPs are also present, however, lower levels in other cellular organelles such as mitochondria, and at the extracellular surface of the plasma membranes.4, 5 It has been reported that mitochondrial CYPs are mainly involved in the metabolism of endogenous compounds and biosynthesis of steroid hormones.6 However, the role of mitochondrial CYPs in drug metabolism still remains unclear. The situation is also the same for CYPs located in the plasma membranes. Although plasma membrane expressed CYPs have been shown to be catalytically active, whether drug metabolism takes place at the surface of the plasma membranes remains unclear.5 In the present, the most important site in drug metabolism related to CYPs in cells is believed to be the ER membranes. Several recent studies have indicated that the lipid bilayers of biomembranes are also involved in drug metabolism processes associated with CYPs as follows. Microsomal CYPs are a membrane protein and have a single N-terminal transmembrane α- helix by which the CYPs are anchored in the lipid bilayer of ER membranes. Since the first report of the crystal structure of a mammalian 3
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CYP,7 the crystal structures of many CYPs has been reported.8-17 These crystal structures revealed that the catalytic active site of CYPs is buried in the hydrophobic inside of the proteins, and that the shape of the fold is conserved over mammalian CYPs. In addition, several tunnels from the active site to the protein surface are recognized in the CYP crystal structures. From this finding, it has been proposed that the access of drugs to the active site and the release of the oxidized drugs from the active site takes place through each different tunnel. These tunnels are also called as channels. All determinations of the crystal structures have been performed for CYPs lacking the transmembrane α-helix. By using the crystal structures, molecular dynamic simulation studies have been conducted to explore whole structure of CYPs with the transmembrane part and the binding mode to a lipid bilayer.18-23 The results of these studies supported the proposal process from the crystal structure of CYPs lacking transmembrane αhelix, suggesting that CYPs have an access channel to the active site and that, the entrances of the channels are located in the hydrophobic regions of the lipid bilayers. In other words, the following process can be assumed; at a first step, CYP-substrate drugs bind to lipid bilayer part of biomembranes and at a second step, the drugs penetrate into the hydrophobic regions of lipid bilayers, and at a final step, the drugs reach the active site through the access channel. After oxidation reaction at the active site, the oxidized drugs are released from the site through another channel (solvent channel).19-23 The studies described above clearly indicates that biomembranes also play a role in drug metabolism associated with CYPs. The lipid bilayer parts of biomembrane are made mainly from phospholipid molecules. Cholesterol is also an essential component of the lipid bilayers. The concentration of cholesterol are widely different in each biomembrane. In eukaryotic cells, the 4
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cholesterol contents are about 3-5 wt% (6-10 mol%) and about 6-10 wt% (12-20 mol%) for the mitochondrial membranes and ER membranes, respectively.24-27 On the other hand, the cholesterol contents are about 10-20 wt% (20-40 mol%) for the plasma membranes and the biomembrane of other subcellular organelle. 24-27 As described above, drug metabolism by CYP takes place mainly in ER membranes. It is naturally expected that living things have developed some methods by which CYP-substrate drugs are effectively introduced to ER membranes containing CYPs. The difference of cholesterol contents of biomembrane leads us to think that lower cholesterol levels of ER membranes are advantageous in the drug metabolism process associated with CYPs. Here we propose that the interaction of CYP-substrate drugs with lipid bilayer regions of biomembranes depends on cholesterol content of the biomembranes. In other words, we postulated that the CYP-substrate drugs mainly interact with the membranes containing lower amounts of cholesterol, i.e., the ER membranes. The ultimate goal is to confirm the above hypothesis by using real biomembranes in living cell systems. Living systems are, however, extremely complex. As a first step, here we explored the hypothesis using a model system. That is, the purpose of this paper is to clarify the effect of cholesterol concentration on the binding or penetration of a CYP-substrate drug to a single phospholipid component lipid bilayers. In this study, we chose chlorzoxazone (CZX, Figure 1) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) as the CYP-substrate drug and a phospholipid component of the model membranes, respectively. CZX is a centrally acting muscle relaxant drug and is known to be metabolized by liver-specific CYP2E1.28 The reason chosen is that the high purity sample of CZX is easily obtained and it is a widely used market drug. POPC was chosen because 5
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phosphatidylcholine (PC) is one of major phospholipid components of the biomembranes and the most PC molecules in biomembranes have one cis-unsaturated fatty acyl chain.29 The interaction of CZX with a single component phosphatidylcholine (PC) bilayers has been investigated by using molecular dynamic simulation technique.20-22 These studies have indicated that the primary location of CZX molecules in dioleoylphosphatidylcholine bilayers is around the interface between the headgroups and the hydrophobic hydrocarbon chain regions. The location position agrees with that of the entrances of the access channels of CYPs bound to membrane. No experimental study, however, has been carried out for the interaction of CXZ with PC bilayers, as far as we know. Thus, we first investigated the interaction of CZX between a single component phosphatidylcholine (PC) bilayers with differential scanning calorimetry (DSC). Next, we examined the effect of cholesterol on the binding of CZX to POPC vesicles by means of UV-visible spectroscopy and X-ray diffraction measurements. On the basis of the results obtained, we report that the binding and insertion of CZX to POPC bilayer depends on the cholesterol concentrations. Our conclusion is that the dependence is biphasic, i.e., high cholesterol concentration (30-50 mol%) tends to prevent CZX from binding and on the other hand, for the case of 5-10 mol% cholesterol concentration, the CZX’s binding and penetration into the bilayer were found to be greater than pure POPC bilayers. This finding agrees with a recent prediction that cholesterol-concentration dependent partitioning of triethylamine into dioleoyl-PC/cholesterol membranes is biphasic, based on a molecular dynamics simulation study performed in order to examine the permeability of lipid membranes for drugs.30
MATERIALS AND METHODS 6
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Materials. Chlorzoxazone (5-chloro-3H-benzooxazol-2-one, CZX) was obtained from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine（ DPPC）was purchased from Avant Polar Lipids Inc. (Alabaster, AL). 1-Palmitoyl-2-oleoyl-snglycero-3-phosphocholine（POPC）was purchased from Avant Polar Lipids Inc. and NOF Corporation (Tokyo, Japan). Both POPCs obtained from the different companies showed the same lamellar repeat spacing within error when multilamellar vesicles were prepared under a fully hydration condition. Poly(vinylpyrrolidone) (PVP) of average molecular weight 40,000 and cholesterol ((3β)-cholest-5-en-3-ol) were purchased from Sigma (St. Louis, MO). All phospholipids used in this study had a purity >99% and were used without further purification. Water used in this study was prepared with a Milli-Q system (Millipore Corp., Bedford, USA). Preparation of Vesicle Samples. Each lipid (POPC, DPPC, and cholesterol) was dissolved in chloroform and the final lipid concentration was 10mM. CXZ was dissolved in a chloroform : methanol mixture (2:1, v/v). The concentration of CZX was 5 mM. Lipid stock solutions and CZX stock solution were mixed in proportion to achieve the desired molar fractions. The solvent was evaporated under a stream of oxygen free dry nitrogen. To remove residual solvents, the samples were kept for 16 h in vacuum. Multilamellar vesicle samples were prepared by suspending the dried lipid film samples in pure water and sonicating for 0.5 h at room temperature for sample containing POPC or at 45 oC for sample containing DPPC, with a bathtype sonicator. The chain-melting transition temperatures of fully hydrated POPC and DPPC bilayers have been reported to be ~ −2 oC and 41.4 oC, respectively.31 The lipid (phospholipid + cholesterol) concentrations were 25 wt% and 100 mM (~ 7-10 wt%) for X-ray diffraction and calorimetry, respectively. 7
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Differential Scanning Calorimetry (DSC). Calorimetric measurements were performed with a DSC6100-Exstar6000 thermal analysis system (Hitachi High-Technologies Corporation (formerly Seiko Instruments Inc.) Chiba, Japan). The heating scan rate was 1.0 oC min−1. Lipid concentrations were 100mM. Fifteen microliters (15 µl) of sample dispersions were put into aluminum sample cells (02190062, PerkinElmer Corporation, Waltham, Massachusetts, U.S.A.) and then were hermetically sealed. The melting transition of high-purity indium was used as a calibration for the instrument. UV-Visible Spectroscopic Measurements. Absorbance measurements were performed with a UV-1800 double-beam UV-visible spectrophotometer (Shimadzu Scientific Instruments, Kyoto, Japan) by using a quartz cuvette with optical path length of 1 cm. CZX solution gave the maximum peak at 280 nm in its absorbance spectrum measured in the range from 250 to 350 nm (see Figure S1 (a) of the Supporting Information). The molar extinction coefficient of CZX at 280 nm was estimated to be 5105 M-1cm-1 from the measurement for 10-100 µM (see Figure S1 (B) of the Supporting Information). To study the binding of CZX to lipid vesicles, absorbance measurements at 280 nm were carried out for the supernatant solutions obtained after centrifugation of lipid-drug vesicle dispersions at 17860 x g for 2 - 5 hours and CZX reference solution (0.1 mM). X-ray Diffraction Measurements. Experiments using a synchrotron X-ray beam were carried out at Photon Factory beamlines (BLs) 10C32 and 6A33 in the High Energy Accelerator Research Organization (Tsukuba, Japan) and RIKEN Structural Biology Beamline I (BL45XU)34 at SPring-8 (Harima, Japan). The wavelengths (λ) of X-ray were 0.09864 nm, 0.1500 nm, and 0.1000 nm for BL10C, BL6A and BL45XU, respectively. The sample-to-detector lengths were ~ 8
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500 mm and ~ 1000 mm for BL10C and BL45XU, respectively. In BL6A, small- and wide-angle diffraction data were recorded separately by using two detectors. The sample-to-detector lengths were ~ 1100 mm and ~ 300 mm for the detector of small-angle and wide-angle, respectively. All diffraction patterns were recorded using an X-ray photon counting two-dimensional pixel array detector PILATUS (DECTRIS, Switzerland)35 with a 172 x 172 µm2 pixel size. The twodimensional diffraction image data were converted to one-dimensional profiles using Fit2d software.36 Diffraction angle was calibrated using the diffraction patterns of silver behenate.37 The sample was mounted on a DSC apparatus for an optical microscope (FP 84, Mettler-Toledo) used as a temperature controller.38 All X-ray diffraction measurements were performed at 25 oC. Reconstruction of Electron Density Profiles. The integrated intensities of ℎth order lamellar diffraction peaks ( ℎ) observed in a small-angle region were obtained by fitting each peak with a Lorentzian line shape function plus a linear function as the background. The absolute values of observed structure factors (| ℎ|) were set equal to ℎ ℎ, i.e., the Lorentzpolarization correction factor for the unoriented samples was approximated to be simply ℎ in the present study.39 The factor is a factor to normalize the data obtained from samples with different lamellar repeat spacings (L). As described below, in order to assign the phase angle of each lamellar diffraction peak, the intensities lamellar diffraction peaks were measured for different samples with different water layer distances, i.e., different hydration levels (swelling experiments). According to the method proposed previously,40 α was defined by
α
= ℎ ℎ ,
9
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where is the minimum value of in the swelling experiment series. If the unit cell of a lipid bilayer structure is centrosymmetric, the one-dimensional electron density profile is calculated from
0 2 2'ℎ = + + ℎcos & (
where is the electron density of water, L is the lamellar repeat spacing and z is the distance from the center of the bilayer.41 The diffraction intensity of zero order cannot be observed in experiments. 0 is given by
0 = ) * − , . .
Because previous studies have shown that the average electron density of POPC bilayers in a liquid crystalline phase is almost the same as that of water, we assumed that the value of 0 is zero within the resolution of this study. Hereafter we will refer a relative electron density calculated from following equation as electron density profile of lipid bilayers.
2 2'ℎ 012 = exp*67ℎ, | ℎ|cos & ( ,
where 7ℎ is a phase angle factor for hth order lamellar diffraction. The value of 7ℎ (' or −') was chosen so that continuous transform curves calculating by using Shannon sampling theorem42 fit all of | ℎ| plotted against the scattering vector 10
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magnitude 8 = 4'sin
