Anal. Chem. 2005, 77, 7080-7083
Creation of a P450 Array toward High-throughput Analysis Kumiko Sakai-Kato,*,† Masaru Kato,‡,§ Hiroshi Homma,| Toshimasa Toyo’oka,‡ and Naoko Utsunomiya-Tate†
Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo, 202-8585, Japan, School of Pharmaceutical Sciences and COE Program in the 21st Century, University of Shizuoka, 52-1 Yada Suruga-ku, Shizuoka, 422-8526, Japan, PRESTO, Japan Science and Technology Agency (JST), Saitama, Japan. and School of Pharmaceutical Sciences, Kitasato University, Shirokane, 5-9-1, Minato-ku, Tokyo, 108-8641, Japan
The rapid metabolism testing of many new chemical entities enables unsuitable candidates to be eliminated from consideration at an early stage of the drug discovery process. We have developed a P450 array toward highthroughput analysis of P450-mediated metabolic reaction. The microsomes containing expressed human P450 enzymes were immobilized on the microassay plate using sol-gel chemistry. A thin-film hydrogel containing microsomes was fabricated using aqueous silicate as a starting material. The TEM image clearly showed that the nanoclusters derived from the silicate formed branched chains, and microsomes were entrapped in the silica network. The different P450 isozymes were immobilized on the microassay plate, and the metabolites by each isozyme were visualized as fluorescent images, which creates opportunity for the inhibitor assays. This method offers several advantages over use of conventional enzyme preparations, including increased storage stability, ease of product isolation from the incubation mixture, and the ability to recover and reuse the enzyme. Because this methodology enabled the development of assay system using P450 that is unstable and involves other enzymes for its function, it can be applicable to various screening assays that require complicated reactions involving many biological components. In biological systems, many important reactions involve various biological components. The development of assay systems using these reactions is often difficult, however, because these reactions involve various biological components, and they are very unstable. One example is the cytochrome P450 (P450)-mediated reaction. Hepatic drug metabolism is crucial for the elimination of many therapeutic drugs. In the process of new drug development, drug metabolism research is very important for minimizing the adverse effect of pharmaceuticals and for maximizing their therapeutic value. Therefore, a combinatorial and screen-based approach to the metabolic evaluation for thousands of compounds is required * To whom correspondence should be addressed. Tel.: +81-424-68-8441. Fax: +81-424-68-8441. E-mail:
[email protected]. † Musashino University. ‡ University of Shizuoka. § Japan Science and Technology Agency. | Kitasato University.
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at an early stage of the drug development. Among the most important drug-metabolizing enzymes are P450s, a group of monooxygenases.1 These enzymes catalyze the biotransformation of lipophilic drugs to more polar compounds that are readily excreted in urine.2 The P450s, a family of closely related isozymes called CYPs, are located mainly in the endoplasmatic reticulum of liver cells. After homogenization of these lamellar membranes, artificial vesicles called microsomes are formed. On the other hand, P450 possesses the greatest industrial promise as a potential biocatalyst because many of the reactions catalyzed by these enzymes are very difficult from an organic synthesis point of view.3 The industrial potential of P450 has not yet been realized, however, because of some limitations, particularly for in vitro applications. First, the metabolic reaction involves various electrontransfer enzymes other than P450, such as reductase, which complicates the reaction. Second, a limitation on the use of P450 in biotechnology has been its thermal instability, which seriously limits its catalytic lifetime even at temperatures below 25 °C. So far, some researchers have developed the immobilization techniques of P450 mainly to improve the stability of the enzyme.4-8 The immobilization of the enzyme has been accomplished by several techniques including covalent bonding onto supports such as cyanogen bromide-activated Sepharose4-6 or entrapment in polyacrylamide gel.7,8 Hara et al. tested three immobilization methods (entrapment, adsorption, and crosslinking) and found that the entrapment gave the best activity.8 These studies were limited, however, to the evaluation of relatively small numbers of experiments due primarily to the lack of methods for high-throughput analysis. We report here the creation of a P450 array toward high-throughput analysis of the P450(1) Spatzenegger, M.; Jaeger, W. Drug Metab. Rev. 1995, 27, 397-417. (2) Hirobe, M.; Kamataki, T. Drug Metabolism; Hirokawa Publishing Co.: Tokyo, 1990. (3) Puchkaev, A. V.; Wakagi, T.; Ortiz de Montellano, P. R. J. Am. Chem. Soc. 2002, 124, 12682-12683. (4) Baess, D.; Janig, G.-R.; Ruckpaul, K. Acta Biol. Med. Ger. 1975, 34, 17451754. (5) Lehman, J. P.; Ferrin, L.; Fenselau, C.; Yost, G. S. Drug Metab. Dispos. 1981, 9, 15-18. (6) Ibrahim, M.; Decolin, M.; Batt, A.-M.; Dellacherie, E.; Siest, G. Appl. Biochem. Biotechnol. 1986, 12, 199-213. (7) Yawetz, A.; Perry, A. S.; Freeman, A.; Katchalski-Katzir, E. Biochim. Biophys. Acta 1984, 798, 204-209. (8) Hara, M.; Iazvovskaia, S.; Ohkawa, H.; Asada, Y.; Miyake, J. J. Biosci. Biotechnol. 1999, 87, 793-797. 10.1021/ac050714y CCC: $30.25
© 2005 American Chemical Society Published on Web 09/29/2005
mediated reaction. The microsomes containing expressed human P450 were immobilized on the microassay plate using sol-gel chemistry. This methodology offers several advantages over the use of conventional microsomal or soluble enzyme preparations. These include increased storage stability, facilitated separation of products from the incubation mixture, and the ability to recover and reuse the enzyme, which facilitate the integration into the high-throughput system. For the expression of P450 activity, other electron-transfer enzymes need to be arrayed near P450 onto the phospholipid membrane. Therefore, the encapsulation of microsomes containing all enzymes in silica micropores was thought to be the best immobilization technique. Sol-gel chemistry has emerged as the immobilization method for a number of proteins.9-12 The protein is encapsulated in the silicate matrix that is formed by polymeric oxo-bridged SiO2 networks. Most of such proteins are, however, entrapped only in single form. Therefore, it is worth noting that active P450 has been entrapped in the silica hydrogel as a form embedded onto the microsome membrane along with other electron-transfer enzymes that are required for the function of P450. EXPERIMENTAL SECTION Entrapment of Microsomes Containing Expressed Human P450. The microsome solution containing expressed human P450 (Daiichi Pure Chemicals Co., Tokyo, Japan) was diluted twice with 0.1 M potassium phosphate buffer, pH 7.4, containing EDTA and dithiothreitol at a concentration of 0.1 mM and glycerol (20%) (preparation buffer). Sodium silicate solution (0.4 M, 0.5 mL, Sigma-Aldrich, Milwaukee, WI) was mixed with Ludox colloidal silica (8.5 M, 0.5 mL, Sigma-Aldrich), and hydrochloric acid (4 M) was added until neutralization. The silicate solution was immediately added to the microsome solution at a volumetric ratio of 5:1 (microsome solution/silicate sol), and the mixture was carefully mixed and sonicated for 5 s to obtain a homogeneous solution. The portions of 10 µL were loaded into a 96-well microassay plate and allowed to gel at 4 °C. On the next day, 50 µL of preparation buffer was carefully applied on the gel to avoid drying. The plates were covered with microplate sealing film, and the gel was aged at 4 °C for 3 days, forming a glass disk that was attached to the bottom of the wells. The gel was stored at 4 °C until use. The tetramethoxysilane (TMOS)-derived materials were prepared using a similar procedure. The monomer solution was obtained by mixing the following reagents just prior to use: (1) 761 µL of TMOS (Tokyo Kasei, Tokyo, Japan), (2) 169 µL of water, and (3) 11 µL of 0.04 N HCl. This monomer solution was stirred for 20 min so that hydrolysis proceeds to form a fully or partially hydrolyzed silane, SiOH4-n(OMe)n. This TMOS hydrolysate was mixed with microsome solution in the same manner as with that using the sodium silicate solution. Transmission Electron Microscope (TEM). Gels were fixed in 2.5% glutaraldehyde containing 0.5% tannin acid in 0.1 M phosphate buffer (pH 7.2) for 70 min. After fixation, the gels were (9) Brinker, C. J.; Scherer, G. W., Sol-gel science: The Physics and Chemistry of Sol-Gel Processing; Academic Press: New York, 1990. (10) Ellerby, L. M.; Nishida, C. R.; Nishida, F.; Yamanaka, S. A.; Dunn, B.; Valentine, J. S.; Zink, J. I. Science 1992, 255, 1113-1115. (11) Flora, K.; Brennan, J. D. Anal. Chem. 1998, 70, 4505-4513. (12) Kato, M.; Sakai-Kato, K.; Matsumoto, N.; Toyo’oka, T. Anal. Chem. 2002, 74, 1915-1921.
Figure 1. (A) Schematic presentation of P450 array. (B) A list of P450 isozymes, substrates, and metabolites used for the assay.
Figure 2. Activities of immobilized CYP3A4 prepared by various conditions. The ratio represents the amount of CYP3A4 solution to aqueous silicate. (CYP solution: aqueous silicate).
immersed in 0.25% sucrose in 0.1 M phosphate buffer (pH 7.2) for 10 min 3 times. Subsequently, the gels were postfixed in 2% OsO4 in 0.1 M phosphate buffer (pH 7.2) containing 0.25% sucrose for 45 min. The samples were then dehydrated by passing them through ethanol series, followed by n-butyl glycidyl ether (QY-1, Nisshin EM, Tokyo, Japan). After dehydration, the samples were embedded in epoxy resin, Epok812 (Oken, Tokyo, Japan). The sections were cut, contrasted with uranyl acetate/lead citrate, and examined in a Hitachi H7000 TEM (Hitachi, Tokyo, Japan). Assay of Entrapped P450. The reaction solution containing 1.3 mM NADP, 3.3 mM glucose 6-phosphate, 0.4 unit/mL glucose6-phosphate dehydrogenase, and 3.3 mM magnesium chloride in 0.1 M potassium phosphate buffer, along with substrate, was preincubated at 37 °C for 10 min. The portions of 50 µL were loaded on gel and incubated at 37 °C for 30 min. In the case of fluorogenic substrate, the product was visualized using a fluorescent microscope ECLIPSE TE 2000U (Nikon, Tokyo, Japan). The Analytical Chemistry, Vol. 77, No. 21, November 1, 2005
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Figure 3. (A) TEM image of immobilized microsome. Bar, 1 µm. (B) Illustration of TEM image A.
activity of CYP3A4 for testosterone was determined by HPLC, Shimadzu LC-VP system (Shimadzu, Tokyo, Japan).13 RESULTS AND DISCUSSION Figure 1 shows a schematic presentation of P450 array. To apply the developed technique to the high-throughput system, microsome-containing gel was fabricated as a thin film on the well bottom of the microassay plate that was pretreated with poly(vinyl acetate) to make the hydrophilic surface. Figure 2 shows the activity of immobilized CYP3A4, which is responsible for the metabolism of many clinically important drugs. For immobilization, the starting silica materials were added to the CYP3A4 solution at the specific ratios, and the mixture was allowed to gel at 4 °C. In the previous report, we prepared the microsome-immobilized column using TMOS as a starting material and we could evaluate the UDP-glucuronyltransferase (UGT) activity.14,15 P450 activity was very low, probably caused by the instability of P450, compared with UGT. Hence, glycerol, EDTA, and dithiothreitol were first added to the CYP solution for stabilization.16 In fact, it was shown in Figure 2 that these additives increased the activity of CYP3A4. TMOS is widely used as a starting material, and methanol is produced as the byproduct. It is well known that methanol inhibits the activity of P450.17 Therefore, another route using aqueous sodium silicate as a starting material was used in order to avoid the production of alcohol.18,19 In this route, SiO2 networks are produced by acidifying aqueous sodium silicate. By using colloidal silica together, silicates condense at the surface of colloidal particles that behave as nucleation sites for condensation. As shown in Figure 2, the activities of CYP3A4 immobilized in aqueous silicate-derived matrix were higher than those in the TMOS-derived matrix. Finally, the volumetric ratio of CYP3A4 solution to aqueous silicate was optimized to 5:1 (Figure 2). The gels at more than 6:1 were not suitable for repeated analysis. It was also found that sonication after mixing of all the solution components increased the activity ∼50%. It probably dispersed microsomes homogeneously or (13) Buters, J. T.; korzekwa, K. R.; Kunze, K. L.; Omata, Y.; Hardwick, J. P.; Gonzalez, F. J. Drug Metab. Dispos. 1994, 22, 688-692. (14) Sakai-Kato, K.; Kato, M.; Toyo’oka, T. Anal. Biochem. 2002, 308, 278284. (15) Sakai-Kato, K.; Kato, M.; Toyo’oka, T. Anal. Chem. 2002, 74, 2943-2949. (16) Kamataki, T., Drug Metabolism Enzyme; Hirokawa Publishing Co.: Tokyo, 2001. (17) Busby W. F., Jr.; Ackermann, J. R.; Crespi, C. L. Drug Metab. Dispos. 1999, 27, 246-249. (18) Nassif, N.; Roux C.; Coradin, T.; Rager, M.-N.; Bouvet, O. M. M.; Livage, J. J. Mater. Chem. 2003, 13, 203-208. (19) Bhatia, R. B.; Brinker, C. J. Chem. Mater. 2000, 12, 2434-2441
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changed the microsome diameters, which caused an increase in the surface area of the microsome and the accessibility of substrates. By these optimizations mentioned above, the activity was increased ∼15 times that of the initial condition. Figure 3 shows the TEM image of the microsome immobilized in the silica matrix. It is clearly observed that the nanoclusters derived from the silicate form branched chains, and microsomes are entrapped in the silica network. Around the microsomes, there are enough spaces for water or substrates to diffuse, which is important for obtaining good enzymatic activity. The water content of this hydrogel calculated from the weight reduction after drying is ∼70%, which coincides with the water content in living organisms. It must be noted that the manner in which microsomes are immobilized in silica networks is clearly observed in TEM images. The Km value of the immobilized CYP3A4 for testosterone was 105 ( 2.5 µM, which was slightly higher than the values in free solution, 60.8 ( 14.2 µM. This result shows that the accessibility of substrates to enzymes in the developed silica matrix was relatively high, even though the enzyme is entrapped in gel matrix. This result is attributed to the fact that the microsomeencapsulated gel was formed in a thin film on the microassay plate, and substrate was easily accessible to enzymes. Moreover, as shown in the TEM image, the surrounding spaces of immobilized microsomes were enough for water and substrates to diffuse. The reproducibility and stability of the activity of immobilized P450 were investigated. The batch-to-batch reproducibility of the enzymatic activity was acceptable, and the relative standard deviation (RSD) was 8.7% (n ) 3), and the day-to-day reproducibility of three consecutive days was also acceptable with a RSD of 10.2%. These values are comparable to those using free solution. After 3-weeks storage at 4 °C, the activity was 98% compared with that before storage. On the other hand, the activity in free solution was 30% that before storage for 1 week and 10% for 3 weeks. These results show that the stability drastically improved by encapsulation. Based on the present immobilization technique, an integrated P450 array may be readily prepared by immobilizing microsomes containing various CYPs. Figure 4 depicts the fluorescence images of the metabolites produced by immobilized P450. The different CYPs were immobilized on wells, and each CYP successfully metabolized each fluorogenic substrate (Figure 4). Figure 4A shows that the immobilized CYP1A1 successfully metabolized ethoxyresorufin to resorufin, whose red fluorescence was visualized. It was also shown that the fluorescence intensity decreased
4C). On a hydrogel without CYP, no or only faint fluorescence was observed (blank). CONCLUSIONS This work is the first in which microsomal P450s are immobilized on a microassay plate toward high-throughput analysis. This array achieved catalysis with good reproducibility and stability. Because this methodology enabled the immobilization of P450 that is unstable and involves other enzymes for its function, it can be applicable to the various screening assays that require complicated reactions involving many biological components. Figure 4. Photographs of fluorescent metabolites on P450 array containing (A) CYP1A1, (B) and (C) CYP3A4, and (D) CYP2C19. Inhibitor concentrations from left to right: (a) R-naphthoflavone 0.016, 0.08, 0.4, 2, and 10 µM; (b) ketoconazole, 2, 10, and 50 µM.
as the concentration of R-naphthoflavone, an inhibitor of CYP1A1, was increased. The green fluorescence by dibenzylfluorescein metabolite on CYP3A4 gel was also decreased with addition of the inhibitor, ketoconazole (Figure 4B). These results indicate that this P450 array can be used for the screening of the inhibitor for each CYP. The immobilized CYP3A4 also metabolizes 7-benzyloxyresorufin to resorufin, and its red light was observed (Figure
ACKNOWLEDGMENT A part of this work was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), HAITEKU (2004), and Nanotechnology Support Project at the Research Center for Ultrahigh Voltage Electron Microscopy, Osaka University. We also gratefully thank Prof. K. Abe for the loan of a fluorescent microscope. The authors acknowledge Prof. R. N. Zare and Dr. M. T. Dulay for the useful comments on this work. Received for review April 25, 2005. Accepted August 23, 2005. AC050714Y
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