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Apr 25, 2017 - Key Laboratory of Liaoning Tumor Clinical Metabolomics, Jinzhou, Liaoning China. §. School of Pharmacy, Key Lab for Basic Pharmacology...
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A Naturally Occurring Isoform-Specific Probe for Highly Selective and Sensitive Detection of Human Cytochrome P450 3A5 Jing-Jing Wu,† Yun-Feng Cao,†,‡ Liang Feng,† Yu-Qi He,§ James Y. Hong,∥ Tong-Yi Dou,† Ping Wang,† Da-Cheng Hao,⊥ Guang-Bo Ge,*,† and Ling Yang# †

Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China Key Laboratory of Liaoning Tumor Clinical Metabolomics, Jinzhou, Liaoning China § School of Pharmacy, Key Lab for Basic Pharmacology of the Ministry of Education, Zunyi Medical University, Guizhou 563000, China ∥ Department of Biopharmaceutical Sciences, University of Illinois, Chicago, Illinois 60612, United States ⊥ School of Environment and Chemical Engineering, Dalian Jiaotong University, Dalian 116028, China # Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China ‡

S Supporting Information *

ABSTRACT: Cytochrome P450 (CYP) 3A5 characterized with polymorphic and extensive expression in multiple tissues is the most important P450 enzyme among the minor CYP3A isoforms. However, a selective and sensitive probe for CYP3A5 remains unavailable. In this study, we identified and characterized a naturally occurring lignan 12 (schisantherin E) as an isoform-specific probe for selective detection of CYP3A5 activity in complex biological samples. With thorough characterization including LC-MS and NMR, we found that 12 can be metabolized by CYP3A5 to generate a major metabolite 2O-demethylated 12. Meanwhile, both reaction phenotyping and chemical inhibition experiments further revealed that CYP3A5 selectively catalyzed the 2-O-demethylation of 12. Specifically, the interactions between the Phe210 residue of CYP3A5 and methyl benzoate of 12 might play key roles in 12-O-demethylation, which was revealed by docking simulation and site-directed mutagenesis studies. These findings are beneficial for exploring the role of CYP3A5 in drug metabolism and pathologic process.

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vincristine.11 As such, clinical dosing control and adverse effects minimization of these drugs call for a methodology to detect CYP3A5 activity with a high-specific readout. Notably, CYP3A5 shows high tissue specificity and correlates to certain disease states. For example, significantly more intense immunohistochemical staining of CYP3A5 can be seen in colorectal cancer tissues than in colorectal normal tissues. Similarly, in primary ovarian cancer tissues, CYP3A5 is present at significantly higher levels than in normal ovarian tissues. In contrast, CYP3A4 expression varies less in either colorectal or ovarian tissues.6,7 In human kidney, only CYP3A5, rather than CYP3A4, mRNA can be detected.12 Renal CYP3A5 generates 6β-hydroxysteroid. Via stimulating the transport of sodium across renal cells, 6β-hydroxysteroid induces sodium retention and eventually leads to sodium-sensitive hypertension.13 Furthermore, CYP3A5 is also the predominant CYP3A isoform expressed in human lung and prostate. The CYP3A5 abundance plays a key role in individual predisposition of lung cancer and androgen-dependent prostate cancer.14−16 In addition, CYP3A5 functions as a tumor suppressor in pathogenesis and metastasis of hepatocellular carcinoma.17

INTRODUCTION Cytochrome P450 (CYP) 3A5 is the best-investigated minor CYP3A isoform, which is intensely involved in the oxidative metabolism of multiple drugs, exogenous carcinogens, and endogenous steroids. It shares 83% homology and similar substrate specificity with CYP3A4, the major CYP isoform.1−4 In comparison with CYP3A4, CYP3A5 is distinct in expression level profile, specificity of tissue distribution, and relationship to disease susceptibility.5−7 However, due to the lack of appropriate specific probes, the role of CYP3A5 activity in drug safety and disease development remains largely unknown. CYP3A5 is characterized with genetic polymorphism. The CYP3A5*3 allele is a major variant, leading to inactivity and subsequent loss of CYP3A5 function, while the CYP3A5*1 allele expresses full-length and active CYP3A5.5 The average allele frequency of CYP3A5*1 is 7.8% in Caucasians, 25.8% in Asians, and rises to 55.8% in Africans (https://www.pharmgkb. org/gene/PA131).8 For individuals possessing one CYP3A5*1 allele, this enzyme accounts for at least 50% of the hepatic CYP3A proteins.5 Thus, a considerable amount of hepatic CYP3A5 protein in CYP3A5*1 individuals is available for metabolizing CYP3A substrates, including some drugs with a narrow therapeutic index such as the immunosuppressants tacrolimus and cyclosporine9,10 as well as antineoplastic agent © 2017 American Chemical Society

Received: January 4, 2017 Published: April 25, 2017 3804

DOI: 10.1021/acs.jmedchem.7b00001 J. Med. Chem. 2017, 60, 3804−3813

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Table 1. Chemical Structure and CYP3A Isozyme Specificity for Dibenzocyclooctadiene Lignans Used in This Studya

a Turnover (pmol/min/pmol CYP) at the compound concentration of 10 μM. +, 1−3; ++, 4−9; +++, 10−15; ++++, 16−29; +++++, ≥30; −, Not detectable.

occurring dibenzocyclooctadiene lignans and derived from Schisandra chinensis. The screening results revealed that compound 12 (schisantherin E) displayed the most remarkable preference for the detection of CYP3A5 isoform. This finding prompted us to further characterize the selectivity and sensitivity of this CYP3A5 probe and explore the relationship between enzyme structure and catalytic specificity by docking simulations and site-directed mutagenesis. Moreover, the feasibility of its application in various biological samples was demonstrated.

However, the detection of CYP3A5 enzyme is limited to the expression levels of its mRNA and protein, which are commonly futile in the detection of the activity and the corresponding role in tissues and various disease states. During the last decades, much attention has been paid in distinguishing the catalytic efficiency of CYP3A4 and CYP3A5 to facilitate more accurate predictions of drug−drug interactions.18 Numerous CYP3A4 isoform-specific substrates have been identified such as quinidine,19 bufalin,20 luciferinisopropyl acetal,21 and Gomisin A.22 In contrast, few substrates selective for CYP3A5 were reported, not to mention their poor sensitivities and the low rates of drug metabolism. For instance, CYP3A5 is the principal enzyme responsible for the hydroxylation of difluorocyclohexane ring of an antiretroviral drug maraviroc, and N-oxidization of a prodrug T5, where their maximum conversion rates via CYP3A5 are 0.93 pmol/min/ pmol CYP and 13.9 pmol/min/pmol CYP, respectively.23,24 Therefore, to combine high selectivity for precise detection of CYP3A5 activity with high catalytic efficiency in complex biological systems is quite alluring and challenging. In this study, we aimed to develop a novel probe that is highly isoform-selective to the CYP3A5 activity in multiple biological systems. In our preliminary study, the metabolic selectivity of compound 1−12 to CYP3A4 and CYP3A5 was evaluated (Table 1). All these compounds are naturally

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RESULTS Identification of Metabolites. To identify the metabolites of 12, in vitro incubation studies were performed. Only one new chromatographic peak was observed in human liver microsomes (HLM) after incubation with 12 in the NADPH generating system (Supporting Information, Figure S1). The metabolite (M) formation was NADPH- and microsomedependent. In addition, no conjugates were observed both in the UDP-glucuronic acid-, 3′-phosphoadenosine-5′-phosphosulfate- and S-adenosyl-L-methionine-generating systems when 12 was incubated with either HLM or S9 fractions (data not shown). The [M + K]+ ion of M at m/z 563 implied that M was a demethylated product of the substrate (m/z 577) (Supporting Information, Figure S1 and Table S1). M was 3805

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further identified as 2-O-demethylated 12 (compound 13, Scheme 1) based on 1 H NMR analysis (Supporting Scheme 1. Chemical Structure of Compound 12 and CYP3A5-Catalyzed Demethylation Reaction

Information, Table S2). The most distinctive spectral changes were peak signals representing H-4, H-6, and OCH3 between 12 and M. The H-4 signal at 6.93 ppm (1H, s) and H-6 at 4.57 ppm (1H, s) shifted upfield to 6.86 ppm (1H, s) and 4.45 ppm (1H, s), respectively, by comparing NMR 1H spectrum of 12 to that of M. Moreover, one OCH3 proton signal disappeared in M, and three OCH3 proton signals presented corresponding shifts (3.75−3.78 ppm, 3.37−3.71 ppm, and 3.05−2.96 ppm, respectively). These observations strongly suggest that demethylation occurred at the 2-O site. Assays with Recombinant Human CYP Isoforms. To elucidate the CYP isoforms involved in the metabolism of 12 in the human body, 16 recombinant human cytochrome P450 (rhCYP) isoforms were tested for their activity in demethylation of 12. In these studies, we used preparations of bacterial membranes containing the respective CYP enzymes coexpressed with human NADPH-cytochrome P450 reductase (Bactosomes) obtained from Cypex (Dundee, UK). As shown in Figure 1, 2-O-demethylation of 12 was predominantly

Figure 2. Effects of selected P450 inhibitors on 2-O-demethylation of 12 in pooled HLM. Results are the mean ± SD from three experiments carried out in duplicate. N.D.: not detectable. *, P < 0.05; **, P < 0.01; ns, P > 0.05.

P450 inactivator ABT completely inhibited 12 demethylation, suggesting that 12 demethylation was highly P450s-specific. Among the 10 selected inhibitors, the most profound effect (52.5% inhibition, P < 0.01) was elicited by ketoconazole, a potent inhibitor of CYP3A enzymes. Similarly, CYP3cide, a selective inhibitor of CYP3A4, inhibited the demethylation of 12 by 49.8% (P < 0.01). Furafylline, a selective inhibitor of CYP1A2, showed 30.6% inhibition to 2-O-demethylation of 12 (P < 0.05). The other CYPs inhibitors showed no significant inhibitory effects on 12 demethylation. These results suggest that CYP3A4 and CYP1A2 could metabolize 12 to a minor extent, although they are not considered as the dominant metabolic enzymes responsible for 2-O demethylation of 12. Correlation Study. In a panel of 13 individual HLMs, the reaction rate of 12 2-O-demethylation exhibited a splendid correlation with the content of CYP3A5 (r2 = 0.9402, P < 0.0001) (Figure 3a). The CYP3A5 activity scaled by the rate of 12 2-O-demethylation in 13 individual HLMs was increased from 23.2 to 158.7 pmol/min/mg with a 53.3% coefficient of variability. In contrast, as shown in Figure 3b, no correlation with the content of CYP3A4 was detected (r2 = 0.046, P = 0.48). The correlation study, together with reaction phenotyping and chemical inhibition assays, confirmed the predominant role of CYP3A5 in 2-O-demethylation of 12. Kinetic Analysis. To characterize the CYP3A5-specific biotransformation of 12, the comparative kinetic experiments were performed by using rhCYP3A4, rhCYP3A5, and HLM. The kinetics behaviors of 2-O-demethylation of 12, occurring in both CYP3A5 and HLM samples, obeyed the Michaelis− Menten kinetics (Supporting Information, Figure S2 and Figure 4). Although the Km value of rhCYP3A5 for 12 was similar to that of rhCYP3A4, the Vmax values obtained with CYP3A5 were almost 25.8-fold higher than that with CYP3A4 (63.4 ± 0.47 pmol/min/pmol CYP vs 2.46 ± 0.01 pmol/min/pmol CYP). Consequently, about 23.1-fold difference in intrinsic clearance (CLint) of 2-O-demethylation of 12 was observed between CYP3A5- and CYP3A4 samples (10.7 μL/min/pmol CYP vs 0.46 μL/min/pmol CYP) (Table 2). These results strongly

Figure 1. Isozymes involved in the 2-O-demethylation reaction of 12. Data are presented as the mean ± SD from three experiments carried out in duplicate. N.D.: not detectable.

catalyzed by CYP3A5 at different substrate concentrations (5 and 50 μM), whereas CYP3A4 displayed very limited or even no catalytic efficiency to this biotransformation. No participation of other CYP isoforms in the demethylation of 12 was detected. Chemical Inhibition Assays. The inhibitory profiles of selective chemical inhibitors for different CYPs on 12 demethylation are shown in Figure 2. The broad-spectrum 3806

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Figure 3. Correlation studies between the formation rate of 13 and the level of CYP3A5 content (a) and CYP3A4 content (b) in a panel of 13 individual HLMs.

the O atom at C-2 site of 12 and the heme iron atom of CYP3A5 (3.39 Å) was smaller than that of CYP3A4 (4.28 Å). Furthermore, Arg105 of both CYP3A4 and CYP3A5 may form hydrogen bond with 12. Notably, π−π stacking interactions may take place between the benzoyl of 12 and Phe210, Phe304 of CYP3A5, while such interactions were not observed between 12 and CYP3A4. Mutation of Residue 210 Alters the Metabolite Formation. To illuminate the predominant role of residue 210 in catalytic activity of CYP3A5 for 12, we mutated this residue in both CYP3A4 and CYP3A5. As shown in Figure 6, a significant decrease of 13 formation was observed when replacing the wild-type CYP3A5 (P < 0.01) by the CYP3A5 F210L mutant. However, 13 formation was markedly increased when the CYP3A4 L210F mutant was used as a substitute of the wild-type CYP3A4 (P < 0.01). These results, together with the results of docking simulation, evidently demonstrate that the CYP3A5 residue Phe210 is, at least in some part, responsible for the high catalytic activity of CYP3A5 in 2-Odemethylation of 12. Detecting CYP3A5 Expression and Activity in Different Cells. We used 12 as a probe to quantify the activities of CYP3A5 in different human tissue microsomes obtained from liver, intestine, kidney, lung, and various tumor cell lines such as HepG2, Caco-2, A549, and HEK293T. It is noteworthy that 12 was nontoxic to HepG2 cells even at a high concentration of 300 μM (Supporting Information, Figure S3). The protein levels of CYP3A5 in these human microsomes and cell lines were quantified by ELISA assays, and the mRNA expressions of CYP3A5 in various cell lines were quantified by real-time PCR. As expected, the activities of 12 2-O-demethylation in four different microsomal preparations and cell lines well agreed with the expression levels of CYP3A5 (Figure 7). The levels of expression and activity of CYP3A5 in the microsomal preparations probed here increase in the following order of the source tissues: liver > intestine > kidney > lung (Figure 7a,b). Among all tested cell lines, hepG2 displayed the highest level of CYP3A5 expression and the highest activity in 2-Odemethylation of 12. This cell line is followed by Caco-2 and A549, whereas the HEK293T cells exhibited the expression of CYP3A5 mRNA but showed neither CYP3A5 activity nor protein presence (Figure 7c,d). These results demonstrate that the activity of O-demethylation of 12 is instrumental in revealing the presence of functional CYP3A5 in different tissues and cell lines.

Figure 4. Michaelis−Menten Kinetic plots of 12 2-O-demethylation catalyzed by rhCYP3A4 and rhCYP3A5. Inset: Eadie−Hofstee plots. Results are the mean ± SD from three experiments carried out in duplicate.

Table 2. Kinetic Parameters of 12 2-O-Demethylation Catalyzed by rhCYP3A5, rhCYP3A4, and Pooled HLMsa enzyme source

Vmax

Km

CLint

pooled HLMs rhCYP3A5 rhCYP3A4 ratio (3A5/3A4)

248.5 ± 3.99 63.4 ± 0.13 2.46 ± 0.01 25.8

4.95 ± 0.37 5.90 ± 0.19 5.30 ± 0.08

50.2 10.7 0.46 23.1

Km values are in μM; Vmax values are in pmol/min per mg for liver microsomes or pmol/min/pmol CYP (CYP3A5 and CYP3A4); Intrinsic clearance (CLint) is obtained by Vmax/Km and is in μL/min/ mg for liver microsomes or μL/min/pmol CYP (CYP3A5 and CYP3A4). Values are the mean ± SD of three determinations performed in duplicate. a

suggest that 12 can serve as a highly selective probe for the detection of CYP3A5 activity in biological samples. The probe reaction of CYP3A5-catalyzed 12 demethylation was thus proposed and shown in Scheme 1. Docking Simulations. Molecular simulation was conducted to explore the interactions between 12 and two CYP isoforms. The ChemScore value of the bioactive pose of 12 with CYP3A5 was similar to that with CYP3A4 (−25.9050 vs −27.6983) (Supporting Information, Table S3), agreeing with the close Km values obtained from CYP3A4 and CYP3A5 in 12 2-O-demethylation. As shown in Figure 5, the distance between 3807

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Figure 5. Docking simulation of 12 into CYP3A4 (a) and CYP3A5 (b). Heme and iron atoms are colored in blue and yellow, respectively.

values indicate that the ability of CYP3A5 to metabolize 12 is over 23-fold higher than that of CYP3A4. It is worth noting that a selective inhibitor of CYP3A5 remains unavailable. We found that the inhibition of the activity of microsomes in 12 demethylation remains incomplete with either the nonspecific inhibitor of CYP3A or the CYP3A4-specific inhibitor. This observation rules out the predominant role of CYP3A4 isoforms in 12 demethylation. The specificity of 12 to CYP3A5 was also confirmed by a close correlation between the level of 12 2-O-demethylation activity and the CYP3A5 protein content. More importantly, the maximal catalytic turnover of CYP3A5 with 12 can be as high as 63.4 pmol/ min/pmol CYP, which is 69-fold and 5-fold higher than those observed with maraviroc (0.93 pmol/min/pmol CYP)23 and T5 (13.9 pmol/min/pmol CYP),24 respectively. This property endows 12 with a high sensitivity in probing the CYP3A5 activity. Furthermore, 12 is a naturally occurring lignan derived from Schisandra chinensis and can be utilized as a noncytotoxic medicinal herb (Supporting Information, Figure S3). The distinctive advantages in accessibility and safety make 12 preferable over other CYP3A5 substrates. It is well understood that the different amino acids in the active site of CYP3A isoforms and the ligand structures affect their catalytic efficiency and selectivity. To illustrate the mechanism of structure-oriented catalytic specificity, a combination of different approaches, including amino acid sequence alignment, homology modeling, and site-directed mutagenesis, is typically employed to elucidate the structural features which govern the regioselectivity of the CYP3A isoforms. For example, a previous study showed that the substitution of the amino acid residues critical for aflatoxin B1 biotransformation by CYP3A4 with those characteristic to CYP3A5 results in the changes in regioselectivity,25 which makes the mutated enzyme more CYP3A5-like. Specifically, this effect was observed with the CYP3A4 mutants P107S, F108L, N206S, L210F, V376T, S478D, and L479T mutations.25 Furthermore, the active-site residue C239 is responsible for CYP3A4 time-dependent inactivation (TDI), as it forms a covalent linkage with raloxifene, leading to the irreversible inhibition of the enzyme. In comparison, the corresponding residue in CYP3A5 is S239 and a reversible inhibition is observed. TDI was successfully engineered out in the CYP3A4 mutant with C239S mutation.26 These reports indicate that the differential residues in the active site of CYP3A5 contribute to the distinct contact with substrates.

Figure 6. Contributions of residue 210 to the differential formation rate of 13 by CYP3A4 and CYP3A5. The metabolite formation was normalized to the total cell protein. **, P < 0.01, compared with wildtype.

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DISCUSSION CYP3A5, which is highly homologous to the CYP3A4, characterized with polymorphism and specific tissue expression, is the most important P450 enzyme among the minor CYP3A isoforms. A CYP3A5-specific probe will be beneficial for differentiating activities of different CYP3A isoforms as well as to uncover the role of CYP3A5 in drug metabolism and certain pathologic processes. In the present study, we discovered a novel selective probe reaction, where the demethylation of 12 is catalyzed by CYP3A5 with high selectivity. The product of this reaction 13 was isolated and identified by LC-MS and 1H NMR as 2-O-demethylated 12. This CYP3A5-specific probe reaction obeys the prototypical Michaelis−Menten kinetics. It was thoroughly characterized in this study by employing recombinant P450s, chemical inhibition assays, and the correlation analysis. The probe reaction, namely CYP3A5-driven 2-O-demethylation of 12, served well as a base for a new method capable of detecting CYP3A5 activity with high selectivity and high conversion rate. First, the high catalytic selectivity of CYP3A5 to 12 was evidenced by kinetics studies with bactosomes containing recombinant P450 enzymes. The intrinsic clearance 3808

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Figure 7. Comparison of the expression levels and activities of CYP3A5 in different human tissues and cell lines. The protein expressions (a) and activities (b) of CYP3A5 in different tissues samples: HLM, human intestinal microsomes (HIM), human lung microsomes (HLuM), and human kidney microsomes (HKM). The protein/mRNA expression levels (c) and activities (d) of CYP3A5 in different tumor cell S9 fraction: HepG2 cell, Caco-2 cell, A549 cell, and HEK293T cell. N.D.: not detectable.

naphthalimide,29 demethylene of Gomisin A,22 and N-dealkylation of propafenone,30 are more specific to CYP1A2 rather than to CYP3A5 as a catalyst. Therefore, we assume that the substrate binding site of CYP1A2 may have some crossreactivity with the CYP3A5 substrates. This cross-reactivity may explain, in some part, why furafylline, an inhibitor of CYP1A2, is capable of suppressing of 2-O-demethylation of 12 to some extent (Figure 3). Moreover, the substrates that are preferentially metabolized by CYP3A5 tend to have a molecular weight over 500 Da, such as tacrolimus,10 vincristine,11 maraviroc,23 and T5.24 With the aim of developing new techniques for CYP3A5 quantification, we used 12 to quantify CYP3A5 activity in various biological samples. CYP3A5 is the predominant P450 isoenzyme in human lung and kidney. Therefore, in probing the activity of 12 demethylation, we used the microsomal preparations and cells obtained from these two tissues along with other samples obtained from liver and intestine. Notably, the CYP3A5 activities measured by using 12 2-O-demethylation as the probe reaction in 13 individual HLMs (male Mongolian, east Asia) exhibited 53.3% variability. This result suggests that at least 50% of individual variability in drug response should be considered when using 12 as a probe for CYP3A5 activity, especially in the absence of individual CYP3A5 polymorphic allele information. Notably, CYP3A5 activity levels in microsomal preparations from different human tissues and tumor cell lines derived from liver, intestine, lung, and kidney could be reliably determined by using 12 as the probe substrate and were commensurate with the respective CYP3A5 expression levels. In this regard, 12 could be used not only as a probe substrate of CYP3A5 activity but also as a biomarker for detecting such specific diseases as colorectal

Accordingly, the observation from our work revealed that in CYP3A5 one additional hydrogen-bonding and two additional π−π stacking interactions are formed between 12 and Arg105, 12 and Phe304, and 12 and Phe210, respectively. Although Arg105 and Phe304 are shared residues in both CYP3A4 and CYP3A5, the hydrogen-bonding is only formed between 12 and Arg105 in CYP3A4. Interestingly, the π−π stacking interaction between the benzoyl group of 12 and Phe210 of CYP3A5 is not present in CYP3A4, where the aromatic Phe210 is substituted with aliphatic Leu210. The critical role of Phe210 in CYP3A5 was further confirmed by the site-directed mutagenesis, where the formation rate of 13 was markedly increased by adopting the L210F mutant of CYP3A4 and significantly decreased when employing the F210L mutant of CYP3A5. These results hint that the bulky benzoyl group might help 12 efficiently orient itself toward the active site of CYP3A5 via the π−π stacking interactions, which could alter the electron cloud density of the aromatic ring and subsequently form the energetically favorable configuration of 12 in CYP3A5 (Figure 5). Our findings can explain, at least in part, why CYP3A5 has the highest catalytic selectivity with 12, as compared to other lignan analogues (Table 1), and may shed new light on developing novel probes with high specificity for certain P450 enzymes. A large number of CYP3A substrates derived from various Traditional Chinese Medicines have been previously screened in our laboratory. The majority of them are substrates of both CYP3A4 and CYP3A5 (Table 1), although some are selectively metabolized by CYP3A4 through hydroxylation reactions.20,22,27,28 In this work, 12 2-O-demethylation was found to be selectively catalyzed by CYP3A5. It is noted that the dealkylation reactions, such as O-demethylation of 83809

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column temperature was kept at 40 °C. 12 and its metabolite were detected at 230 nm. The metabolite concentrations were estimated against a standard curve with a linear range of 0.05−30 μM of 12, with a correlation coefficient >0.999. A Shimadzu LC-MS-2010EV (Kyoto, Japan) instrument with an ESI interface was used for the identification of 12 and its metabolite. Mass detection was performed in both positive-ion mode (ESI+) and negative ion mode (ESI−) from m/z 300 to 700. The detector voltage was set at +1.75 kV and −1.55 kV for positive and negative ion detections, respectively. The curved desolvation line (CDL) temperature and the block heater temperature were both set at 250 °C. Other mass spectrometry (MS) detection conditions were as follows: the interface voltage was 4 kV, CDL voltage was 40 V, nebulizing gas (N2) flow was 1.5 L/min, and drying gas pressure was 0.06 MPa. Data processing was performed using LC-MS Solution software, version 3.41. Incubation Conditions. The incubation mixture, with a total volume of 200 μL, consisted of 100 mM potassium phosphate buffer (pH 7.4), a NADPH-generating system (1 mM NADP+, 10 mM glucose-6-phosphate, 1 unit/mL of glucose-6-phosphate dehydrogenase, and 4 mM MgCl2), and human liver microsomes or CYPs. In all of the experiments, 12 (20 mM dissolved in methanol previously) was serially diluted to the required concentrations and the final concentration of methanol did not exceed 1% (v/v) in the mixture. After preincubation at 37 °C for 3 min, the reaction was initiated by adding an NADPH-generating system; the reaction was incubated at 37 °C in a shaking water bath. The reaction was terminated by the addition of 200 μL of ice-cold methanol. The mixture was kept on ice until it was centrifuged at 20000g for 20 min at 4 °C. Aliquots of supernatants were stored at −20 °C until analysis. Control incubations without an NADPH-generating system, without a substrate, or without CYP enzyme sources were carried out to ensure that metabolite formation was CYP- and NADPH-dependent. All of the incubations throughout the study were carried out in three experiments performed in duplicate with standard deviation (SD) values generally below 10%, and the results are expressed as the mean ± SD. Reaction Phenotyping Assays with Recombinant P450s. Sixteen preparations of bacterial membranes (Bactosomes) containing various recombinant CYP isoforms (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4F2, and CYP4F3B) coexpressed with human NADPH-cytochrome P450 reductase were used to screen the involved isoform(s) for the demethylation of 12 in HLM. The incubations were carried out under the above-mentioned incubation conditions with each CYP isoform. Two substrate concentrations (5 and 50 μM) were used, and 12 was incubated with each of the recombinant CYPs (40−80 nM) at 37 °C for 30 min. The reaction mixtures were centrifuged to precipitate the protein as described previously. The supernatants were then analyzed by UFLCDAD to quantify the metabolites of 12. Chemical Inhibition Assays. Chemical inhibition studies were performed by adding specific inhibitors for different CYP isoforms to the incubation mixture of 12 to verify the involved enzyme(s). In brief, 12 (5 μM, relevant to the Km value) was incubated in HLM (0.2 mg protein/mL) with an NADPH-generating system in the absence (control) or presence of known CYP isoform-specific inhibitors. The inhibitors and their concentrations were as follows: sulfaphenazole (10 μM) for CYP2C9,31 quinidine (10 μM) for CYP2D6,31 clomethiazole (50 μM) for CYP2E1,31 ketoconazole (1 μM) for CYP3A,31 omeprazole (20 μM) for CYP2C19,32 and montelukast (2 μM) for CYP2C8.33 Inhibition by furafylline (10 μM) for CYP1A2,31 8methoxypsoralen (2.5 μM) for CYP2A6,34 TEPA (50 μM) for CYP2B6,35 ABT (500 μM) for broad CYPs,36 and CYP3cide (1 μM) for CYP3A437 were assayed by preincubation with an NADPHgenerating system at 37 °C for 25 min. Correlation Studies. The formation rates of the metabolite described for 12 (5 μM, near Km value) were determined in a panel of HLMs prepared from 13 individual donors. The incubation conditions were described as mentioned above. These values were compared with the levels of CYP3A5 or CYP3A4 in 13 individual HLMs. Briefly,

cancer, ovarian cancer, lung cancer, androgen-dependent prostate cancer, and sodium-dependent hypertension, etc.

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CONCLUSION In summary, we reported a dibenzocyclooctadiene derivative of lignan (schisantherin E, 12) as the first naturally occurring probe for highly selective detection of human CYP3A5. Owing to its excellent selectivity toward CYP3A5 as well as the high catalytic efficiency of CYP3A5 with 12, this substrate can be used for the quantification of changeable levels of CYP3A5 activity in various biological specimens including a variety of human tissues and cell lines. Moreover, the results obtained from the analysis of the structural basis of catalytic specificity of CYP3A5 for 12 with docking simulations and site-directed mutagenesis indicated that the bulky benzoyl group of 12 may be important for orienting the molecule of 12 in the active site of CYP3A5 in a more efficient way as compared to the molecules of other lignan derivatives. These findings suggest that 12 may be used as a promising tool for exploring the role of CYP3A5 in xenobiotic metabolism and assessment of potential drug interactions.

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EXPERIMENTAL SECTION

Chemicals and Reagents. Compound 12 (schisantherin E) was purchased from Sichuan WeiKeqi Biological Technology Co., Ltd. (Chengdu, China). 1-Aminobenzotriazole (ABT), sulfaphenazole, quinidine, clomethiazole, furafylline, 8-methoxypsoralen, omeprazole, CYP3cide, glucose-6-phosphate dehydrogenase, NADP+, D-glucose-6phosphate, and trypsin (TPCK-treated, from bovine pancreas) were purchased from Sigma (St. Louis, MO, USA). Ketoconazole was obtained from ICN Biomedicals, Inc. (Aurora, Ohio, USA). Montelukast was purchased from Beijing Aleznova Pharmaceutical (Beijing, China). Triethylenethiophosphoramide (TEPA) was purchased from Acros Organics (Geel, Belgium). Human CYP3A5 ELISA kit was from HZbscience (Shanghai, China). The human cell lines hepG2, Caco-2, and A549 were purchased from the Committee on Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). COS-7 cell line was purchased from the Biowit Technologies Co., Ltd. (Shenzhen, China). HEK293T cell line is a generous gift from Professor Yang Liu, Scientific Research Center for Translational Medicine, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (Dalian, China). Dulbecco’s Modified Eagle’s Medium (DMEM) and 1640 medium and fetal calf serum (FCS) were purchased from Invitrogen. The cell counting kit-8 (CCK-8) was purchased from Solarbio (Beijing, China). BCA protein assay kit was purchased from Beyotime Biotechnology Co., Ltd. (Jiangsu, China). All other reagents were of the highest commercially available grade. Enzyme Source. Sixteen rhCYP isoforms, including CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4F2, and CYP4F3B, were purchased from Cypex (Dundee, UK). Mixed HLM, HIM, HLuM, and HKM were obtained from BioreclamationIVT (MD, USA). A panel of 13 HLMs from male Mongolian individuals was obtained from Research Institute for Liver Diseases Co. Ltd. (Shanghai, China). All of the microsomal samples and rhCYP isoforms were stored at −80 °C until use. Analytical Instruments and Conditions. The UFLC system was equipped with a CBM-20A communications bus module, a SIL20ACHT autosampler, two LC-20AD pumps, a DGU-20A3 vacuum degasser, a CTO-20AC column oven, and a SPD-M 20A diode array detector (DAD). A Hypersil ODS (C18) analytical column (150 mm × 2.1 mm, 3 μm, Thermo Scientific) with a Hypersil ODS (C18) guard column (150 nm × 2.1 mm, 3 μm, Thermo Scientific) was used to separate 12 and its metabolite. The mobile phase consisted of CH3OH (A) and water (B) with the following gradient profile: 0−2 min, 50% B; 2−9 min, 50%−15% B; 9−12 min, 15%−5% B; and 12− 16.5 min, balanced to 50% B. The flow rate was 0.4 mL/min, and the 3810

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microsomes were digested by trypsin at 37 °C for 14 h using an enzyme-to-substrate ratio of 1:50 as previously described.38 Then, the digested sample was centrifuged for 10 min at 13000g, and the supernatant was spiked with a stable isotope labeled peptide for LCMS/MS analysis using the multiple reaction monitoring (MRM) mode and an isotope labeled peptide from the internal standards. Specific peptides of SLGPVGFMK (for CYP3A5) and EVTNFLR (for CYP3A4) were selected for their quantification by using transition ions with ratios of 468.3/678.5 and 439.7/549.3, respectively. The correlation parameter was expressed by the linear regression coefficient (r). P < 0.05 was considered statistically significant. Kinetics Analysis. To estimate the kinetic parameters of 12 2-Odemethylation in different enzyme sources, the incubation conditions were optimized to ensure that the formation rates of 13 were in the linear range in relation to the incubation time and protein concentration at 37 °C. 12 was serially diluted to the required concentrations (2, 5, 10, 20, 50, 100, 200 μM), and the final concentration of methanol was 1% (v/v). 12 was incubated with HLM (0.4 mg/mL), rhCYP3A4 (40 nM), or rhCYP3A5 (20 nM) for 20, 20, and 5 min, respectively. All of the incubations were carried out in three independent experiments in duplicate. The apparent Km and Vmax values were calculated from the nonlinear regression analysis of experimental data, and the results were graphically represented by Eadie−Hofstee plots. The kinetic constants were estimated by the software program Prism (version 5.0.1, GraphPad, San Diego, CA) and are reported as the mean ± SD of the parameter estimate. In Vitro Biosynthesis and Isolation of Major Metabolite. The predominant metabolite 2-O-demethylated 12 was biosynthesized using cDNA-expressed rhCYP3A5 enzyme. The incubation system was scaled up to 200 mL. 12 (200 μM) was incubated with rhCYP3A5 (final enzyme concentration, 50 nM) and the NADPH-generating system (1 mM NADP+, 10 mM glucose-6-phosphate, 1 unit/mL of glucose-6-phosphate dehydrogenase, and 4 mM MgCl2) for 4 h at 37 °C. Under these conditions, approximately 50% of 12 was converted to 2-O-demethylated 12. Methanol (200 mL) was added to the reaction mixture to precipitate the protein. After centrifuging at 20000g for 20 min at 4 °C, the supernatant was separated and extracted with ethyl acetate (200 mL × 3). The organic layer was combined and dried in vacuo, the residue was redissolved in methanol (1.2 mL), and the solution was injected into the LC column. The preparative HPLC system (SHIMADZU, Kyoto, Japan) consisted of a SCL-10A system controller, two LC-10AT pumps, a SIL-10A auto injector, a SPD-10AVP UV detector, and a C18 column (4.6 mm × 250 mm, 10 μm) and was used to separate 12 and its metabolite. The mobile phase was 48% acetonitrile in water. The eluent was monitored at 254 nm with a flow rate of 5.0 mL/min, and the fractions containing 2-O-demethylated 12 were collected and dried in vacuo. The purity of the target compounds was determined to be >95% by analytical UFLC system, with UV detection at 230 and/or 254 nm. NMR Spectrometry. NMR spectra were measured with a 500 MHz Bruker ARX-600 spectrometer using tetramethylsilane as the reference for 1H NMR. The compounds were dissolved in DMSO-d6, and the experiments were conducted at 22 °C. Chemical shifts are reported in parts per million (ppm). Docking Simulation. To illustrate the molecular mechanism of the selective catalysis of CYP3A5 for 12, the docking simulation was performed using the knowledge-based homology modeling package from Advanced Protein Modeling that was distributed within SYBYL (X-1.1). The homology model of CYP3A5 was constructed based on the template structure of CYP3A4 (PDB 3TJS) as previously described.22 With the established 3D-structure of 3A5 as well as the crystal structure of 3A4, the bioactive binding conformations of 12 were generated using Surflex-Dock, which were evaluated by an empirical function ChemScore, one of the most suitable scoring functions for the P450s superfamily.39 The docking results were further visualized using PyMOL Molecular Graphics System, version 0.99 (DeLano Scientific LLC). Site-Directed Mutagenesis of CYP3A4/5 and Activity Assays. COS-7 cells were used for the expression of CYP3A5 and CYP3A4 as previously described.23 Briefly, COS-7 (1 × 106/well) cells were

cultured in 1640 medium containing 10% FCS as a monolayer in 6well plates. Plasmids pCMV6-XL4 containing CYP3A5 and CYP3A4 full-length cDNAs (TrueClones) were obtained from OriGene (Rockville, MD). Mutations, plasmid DNA isolation, and DNA sequencing were performed by Suzhou Convenience Biotechnology Co., Ltd. (China). The 4 μg plasmid constructs were transfected into COS-7 cells per well using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction in 6-well plates. Then 24 h after transfection, the cells were first washed twice with PBS (2 mL) then scraped from the wells in the presence of PBS (1 mL). Following centrifugation for 5 min at 200g, the cell pellets were gently resuspended in 100 mM potassium phosphate buffer (pH7.4). The cells were disrupted by sonication on ice for 3 × 3 s pulses, with 10 s intervals between two pulses, with the sonifier (JYD- 650, Shanghai Zhixin Instrument Co., China) power setting at 200 W. Following sonication, the cells suspension was centrifuged at 9000g for 20 min at 4 °C and the supernatant fraction (S9) was obtained. The total protein concentration of S9 was measured using the BCA protein assay kit. Then, 12 and CYP3A mutant cells S9 at a final concentration of 50 μM and 5 mg/mL, respectively, were added to 200 μL of incubation mixture as above-described. After 60 min of incubation, 200 μL of methanol was added, vortexed, and centrifuged at 20000g for 20 min at 4 °C. Supernatants were stored at −20 °C until analysis. Analysis of CYP3A5 mRNA Expression in Different Cell Lines. Four human tumor cell lines, hepG2, Caco-2, A549, and HEK293T, derived from human liver, intestine, lung, and kidney, respectively, were separately cultured in 6-well plates with complete DMEM containing 10% FCS and collected by trypsin when they reached 90% confluence. The effects of 12 against the viability of treated cells were assessed using a CCK-8 assay kit (Solarbio, Beijing, China). The CYP3A5 expression in different human tumor cell lines was determined with real-time RT-PCR analysis. Briefly, the total RNA was isolated using the RNAiso Plus reagent with gDNA Eraser (Takara) according to the manufacturer’s protocol. The yield and purity were measured by the ratio of A260/A280 using an ultraviolet spectrophotometer. RNA of 500 ng was subjected to the reverse transcription in a 20 μL reaction mixture using a PrimeScript RT reagent Kit (Takara). After the synthesis of cDNA, the real-time PCR was conducted with a SYBR Premix Ex TaqII kit (Takara) to quantify the transcript levels of CYP3A5. The primer sequences for real-time PCR are listed in Supporting Information, Table S4. The relative expression level for CYP3A5 was normalized by the CT value of the human housekeeping gene GAPDH (2−ΔΔCT formula). Data was analyzed by Applied Biosystems StepOne Real-Time PCR System software version 2.0. Detection of the Activity and Protein Expression of CYP3A5 in Various Cell Lines. For the preparation of cell S9 fractions, HepG2, Caco-2, A549, and HEK293T cells were cultured in 75 cm2 flasks and collected by trypsin when they reached 90% confluence, respectively. Then cells were washed, resuspended, and sonicated in ice-cold PBS buffer. The S9 fractions were prepared following centrifugation at 9000g for 20 min. Supernatants were collected, and protein contents were assessed by the BCA reagent assay (Beyotime, Jiangsu, China). Then the protein levels of CYP3A5 in these human cell lines were quantified by the human CYP3A5 ELISA kit (HZbscience). Meanwhile, 12 (50 μM) was used as a probe to quantify the activities of CYP3A5 in four various cell lines as abovedescribed. After 60 min of incubation, the reactions were terminated by addition of 200 μL of ice-cold methanol. The mixture was kept on ice until it was centrifuged at 20000g for 20 min at 4 °C. Aliquots of supernatants were stored at −80 °C until analysis. Statistical Analysis. All statistical analyses were done by the program Prism (version 5.0.1, GraphPad, San Diego, CA). P < 0.05 were considered as significant in the two-tailed Student t-test. 3811

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Relling, M.; Brimer, C.; Yasuda, K.; Venkataramanan, R.; Strom, S.; Thummel, K.; Boguski, M. S.; Schuetz, E. Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression. Nat. Genet. 2001, 27, 383−391. (6) Kumarakulasingham, M.; Rooney, P. H.; Dundas, S. R.; Telfer, C.; Melvin, W. T.; Curran, S.; Murray, G. I. Cytochrorne P450 profile of colorectal cancer: Identification of markers of prognosis. Clin. Cancer Res. 2005, 11, 3758−3765. (7) Downie, D.; McFadyen, M. C.; Rooney, P. H.; Cruickshank, M. E.; Parkin, D. E.; Miller, I. D.; Telfer, C.; Melvin, W. T.; Murray, G. I. Profiling cytochrome P450 expression in ovarian cancer: identification of prognostic markers. Clin. Cancer Res. 2005, 11, 7369−7375. (8) Whirl-Carrillo, M.; McDonagh, E. M.; Hebert, J. M.; Gong, L.; Sangkuhl, K.; Thorn, C. F.; Altman, R. B.; Klein, T. E. Pharmacogenomics knowledge for personalized medicine. Clin. Pharmacol. Ther. 2012, 92, 414−417. (9) Dai, Y.; Iwanaga, K.; Lin, Y. S.; Hebert, M. F.; Davis, C. L.; Huang, W.; Kharasch, E. D.; Thummel, K. E. In vitro metabolism of cyclosporine A by human kidney CYP3A5. Biochem. Pharmacol. 2004, 68, 1889−1902. (10) Dai, Y.; Hebert, M. F.; Isoherranen, N.; Davis, C. L.; Marsh, C.; Shen, D. D.; Thummel, K. E. Effect of CYP3A5 polymorphism on tacrolimus metabolic clearance in vitro. Drug Metab. Dispos. 2006, 34, 836−847. (11) Dennison, J. B.; Kulanthaivel, P.; Barbuch, R. J.; Renbarger, J. L.; Ehlhardt, W. J.; Hall, S. D. Selective metabolism of vincristine in vitro by CYP3A5. Drug Metab. Dispos. 2006, 34, 1317−1327. (12) Koch, I.; Weil, R.; Wolbold, R.; Brockmoller, J.; Hustert, E.; Burk, O.; Neussler, A.; Neuhaus, P.; Eichelbaum, M.; Zanger, U.; Wojnowski, L. Interindividual variability and tissue-specificity in the expression of cytochrome P450 3A mRNA. Drug Metab. Dispos. 2002, 30, 1108−1114. (13) Givens, R. C.; Lin, Y. S.; Dowling, A. L.; Thummel, K. E.; Lamba, J. K.; Schuetz, E. G.; Stewart, P. W.; Watkins, P. B. CYP3A5 genotype predicts renal CYP3A activity and blood pressure in healthy adults. J. Appl. Physiol. 2003, 95, 1297−1300. (14) Yeh, K. T.; Chen, J. C.; Chen, C. M.; Wang, Y. F.; Lee, T. P.; Chang, J. G. CYP3A5*1 is an inhibitory factor for lung cancer in Taiwanese. Kaohsiung J. Med. Sci. 2003, 19, 201−207. (15) Moilanen, A. M.; Hakkola, J.; Vaarala, M. H.; Kauppila, S.; Hirvikoski, P.; Vuoristo, J. T.; Edwards, R. J.; Paavonen, T. K. Characterization of androgen-regulated expression of CYP3A5 in human prostate. Carcinogenesis 2007, 28, 916−921. (16) Anttila, S.; Hukkanen, J.; Hakkola, J.; Stjernvall, T.; Beaune, P.; Edwards, R. J.; Boobis, A. R.; Pelkonen, O.; Raunio, H. Expression and localization of CYP3A4 and CYP3A5 in human lung. Am. J. Respir. Cell Mol. Biol. 1997, 16, 242−249. (17) Jiang, F.; Chen, L.; Yang, Y. C.; Wang, X. M.; Wang, R. Y.; Li, L.; Wen, W.; Chang, Y. X.; Chen, C. Y.; Tang, J.; Liu, G. M. Y.; Huang, W. T.; Xu, L.; Wang, H. Y. CYP3A5 functions as a tumor suppressor in hepatocellular carcinoma by regulating mTORC2/Akt signaling. Cancer Res. 2015, 75, 1470−1481. (18) Yamazaki, H.; Niwa, T.; Murayama, N.; Emoto, C. Comparison of kinetic parameters for drug oxidation rates and substrate inhibition potential mediated by cytochrome P450 3A4 and 3A5. Curr. Drug Metab. 2008, 9, 20−33. (19) Nielsen, T. L.; Rasmussen, B. B.; Flinois, J. P.; Beaune, P.; Brosen, K. In vitro metabolism of quinidine: The (3S)-3-hydroxylation of quinidine is a specific marker reaction for cytochrome P-4503A4 activity in human liver microsomes. J. Pharmacol. Exp. Ther. 1999, 289, 31−37. (20) Ge, G. B.; Ning, J.; Hu, L. H.; Dai, Z. R.; Hou, J.; Cao, Y. F.; Yu, Z. W.; Ai, C. Z.; Gu, J. K.; Ma, X. C.; Yang, L. A highly selective probe for human cytochrome P450 3A4: isoform selectivity, kinetic characterization and its applications. Chem. Commun. 2013, 49, 9779−9781. (21) Li, A. P. Evaluation of Luciferin-Isopropyl Acetal as a CYP3A4 substrate for human hepatocytes: effects of organic solvents,

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00001. 1 H NMR spectrum of 12 and 13. Chemical structure and CYP3A isozyme specificity of lignans. Representative UFLC profiles and mass spectra of 12 and its metabolite. Kinetic plots of 12 2-O-demethylation catalyzed by HLM. Cytotoxic effects of 12 on hepG2 cell line (PDF) Molecular formula strings (CSV) Accession Codes

The crystal complex of CYP3A4 and homology model of CYP3A5 with 12 can be accessed using PDB code 3TJS. Authors will release the atomic coordinates and experimental data upon article publication.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-411-84379317. Fax: +86-411-84676961. E-mail: [email protected]. ORCID

Jing-Jing Wu: 0000-0003-0466-3058 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was financially supported by grants from the National Natural Science Foundation of China (81403003, 81573501, 81402985, and 81672961), and Projects of Shenyang Science & Technology Plan (F13-221-9-17). We especially thank Dr. Yang Liu, Scientific Research Center for Translational Medicine, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, for providing HEK293T cells.

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ABBREVIATIONS USED ABT, 1-aminobenzotriazole; BCA, bicinchoninic acid; CCK-8, cell counting kit-8; CDL, curved desolvation line; CLint, intrinsic clearance; CYP, cytochrome P450; DMEM, Dulbecco’s Modified Eagle’s Medium; FCS, fetal calf serum; HIM, human intestine microsome; HKM, human kidney microsomes; HLM, human liver microsomes; HLuM, human lung microsomes; rhCYP, recombinant human cytochrome P450; TEPA, triethylenethiophosphoramide; UFLC-DAD, ultrafast liquid chromatography-diode array detector; Vmax, apparent maximum reaction velocity

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REFERENCES

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