Inhibition of CYP1 by berberine, palmatine, and jatrorrhizine: Selectivity, kinetic characterization, and molecular modeling

Toxicology and Applied Pharmacology 272 (2013) 671–680

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Inhibition of CYP1 by berberine, palmatine, and jatrorrhizine: Selectivity, kinetic characterization, and molecular modeling Sheng-Nan Lo a,b, Yu-Ping Chang a, Keng-Chang Tsai a, Chia-Yu Chang a,d, Tian-Shung Wu c,⁎, Yune-Fang Ueng a,b,d,⁎⁎ a

National Research Institute of Chinese Medicine, Taipei 112, Taiwan, ROC Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan, ROC Department of Chemistry, National Chung-Kung University, Tainan 701, Taiwan, ROC d Institute of Medical Sciences, Taipei Medical University, Taipei 101, Taiwan, ROC b c

a r t i c l e

i n f o

Article history: Received 27 March 2013 Revised 9 July 2013 Accepted 12 July 2013 Available online 22 July 2013 Keywords: Berberine CYP1B1 Selective inhibition

a b s t r a c t Cytochrome P450 (P450, CYP) 1 family plays a primary role in the detoxification and bioactivation of polycyclic aromatic hydrocarbons. Human CYP1A1, CYP1A2, and CYP1B1 exhibit differential substrate specificity and tissue distribution. Berberine, palmatine, and jatrorrhizine are protoberberine alkaloids present in several medicinal herbs, such as Coptis chinensis (Huang-Lian) and goldenseal. These protoberberines inhibited CYP1A1.1- and CYP1B1.1-catalyzed 7-ethoxyresorufin O-deethylation (EROD) activities, whereas CYP1A2.1 activity was barely affected. Kinetic analysis revealed that berberine noncompetitively inhibited EROD activities of CYP1A1.1 and CYP1B1.1, whereas palmatine and jatrorrhizine caused either competitive or mixed type of inhibition. Among protoberberines, berberine caused the most potent and selective inhibitory effect on CYP1B1.1 with the least Ki value of 44 ± 16 nM. Berberine also potently inhibited CYP1B1.1 activities toward 7-ethoxycoumarin and 7-methoxyresorufin, whereas the inhibition of benzo(a)pyrene hydroxylation activity was less pronounced. Berberine inhibited the polymorphic variants, CYP1B1.3 (V432L) and CYP1B1.4 (N453S), with IC50 values comparable to that for CYP1B1.1 inhibition. Berberine-mediated inhibition was abolished by a mutation of Asn228 to Thr in CYP1B1.1, whereas the inhibition was enhanced by a reversal mutation of Thr223 to Asn in CYP1A2.1. This result in conjugation with the molecular modeling revealed the crucial role of hydrogenbonding interaction of Asn228 on CYP1B1.1 with the methoxy moiety of berberine. These findings demonstrate that berberine causes a selective CYP1B1-inhibition, in which Asn228 appears to be crucial. The inhibitory effects of berberine on CYP1B1 activities toward structurally diverse substrates can be different. © 2013 Elsevier Inc. All rights reserved.

Introduction The cytochrome P450 (P450, CYP) 1 family plays a crucial role in the detoxification and bioactivation of xenobiotics, such as benzo(a)pyrene and the physiologically relevant compounds, such as estradiol. CYP1A1 and CYP1A2 share approximately 70% amino acid sequence identity and CYP1B1 shares about 40% identity with CYP1A1 and CYP1A2 (Sutter et al., 1994). CYP1 members exhibit differences in tissue distribution, substrate preference, and region-selectivity. CYP1A2 is constitutively expressed in human liver, whereas CYP1A1 and CYP1B1 are primarily localized in the extrahepatic tissues. Relatively high expression levels Abbreviations: AHH, benzo(a)pyrene hydroxylase; P450, CYP, cytochrome P450; ECOD, 7-ethoxycoumarin O-deethylation; EROD, 7-ethoxyresorufin O-deethylation; MROD, 7-methoxyresorufin O-demethylation. ⁎ Co-corresponding author. ⁎⁎ Correspondence to: Y-F. Ueng, Laboratory of Drug Metabolism, National Research Institute of Chinese Medicine, 155-1, Li-Nong Street, Sec. 2, Taipei 112, Taiwan, ROC. Fax: + 886 2 28264266. E-mail address: [email protected] (Y.-F. Ueng). 0041-008X/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2013.07.005

of CYP1B1 were found in steroidogenic tissues including the prostate, ovary, adrenal, uterus, and mammary (Gajjar et al., 2012; Shimada et al., 1996). CYP1A1 was the most effective CYP1 member in the activation of benzo(a)pyrene genotoxicity and CYP1B1 was more effective than CYP1A1 and CYP1A2 in the activation of 6-nitrochrysene and 5methylchrysene-1,2-diol (Shimada et al., 1996). In the hydroxylation of estradiol, CYP1A1 and CYP1A2 mainly produce the 2-hydroxylation metabolite, whereas CYP1B1 was more effective in the production of the genotoxic metabolite 4-hydroxyestradiol (Lee et al., 2003). Higher expression levels of CYP1B1 have been found in peri-tumor or tumor tissues in estrogen-associated cancers, such as breast and endometrial cancers (Gajjar et al., 2012; Hevir et al., 2011). Due to the selective tissue distribution and the detrimental bioactivation effect of CYP1B1, researchers have made efforts to develop a selective CYP1B1 inhibitor, such as the synthetic derivatives of stilbene, for cancer chemoprevention (Gajjar et al., 2012; Mikstacka et al., 2012). For example, the potent CYP1B1 inhibitor 2,3′,4,5′-tetramethoxystilbene (TMS) reduced the CYP1B1-activated mutagenecity of 2-amino-3,5-dimethylimidazo[4,5f]quinoline (MeIQ) in a bacterial test system (Chun et al., 2001) and

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decreased the formation of estrogen–DNA adduct in female mice (Kim et al., 2011). Recently, CYP1B1 has been reported to make an important contribution to angiotensin II-induced hypertension and reactive oxygen species (ROS) production (Jennings et al., 2010). Angiotensin II-mediated increase of blood pressure and ROS in Cyp1b1+/+ mice were significantly higher than those in Cyp1b1−/− mice. The stimulatory effects of angiotensin II on NADPH oxidase activity and the phosphorylation of ERK1/2, p38MAPK, and c-Src were reduced by Cyp1b1 gene disruption in mice (Jennings et al., 2012a). Angiotensin II-induced proteinuria, albuminuria, and the elevation of plasma creatinine concentration could be reduced by Cyp1b1 gene disruption. Modulation of the metabolism of arachidonic acid is one of the primary factors for hypertension and ROS production (Malik et al., 2012). Human and mouse CYP1B1 catalyzed the oxidation of arachidonic acid to form hydroxyeicosatetraeonic acids (HETEs) and epoxyeicosatrienoic acids (EETs) including 12- and 20-HETEs (Choudhary et al., 2004). In Cyp1b1+/+ but not Cyp1b1−/− mice, angiotensin II-treatment increased renal Cyp1b1 activity and 12and 20-HETE levels (Jennings et al., 2012a). HETEs may contribute to the Cyp1b1-dependent effect of angiotensin II in mice. In Spargue– Dawley rats, the angiotensin II-induced hypertension and associated renal injury were accompanied with an increase of CYP1B1 activity (Jennings et al., 2012b). However, angiotensin II did not alter the renal levels of several HETEs/EETs including 12- and 20-HETE in rats (Malik et al., 2012). The species difference in the contribution of CYP1B1 to HETEs/EETs generation remained unclear. Although the HETEs/EETs levels remained unchanged, renal CYP1B1 activity was decreased by TMS in rats co-treated with either vehicle or angiotensin II (Jennings et al., 2012b). TMS ameliorated angiotensin II-induced hypertension and associated renal injury in rats and mice (Jennings et al., 2010). TMS reduced the angiotensin II- and arachidonic acid-induced proliferation, migration, and hypertrophy of rat vascular smooth muscle cells (Jennings et al., 2012a, b; Malik et al., 2012). Although we could not exclude the CYP1B1-independent beneficial effect of TMS (Jennings et al., 2010), these reports suggest that an inhibitor of CYP1B1 can be the target for ameliorating hypertension and associated pathophysiological disorders. Single nucleotide polymorphyisms (SNPs) have been identified in the CYP1B1 gene. Among the CYP1B1 genotypes, CYP1B1*3 (CYP1B1V432L) was the most frequent variant in Asians (Leu/Leu: ~71%, classified as the wild type in the Human Cytochrome P450 (CYP) Allele Nomenclature Database) (Paracchini et al., 2007). In Caucasians, the distributions of Val/Val, Val/Leu, and Leu/Leu were roughly equal. To date, no conclusive association has been identified between CYP1B1*3 and breast cancer risk from either case–control studies or pooled and meta-analysis of published results (Bailey et al., 1998; Paracchini et al., 2007; Yao et al., 2010). In reconstituted systems of purified CYP1B1 variants, CYP1B1.3 and CYP1B1.4 (N453S) had about 3-fold higher catalytic efficiencies (kcat/Km) in estradiol 4-hydroxylation than the wild type CYP1B1.1 (Hanna et al., 2000). CYP1B1.3 and CYP1B1.4 had similar kcat values to CYP1B1.1, but lower Km values. However, in the yeast expression system, the intrinsic estradiol clearance (Vmax/Km) through either 2- or 4-hydroxylation was similar with all variants (Aklillu et al., 2002). In the production of benzo(a)pyrene 7,8-dihydrodiol from benzo(a)pyrene oxidation, CYP1B1.4 had lower clearance than CYP1B1.1, whereas the activity of CYP1B1.3 was similar to that of CYP1B1.1 (Aklillu et al., 2005). These results suggested that the CYP1B1 SNPs did not consistently influence activity toward structurally diverse substrates. The influence of SNPs on the susceptibility of CYP1B1 variants to inhibitors has not been reported. Protoberberine alkaloids represent a group of natural products with a core structure of dihydroisoquinolino[2,1-b]isoquinolin and have shown multiple pharmacological activities including anti-cancer and anti-inflammation effects (Grycova et al., 2007; Kulkarni and Dhir, 2010). Berberine, palmatine, and jatrorrhizine are protoberberines that have been identified in several medicinal plants including

C. chinensis and Berberis aristata. Berberine was the most abundant protoberberine in goldenseal (Hydrastis canadensis), which has been widely used as an immunostimulant in several countries including the USA (Douglas et al., 2010). Berberine has shown various pharmacological activities including anti-cancer, anti-inflammation and cholesterol-reducing effects (Li et al., 2011; Tang et al., 2009). In anesthetized normotensive rats, intravenous administration of 1 μg/kg berberine decreased the mean blood pressure (Kang et al., 2003). This hypotensive effect of berberine was attributed to the inhibition of angiotensin-converting enzyme and the stimulation of nitric oxide production. However, the potential involvement of CYP1B1 in the hypotensive effect of berberine has not been explored. The effect of berberine on CYP1B1 could be interesting. In a recombinant enzyme system, berberine inhibited CYP1A1 activity with a half-maximal inhibitory concentration value (IC50) of 2.5 μM (Vrzal et al., 2005). However, the kinetics of this CYP1A1 inhibition were not characterized. Although berberine (10 μM) was suggested to be able to inhibit CYP1A2 activity in CYP1A2 overexpressing Huh-7 cells (Chu et al., 2011; Zhao et al., 2012), its IC50 for inhibition of supersomes expressing human CYP1A2 was greater than 70 μM, much higher than the blood concentration of berberine in humans (Zeng and Zeng, 1999; Zhao et al., 2012). In volunteers taking a 3-times-per-day oral dose of 300 mg berberine for 2 weeks, the pharmacokinetic parameters of a CYP1A2 substrate caffeine were not significantly different from the values of the placebo control group (Guo et al., 2012), suggesting that berberine did not affect CYP1A2 activity in vivo. Therefore, to clarify the inhibitory effects of protoberberines on CYP1 members, we studied the selectivity and kinetic properties of the CYP1 inhibitory effects of berberine, palmatine, and jatrorrhizine. With the aid of molecular modeling, site-directed mutagenesis was performed to identify the crucial amino acid residue(s) responsible for the selective inhibition of CYP1 members. Materials and methods Chemicals and enzymes. Berberine chloride, caffeine, 1,7-dimethylxanthine, 7-ethoxycoumarin, 7-ethoxyresorufin, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, 7-hydroxycoumarin, 7methoxyresorufin, β-nicotinamide adenine dinucleotide phosphate (NADP+) sodium salt, and the reduced form of NADP+ (NADPH) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Palmatine and jatrorrhizine, which were isolated and purified from Fibraurea tinctoria as described before (Su et al., 2007). Preparation of bacterial membranes expressing human CYP1 enzymes, CYP1B1 variants, and CYP1A2 mutants. The constructs of wild type CYP1A1*1, CYP1A2*1, and CYP1B1*1 with N-terminal modification were generously provided by Dr. F. Peter Guengerich (Nashville, TN, USA) (Parikh et al., 1997; Shimada et al., 1998a). Bicistronic human constructs consisting of the P450 coding sequence, followed by that of NADPH-P450 reductase, were transformed into Escherichia coli DH5α by electroporation (Gene pulser II, Bio-Rad, Hercules, CA, USA). SNPs were introduced into the respective wild-type CYP1B1*1 or CYP1A2*1 cDNAs by using the primer-directed enzymatic amplification method, according to the manufacturer's instructions for the QuickChange Lightning site-directed mutagenesis kit (Stratagene, an Agilent Technologies Company, La, Jolla, CA, USA). Oligonucleotide primer sets used for the mutagenesis were designed with a primer containing a mutated base and the sequences of the mutated constructs were determined. Primers used were CYP1B1.3: forward, 5′-CCGGGTTAGGCCACTTCAGTGGGTC ATGATTCAC-3′ and reverse: 5′-GTGAATCATGACCCACTGAAGTGGCC TAACCCGG-3′; CYP1B1.4: forward: 5′-GGTCAGGTCCTTGCTGATGAGGC CATCC-3′ and reverse: 5′-GGATGGCCTCATCAGCAAGGACCTGACC-3′; CYP1B1N228T: forward, 5′-GTGAGCTGCTCAGCCACACCGAAGAGTTC-3′ and reverse, 5′-GAACTCTTCGGTGTGGCTGAGCAGCTCAC-3′; and

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CYP1A2T223N: forward, 5′-CAGCCTCGTGAAGAACAATCATGAGTTCGT GG-3′ and reverse, 5′-CCACGAACTCATGATTGTTCTTCACGAGGCTG-3′. Enzyme expression and membrane preparation were performed following the methods of Shimada et al. (1998a) and Parikh et al. (1997), respectively. For the expression of CYP1B1, bacteria were incubated in a Luria–Bertani medium containing 0.1% glucose and 100 μg/ml ampicillin. The overnight incubation was further incubated in the Terrific Broth medium containing 1 mM thiamine, a mixture of trace elements, 100 μg/ml ampicillin, 1 mM IPTG and 0.5 mM δ-amino levulinic acid. For the expression of CYP1A1 and CYP1A2, glucose and δ-amino levulinic acid were not included. E. coli carrying the constructs of CYP1 variants and mutants were incubated under the same condition as the respective wild type enzymes. The mean P450 expression yields of E. coli transformed with CYP1A1.1, CYP1A2.1, and CYP1B1.1 constructs were 4.1, 37.1, and 36.8 nmol P450/l culture, respectively. The mean P450 concentrations of CYP1A1.1, CYP1A2.1, and CYP1B1.1 membrane fractions were 1.90, 2.13, and 3.18 nmol/ml, respectively. Determination of CYP1 and NADPH-cytochrome c reductase activities. Membrane fraction of E. coli expressing individual human CYP1 enzymes was used for activity determination. P450 content was determined using the spectrophotometric methods (Omura and Sato, 1964). Benzo(a) pyrene hydroxylation (arylhydrocarbon hydroxylase, AHH) activity was determined by fluorometric determination following the method of Nebert and Gelboin (1968) and 3-hydroxybenzo(a)pyrene was used as the standard for quantification of the hydroxylation metabolites. Caffeine 3-demethylation activity was determined by HPLC analysis with the detection of absorbance at 280 nm (Lee et al., 1994). 7-Ethoxycoumarin O-deethylation (ECOD) activity was determined following the method of Greenlee and Poland (1978). 7-Ethoxyresorufin O-deethylation (EROD) and 7-methoxyresorufin O-demethylation (MROD) activities were determined by measuring the fluorescence of resorufin (Pohl and Fouts, 1980). Activities were determined using 100 pmol P450/ml in the caffeine 3-demethylation assay and 5 pmol/ml P450 in the other assays. Substrate concentrations used in the assays were 0.5 μM benzo(a)pyrene, 2 μM 7-ethoxyresorufin, 20 μM 7-methoxyresorufin, and 500 μM 7-ethoxycoumarin. The cytochrome c reduction activity of NADPH-P450 reductase (NADPHcytochrome c reductase) was determined following the method of Phillips and Langdon (1962). Berberine chloride, palmatine, and jatrorrhizine were dissolved in dimethylsulfoxide (DMSO). The final concentration of DMSO was 1% in the reaction mixtures with or without protoberberines. Intrinsic fluorescence measurements. Binding of berberine to P450 in membrane fractions was monitored by determining the decrease of intrinsic fluorescence intensity (fluorescence quenching) using a method modified from Liu et al. (2012), in which P450-expressed supersomes were used. CYP1B1 and CYP1B1N228T-expressed bacterial membrane fractions were diluted to 0.2 μM P450 using 0.1 M potassium phosphate buffer (pH 7.4). The emission fluorescence spectra of CYP1B1 and CYP1B1N228T were measured between 280 and 400 nm (excitation wavelength at 295 nm). The emission wavelength with maximal fluorescence intensity was used for the following determination of fluorescence quenching. Fluorescence intensity of bacterial membrane fractions expressing CYP1B1 and CYP1B1N228T was measured at 336 and 343 nm, respectively. The reduction of fluorescence intensity by the addition of increasing concentrations of berberine (I) was calculated. The dissociation constants (Kd) were estimated by nonlinear regression analysis (SigmaPlot software) according to the equation for single binding site: F0 − F = ΔFmaxI/(Kd + I), where F0 and F are the fluorescence intensities before and after the addition of berberine, respectively. ΔFmax is the maximal value of (F0–F) (Sampedro et al., 2007). The estimates of variance (denoted by ±) are presented from the analysis of individual sets of data.

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Computer modeling and berberine docking. The MODELER v.9.4 program in Discovery Studio v.3.5 (Accelrys Software Inc., USA) was used to generate a CYP1A1 homology model using the crystallographic structures of CYP1A2 (PDB ID: 2HI4) and CYP1B1 (PDB ID: 3PM0) as structural templates. Models of berberine in complex with CYP1 enzymes were generated through docking berberine to the active site of CYP1A1, CYP1A2 and CYP1B1 with the Leu432 variant (Fig. 4). In order to predict the position of berberine in the active site, we implemented the docking program (GOLD Genetic Optimization for Ligand Docking) (Cambridge Crystallographic Data Center (CCDC), version 3.1.1) with the Goldscore scoring function. Before docking, a 3D structure of berberine was extracted from the crystal structure of the multidrug binding transcriptional repressor QacR bound to the natural drug berberine (PDB ID: 1JUM). GOLD was used to dock berberine into the proteins with the flexible docking option turned on. Initially, 100 independent genetic algorithm cycles of computation were carried out with ligand torsion angles varying between −180° and 180°. The search efficiency was set at 200% to ensure the most exhaustive search for the docking conformational space. All other parameters were kept the same as the default settings. Finally, from the 100 docking conformations of berberine, the top one was chosen to explore the “inhibitor-bond” conformations in the CYP1 active site using Goldscore within the GOLD program. The molecular models of berberine were displayed using the PyMOL software (http://www.pymol.org). Data analysis. The concentrations of protoberberines required for 50% inhibition of catalytic activities (IC50) were calculated by curve fitting (start at 0, defined end, Grafit, Erithacus Software Ltd., Staines, UK). For competitive inhibition, kinetic analysis of P450 activity was done following Michaelis–Menten kinetic property. Vmax and I are the maximal velocity and protoberberine concentration, respectively. Ki and KI are the inhibitor constants for the binding of an inhibitor to P450 and P450-substrate complex, respectively. For noncompetitive inhibition, Ki is equivalent to KI. Values of velocities (v) at various substrate concentrations (S) were fitted by nonlinear least-squares regression without weight according to the Michaelis–Menten equation: competitive inhibition: v = Vmax · S/{S + Km[1 + (I/Ki)]}; noncompetitive inhibition: v = Vmax · S/ [1 + (I/Ki)][S + Km]; mixed type of inhibition: v = Vmax · S/{S[1 + (I/KI)] + Km[1 + (I/Ki)]} (Sigma Plot, Jandel Scientific, San Rafael, CA, USA). Estimates of variance and the coefficient of variation (CV, %) of the estimates of variance are presented from the analysis of individual sets of data. Results Inhibition of CYP1 activities by berberine, palmatine, and jatrorrhizine To study the inhibition of CYP1 enzymes by protoberberines (Scheme 1A), 7-ethoxyresorufin (Scheme 1B) was used as the common substrate of CYP1A1, CYP1A2, and CYP1B1. Wild type CYP1A1.1, CYP1A2.1, and CYP1B1.1 had EROD activities of 73.7 ± 3.7, 7.93 ± 1.1, and 12.5 ± 0.9 nmol/min/nmol P450, respectively (mean ± SE of 3–7 determinations). Among protoberberines, berberine caused the most potent inhibition of CYP1B1.1 activity with an IC50 value of 94 ± 8 nM, which was 8% and less than 0.2% of those for CYP1A1.1 (1.38 ± 0.12 μM) and CYP1A2.1 (N60 μM) inhibition, respectively (Fig. 1). To confirm the lack of potent inhibition of CYP1A2 by berberine, effect on CYP1A2 activity was further examined using two marker substrates, caffeine and 7-methoxyresorufin. Berberine decreased the caffeine 3-demethylation and MROD activities of CYP1A2 with IC50 values higher than 60 μM (Supplementary Fig. 1). This result indicated that berberine selectively inhibited CYP1B1.1. Palmatine and jatrorrhizine did not show this selectivity for CYP1B1 inhibition. Palmatine preferentially inhibited CYP1A1.1 activity with an IC50 value of 8.71 ± 0.55 μM, which was lower than that (37.2 ± 4.2 μM) for CYP1B1.1 inhibition. Jatrorrhizine inhibited CYP1A1.1 and CYP1B1.1

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1 O

A

2

14a

O3

14 12 11

12a

13

13b 13a

4 4a

10 H3 CO

3a

8a

9

8

OCH3

N 7

+

5 6

Berberine OCH3

OCH3

OH

OCH3

N

H3CO OCH3

B

+

N

H3 CO OCH3

Palmatine

O

O

O

O

+

Jatrorrhizine

O

O

N

7-Ethoxycoumarin

O

O

7-Ethoxyresorufin

O

N

7-Methoxyresorufin

Benzo(a)pyrene

Scheme 1. Chemical structures of protoberberines (A) and the substrates of CYP1B1 (B).

activities to similar extents. The IC50 values of jatrorrhizine for the inhibition of CYP1A1.1 and CYP1B1.1 activities were 2.17 ± 0.08 and 1.71 ± 0.08 μM, respectively. However, the decrease in CYP1A2.1 activity was less than 30% when the protoberberine concentration was increased up to 60 μM (IC50 N 60 μM). These results indicated preferential inhibition of CYP1A1 and CYP1B1 activities by protoberberines, with CYP1B1.1 activity most inhibited by berberine. CYP1A2-catalyzed EROD activity was barely inhibited by the protoberberines tested. Thus, the kinetic properties of CYP1A1 and CYP1B1 inhibition by protoberberines were studied.

jatrorrhizine caused a mixed type of inhibition (Fig. 2). The Ki value of berberine-mediated CYP1A1.1 inhibition was lower than those of palmatine and jatrorrhizine (Ki and KI, Table 1). Berberine, palmatine, and jatrorrhizine also showed different CYP1B1.1inhibitory behaviors (Fig. 3). Berberine noncompetitively inhibited CYP1B1.1 activity with a low Ki value (Table 1), while its Ki for CYP1A1 inhibition was 15-fold higher. However, palmatine and jatrorrhizine caused competitive and mixed type of inhibition, respectively. These protoberberines showed differential inhibitory kinetics for the inhibition of CYP1A1.1 and CYP1B1.1.

Kinetic analysis of the inhibition of CYP1A1.1 and CYP1B1.1 by berberine, palmatine, and jatrorrhizine

Docking of berberine into CYP1A1, CYP1A2, and CYP1B1

Kinetic analysis was performed using non-linear regression. The coefficient of variation of the estimates of variance generated from kinetic analyses of all sets of data was 13 ± 9% (mean ± SD). The correlation coefficients (r) were 0.970–0.999. CYP1A1-catalyzed EROD activity had Km and Vmax values of 2.08 ± 0.58 μM and 204.9 ± 29.3 nmol/min/nmol P450, respectively (mean ± SE of 3 individual sets of data). CYP1B1-catalyzed EROD activity had Km and Vmax values of 0.48 ± 0.12 μM and 16.8 ± 3.1 nmol/min/nmol P450, respectively (mean ± SE of 3 individual sets of data). Berberine caused a noncompetitive inhibition of CYP1A1.1, whereas palmatine and

To help the identification of crucial amino acid residue(s) for the selective inhibition of CYP1B1, computer modeling of the binding of berberine to CYP1 enzymes was performed (Fig. 4). The dockings of berberine to the active sites of CYP1 members had the GOLD fitness (score) in the order of CYP1B1 (63.78) N CYP1A1 (58.89) N CYP1A2 (46.64). The binding of berberine to CYP1 with a higher score had a lower IC50 value in activity inhibition (Fig. 1). The methylenedioxy ring of berberine located closely to the heme moiety of all CYP1 members. Berberine formed π–π interaction with Phe231 in CYP1B1, Phe224 in CYP1A1, and Phe226 in CYP1A2. There was no obvious difference between CYP1 members in forming hydrophobic interactions with berberine.

S.-N. Lo et al. / Toxicology and Applied Pharmacology 272 (2013) 671–680

A

80 60

CYP1A1 CYP1A2 CYP1B1

0 0.001

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B

40 20 0 0.001

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120 100

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100

Jatrorrhizine, µM Fig. 1. Inhibition of human CYP1A1.1 (○)-, CYP1A2.1 (▲)-, and CYP1B1.1 (●)-catalyzed 7-ethoxyresorufin O-deethylation activities by the protoberberines, berberine, palmatine, and jatrorrhizine. Data represent the mean and mean ± SE of 2 and 3–5 determinations, respectively.

However, in CYP1B1, the methoxy moieties of berberine were positioned near Asn228 and Gln332 (Fig. 4A). The corresponding amino acid residues were Asn221 and Phe319 in CYP1A1 (Fig. 4B) and Thr223 and Phe319 in CYP1A2 (Fig. 4C). Asn and Gln were predicted to form hydrogen-bonding interactions with 2 methoxy moieties of berberine. Although Thr has a hydroxyl side chain, the crystallographic structure of CYP1A2 (PDB ID: 2HI4) showed that this side chain was oriented in an unfavorable direction for the hydrogen-bonding interaction of Thr223 with berberine. The phenyl group of Phe319 of CYP1A1 and CYP1A2 was oriented away from the isoquinoline moiety and did not form a π–π interaction with berberine. Thus, the methoxy moieties of berberine formed two and one hydrogen-bonding interactions with CYP1B1 and CYP1A1, respectively. However, there was no hydrogen bond formation between the methoxy moieties of berberine and CYP1A2. These docking results suggested that Asn228 and Gln332 might be of significance for the selective inhibition of CYP1B1 by berberine. Inhibition of CYP1B1.1-catalyzed oxidations of structurally diverse substrates by berberine To examine the inhibitory effects of berberine on CYP1B1.1 activities with structurally diverse substrates, its oxidation activities toward 7-ethoxycoumarin, 7-methoxyresorufin, and benzo(a)pyrene (Scheme 1B) were determined. The inhibition of these activities

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2

4 6 8 10 12 1/S, µM-1

Fig. 2. Kinetic analysis of the inhibition of 7-ethoxyresorufin O-deethylation activity by berberine (upper panel), palmatine (middle panel), and jatrorrhizine (bottom panel) in a recombinant CYP1A1.1 system. (A), (C), and (E) show the velocity (v) versus 7ethoxyresorufin concentration (S) plots of activity in the presence of various concentrations of protoberberines as indicated. (B), (D), and (F) show the Lineweaver–Burk plots. Data represent the mean ± SE of 3 determinations. Solid lines represent the lines of the best fits obtained from the non-linear regression as described in the Materials and methods section.

was compared to that of EROD activity. The mean ECOD and MROD activities of CYP1B1.1 were 6.69 and 4.23 nmol/min/nmol P450, respectively. Like 7-ethoxyresorufin, 7-ethoxycoumarin and 7-methoxyresorufin (Scheme 1B) have oxygen atoms in the polycyclic rings and side chain, which might form hydrogen-bonding interactions with CYP1 (Lewis et al., 1999). However, benzo(a)pyrene did not exhibit this property. Berberine inhibited ECOD and MROD activities with IC50 values of 251 ± 38 and 43 ± 5 nM, respectively (Fig. 5A). The IC50 values were lower than the respective substrate concentrations. The AHH activities at 0.5 and 1 μM benzo(a)pyrene were 4.06 ± 0.31 and 5.51 ± 0.65 nmol/min/nmol P450, respectively. Berberine inhibited AHH activity with an IC50 value of 33.9 ± 1.7 μM when 0.5 μM benzo(a)pyrene was used in the activity determination. The IC50 value was 68-fold

S.-N. Lo et al. / Toxicology and Applied Pharmacology 272 (2013) 671–680

CYP1B1.1

Noncompetitive

Noncompetitive

Palmatine

Jatrorrhizine

Ki, nM

679 ± 106

44 ± 16

Type of inhibition

Mixed

Competitive

Ki, μM KI, μM

12.77 ± 1.33 6.48 ± 3.03

5.64 ± 0.41 –a

Type of inhibition

Mixed

Mixed

Ki, μM KI, μM

4.98 ± 0.39 3.20 ± 0.73

0.47 ± 0.05 9.93 ± 3.86

Activity determination was performed in the assays with 3 determinations. Data show the mean ± SE of the inhibitory constants obtained from the non-linear regression analysis of velocity versus substrate concentration plots of assays with 3 different inhibitor concentrations. a Competitive inhibition does not include this item.

higher than the benzo(a)pyrene concentration in the assay. When the benzo(a)pyrene concentration was increased to 1 μM, berberine had an IC50 value higher than 60 μM (data not shown). Compared to 7-ethoxyresorufin, 7-ethoxycoumarin, and 7-methoxyresorufin, benzo(a)pyrene did not have the alkoxyl side chain and the hydroxylation of benzo(a)pyrene was less susceptible to the inhibitory effect of berberine. These results revealed the differential inhibitory effects of berberine on CYP1B1.1 activities toward structurally diverse substrates. Inhibitory effects of berberine on CYP1B1.3 and CYP1B1.4 variants

12

The inhibition of CYP1A2T223N and CYP1B1N228T by berberine Based on the docking results, CYP1 members showed differences in hydrogen-bonding interactions with 2 methoxy moieties of berberine. In addition, we chose not to make radical changes to the size and polarity of amino acid residues (i.e. Gln332 of CYP1B1 and Phe319 of CYP1A1 and CYP1A2) because a detectable EROD activity in the CYP1 mutant was essential for the inhibition studies. Thus, we focused on Asn228 of berberine-sensitive CYP1B1, which corresponded to Thr223 of berberine-resistant CYP1A2. To confirm the importance of Asn228 for selective CYP1B1 inhibition, we conducted side-directed single-point mutation to obtain CYP1B1N228T and CYP1A2T223N mutants and the effects of berberine on the EROD activities of these mutants were determined. It was remarkable that the N228T mutation decreased the CYP1B1 expression yield and activity (as described above) to 0.438 nmol P450/l culture and 0.72 ± 0.01 nmol/min/nmol P450 (mean ± SE of 3 determinations), respectively. CYP1B1N228T showed resistance to the inhibitory effect of berberine (Fig. 5C). In contrast, the expression yield and EROD activity of CYP1A2T223N mutant were not lower than the respective values for CYP1A2.1. CYP1A2T223N was expressed in E. coli with a yield of 24.9 nmol P450/l culture and its EROD activity was 16.7 ± 0.8 nmol/min/nmol P450 (mean ± SE of 5 determinations). The IC50 value of berberine was reduced from N60 μM for CYP1A2.1 inhibition to 45.8 ± 7.2 μM for CYP1A2T223N

0 µM 0.05 µM 0.1 µM 0.3 µM

10 8

10 8

6

6

4

4

2

2

0

0 0.0 0.5 1.0 1.5 2.0 2.5

-2 0

2

7-Ethoxyresorufin, µM

Palmatine

C 25 20

4 6 8 10 12 1/S, µM-1

D 2.5

0 µM 10 µM 20 µM 50 µM

0 µM 10 µM 20 µM 50 µM

2.0 1.5

15 1.0

10 0.5 5

0.0 -0.5

0 0.0 0.5 1.0 1.5 2.0 2.5

-2 0 2 4 6 8 10 12 1/S, µM-1

7-Ethoxyresorufin, µM

Jatrorrhizine

E 7-Ethoxyresorufin O-deethylation v, nmol/min/nmol P450

14

To determine the inhibitory effects of berberine on CYP1B1 variants, bacterial membranes expressing CYP1B1.3 and CYP1B1.4 were prepared and the inhibition of EROD activity was studied. The EROD activities of CYP1B1.3 and CYP1B1.4 were 7.9 ± 1.6 and 16.7 ± 2.1 nmol/min/nmol P450 (mean ± SE of 3–4 determinations), respectively. CYP1B1.3 and CYP1B1.4 had slightly lower and higher EROD activities than wild type CYP1B1.1 (12.5 ± 0.9 nmol/min/nmol P450), respectively. Berberine inhibited CYP1B1.3- and CYP1B1.4-catalyzed EROD activities with IC50 values of 71 ± 4 and 65 ± 7 nM, respectively (Fig. 5B). These values were close to that for CYP1B1.1 inhibition.

12

1/v

CYP1A1.1

Type of inhibition

14

B

0 µM 0.05 µM 0.1 µM 0.3 µM

1/v

Inhibition

Berberine

16

12 10

0 1 3 5

F 3.0

µM µM µM µM

8

2.0 1.5

6

1.0

4

0.5

2

0.0

0 0.0 0.5 1.0 1.5 2.0 7-Ethoxyresorufin, µM

0 µM 1 µM 3 µM 5 µM

2.5

1/v

Protoberberines

Berberine

A 7-Ethoxyresorufin O-deethylation v, nmol/min/nmol P450

Table 1 Inhibitory kinetic parameters for the inhibition of CYP1A1.1- and CYP1B1.1-catalyzed 7ethoxyresorufin O-deethylation activity by protoberberines.

7-Ethoxyresorufin O-deethylation v, nmol/min/nmol P450

676

-0.5 -2 0

2

4 6 8 10 12 1/S, µM-1

Fig. 3. Kinetic analysis of the inhibition of 7-ethoxyresorufin O-deethylation activity by berberine (upper panel), palmatine (middle panel), and jatrorrhizine (bottom panel) in a recombinant CYP1B1.1 system. (A), (C), and (E) show the velocity (v) versus 7-ethoxyresorufin concentration (S) plots of activity in the presence of various concentrations of protoberberines as indicated. (B), (D), and (F) show the Lineweaver– Burk plots. Data represent the mean ± SE of 3 determinations. Solid lines represent the lines of the best fits obtained from the non-linear regression as described in the Materials and methods section.

inhibition (Fig. 5C), revealing that the inhibitory effect of berberine on CYP1A2 was enhanced by the mutation of Thr223 to Asn. Thus, these data demonstrated that Asn228 in CYP1B1 was an important residue for berberine-mediated inhibition. Quenching of CYP1B1.1 and CYP1B1N228T fluorescence with berberine Upon excitation at 295 nm, bacterial membranes expressing recombinant CYP1B1.1 and CYP1B1N228T had the intrinsic fluorescence emission peaks at 336 nm and 343 nm, respectively (Fig. 6A). N228T mutation caused a 7-nm red shift. The dissociation constant (Kd) was calculated by determining the fluorescence quenching, presumably reflecting the binding of berberine to P450. The plots of fluorescence quenching versus berberine concentrations were hyperbolic (Fig. 6B).

S.-N. Lo et al. / Toxicology and Applied Pharmacology 272 (2013) 671–680

677

Fig. 4. The docking results of berberine with CYP1B1 (A), CYP1A1 (B), and CYP1A2 (C). Molecular modeling of the binding of berberine (yellow sticks) to the putative active site of CYP1 was performed as described in the Materials and methods section. The left and right pictures of each panel display the 3-D and 2-D structural docking patterns, respectively. The nitrogen and oxygen atoms are shown in dark blue and red colors, respectively. The heme prosthetic group of P450 is shown in magenta color with an iron colored white. The hydrogen bond formation and the electrostatic interaction between berberine and the amino acid residues of apoprotein of CYP1 are shown in green and light blue dashed lines, respectively. In the left picture, the rings with the same color show a group of moieties contacted with the type of interaction as indicated.

The apparent Kd values for the interaction of berberine with CYP1B1.1 and CYP1B1N228T were estimated to be 14.6 ± 1.8 and 26.3 ± 8.0 μM, respectively. The binding to CYP1B1N228T had a higher Kd value than CYP1B1.1, suggesting that N228T mutation reduced the binding affinity of berberine to CYP1B1. Discussion Berberine has been reported to inhibit CYP2D6 and CYP3A4 activities with respective IC50 values of approximately 45 and 400 μM in

human liver microsomes (Chatterjee and Franklin, 2003). Berberine at a concentration of up to 100 μM caused no more than a 50% decrease in CYP1A2, CYP2C9, CYP2E1, CYP3A4 CYP2C8, or CYP2C19 activity in human liver microsomes (Chatterjee and Franklin, 2003; Han et al., 2011). In yeast supersomal systems expressing human P450 and NADPH-P450 reductase, berberine inhibited CYP2D6 and CYP3A4 activities with IC50 values of 7.4 and 48.9 μM, respectively (Zhao et al., 2012). As described in the report of Zhao et al. (2012), an oral dose of 300 mg berberine generated a maximal plasma concentration (Cmax) of 0.39 μg/ml (1.16 μM) in healthy volunteers. In patients with

S.-N. Lo et al. / Toxicology and Applied Pharmacology 272 (2013) 671–680

A

CYP1B1.1 activity % of control

100

800

EROD ECOD MROD AHH

Fluorescence unit

120

80 60 40 20

A

800

Fluorescence quenching, F0-F

678

CYP1B1.1 CYP1B1 CYP1B1NT

600

400

200

CYP1B1N228T

0

7-Ethoxyresorufin O-deethylation % of control

0.01 120

B

100

1

10

100 0 300

CYP1B1.1 CYP1B1.3 CYP1B1.4

CYP1B1 CYP1B1N228T

600

400

200

0 350

Wavelength, nm

400

0

10 20 30 40 50 60

Berberine, µM

Fig. 6. CYP1B1.1 and CYP1B1N228T fluorescence quenching by berberine binding in recombinant enzyme systems. (A) Fluorescence spectra of bacterial membranes expressing CYP1B1.1 and CYP1B1N228T. Fluorescence was measured at an excitation wavelength of 295 nm. The wavelength of maximal fluorescence emission was 336 nm and 343 nm for CYP1B1.1 and CYP1B1N228T, respectively. (B) The plots of CYP1B1.1 and CYP1B1N228T fluorescence changes versus beberine concentrations. Fluorescence was measured in the absence (F0) and presence (F) of increasing concentrations of berberine. The solid lines show the results of fitting the data by nonlinear regression.

80 60 40 20 0 0.01

140

7-EthoxyresorufinO-deethylation % of control

0.1

B

0.1

1

10

100

1

10

100

C

120 100 80 60

1B1.1 1A2.1 1B1N228T 1A2T223N

40 20 0 0.01

0.1

Berberine, µM Fig. 5. Inhibitory effects of berberine on activities of CYP1B1.1 toward structural diverse substrates (A) and on 7-ethoxyresorufin O-deethylation (EROD) activities of CYP1B1 variants (B) and CYP1A2T223N (●) and CYP1B1N228T (○) mutants (C). CYP1B1.1-catalyzed 7-ethoxycoumarin O-deethylation (ECOD), 7-methoxyresorufin O-demethylation (MROD), and benzo(a)pyrene hydroxylation (AHH) activities were determined. The variants CYP1B1.3 and CYP1B1.4 as well as the mutants were expressed in E. coli DH5α and EROD activity was determined. Data represent the mean and mean ± SE of 2 and 3–4 determinations, respectively. For the purpose of comparison, the decreases of the EROD activity of either CYP1A2.1 or CYP1B1.1 is shown in each figure using a dashed line.

congestive heart failure, the mean values of groups with high (N 296 nM, 31 patients) and low (b 296 nM, 25 patients) plasma berberine concentrations was 188 and 511 nM after a 2-week treatment with an daily oral dose of 1.2 g berberine, respectively (Zeng and Zeng, 1999). In rats treated with 3 g/kg Xiexin decoction, which contained Rhei Rhizoma, Raqdix Scutellaria, and Coptidis Rhizoma (the roots of Coptidis chinensis), the plasma Cmax of berberine, palmatine, and jatrorrhizine were 0.03, 0.01, and 0.01 μM, respectively (Zan et al., 2011). Because the IC50 values of berberine for the inhibition of these P450 enzymes were greater than the plasma concentrations described above, it would be difficult for berberine to cause inhibition of these enzymes in the clinical situation. In agreement with previous reports (Guo et al., 2012; Zhao et al., 2012), our findings also showed that CYP1A2 was resistant to inhibition by berberine, palmatine, and jatrorrhizine. Our findings revealed that berberine selectively inhibited CYP1B1 with an IC50 value that

was more than 10-fold lower than that for CYP1A1 inhibition. Compared with berberine, palmatine and jatrorrhizine showed less selectivity between CYP1A1 and CYP1B1 inhibition. Kinetic analyses revealed that these protoberberines showed differential inhibitory kinetic behaviors. Berberine appeared to have the least Ki value (44 nM) for the inhibition of CYP1B1 through noncompetitive inhibition, indicating that it bound to both CYP1B1 and CYP1B1-7ethoxyresorufin complex. Compared with the synthetic stilbene derivative 2,3′,4,5′-tetramethoxystilbene (Ki = 3 nM) (Chun et al., 2001), the Ki value of berberine was relatively high. However, the IC50 and Ki values of berberine for the inhibition of CYP1B1 were within or close to the range of blood concentrations in humans and rats taking berberine or berberine-containing decoction (Zan et al., 2011; Zeng and Zeng, 1999; Zhao et al., 2012). It is of interest to further evaluate the benefit of berberine through CYP1B1 inhibition in vivo and the involvement of CYP1B1 in the anti-hypertensive effect of berberine, such as the production of HETEs/EETs. Hydrogen-bonding and π–π interactions were proposed to be important in the binding of alkoxy derivatives of heterocyclic compounds to CYP1 enzymes (Lewis et al., 1999). Compared with the alkoxy polycyclic substrates, benzo(a)pyrene showed higher hydrophobicity and did not exhibit any moieties suitable for the formation of hydrogen bonds. The lack of tendency to form hydrogen-bonding interactions may move benzo(a)pyrene to a binding site away from the berberinebinding site, which renders it resistant to inhibition. However, docking analysis of the binding of benzo(a)pyrene to CYP1B1 has not been resolved. Other factors affecting the inhibitory effect of berberine on AHH activity remain unclear. Using site-directed mutagenesis in conjunction with molecular modeling, our findings supported the importance of Asn228 on CYP1B1 for berberine-mediated inhibition, corresponding to Asn221 on CYP1A1 and Thr223 on CYP1A2. The side chain of Asn has one keto and one amine group, which are available for the formation of 2 hydrogen bonds. However, Thr had only one oxygen atom in the hydroxyl moiety of the side chain. Asn228, Asn221, and Thr223 were all positioned near the methoxy moiety of berberine and Asp on P450 as indicated in Fig. 4. In CYP1A2, Thr223 formed a hydrogen bond with Asp320 (Wang et al., 2011), while Asn228 (CYP1B1) and Asn221 (CYP1A1) could interact with both the Asp nearby and the methoxy moiety of berberine. Results of the binding-induced fluorescence quenching indicated that mutation of Asn228 in CYP1B1 to Thr reduced the binding affinity of berberine. The present study indicated that the Gln332 of CYP1B1 formed an additional hydrogen-bonding

S.-N. Lo et al. / Toxicology and Applied Pharmacology 272 (2013) 671–680

interaction with berberine and may therefore increase the inhibitory susceptibility of CYP1B1. α-Naphthoflavone is known as a potent but nonselective inhibitor of CYP1A2 (IC50: 6 nM) and CYP1B1 (IC50: 5 nM) (Shimada et al., 1998b). The predicted berberinebinding was superimposed with the crystallographic structure (PDB ID: 2HI4) of P450 with α-naphthoflavone (Supplementary Fig. 2). The oxygens of the pyran-4-one moiety in α-naphthoflavone could form hydrogen bond interactions with both CYP1A2 and CYP1B1. In contrast, more hydrogen bond interactions were predicted to be formed between the methoxy moieties of berberine and CYP1B1, which may be associated with the preferential CYP1B1 inhibition by berberine. Li et al. (2011) proposed that the binding of berberine to CYP1A2 showed a reasonable docking score and berberine appeared to be a CYP1A2 substrate. Although berberine was a CYP1A2 substrate, our study revealed that it was not a potent inhibitor of CYP1A2. The CYP1B1.3 and CYP1B1.4 variants had mutated amino acids that were at positions away from the berberine-binding site (Supplementary Fig. 3). The differences between IC50 values for berberine-mediated inhibition of CYP1B1.1, CYP1B1.3 and CYP1B1.4 were less than 40%. Because of a difference of over 2-fold has been generally considered to show clinical significance, these variants might have little influence on the potency of berberine inhibition. In conclusion, our study showed that berberine preferentially inhibited the extrahepatic CYP1B1.1 and its variants CYP1B1.3 and CYP1B1.4. The formation of a hydrogen bond between Asn228 and berberine was crucial for berberine-mediated CYP1B1 inhibition. Activities toward structurally diverse substrates may show distinct susceptibilities to berberine-mediated inhibition. The selective CYP1B1 inhibition reduced the chance to cause interaction with drug substrates, which were mainly metabolized by hepatic P450s. The clinical benefits of berberine in reducing CYP1B1-activated genotoxicity or other pathological effects, including those on hypertension, provide interesting venues for future investigations. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.taap.2013.07.005.

Conflict of interest The authors declare that there are no conflicts of interest.

Acknowledgments Mr. S.-N. Lo was a graduate student, who did the majority of this P450 study supervised by Dr. Y.-F. Ueng. This work has been supported partly by a grant NSC101-2320-B-077-001-MY3 from National Science Council, Taipei and partly by National Research Institute of Chinese Medicine, Taipei. We are grateful to the National Center for HighPerformance Computing for allowing us to use their computer facilities.

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