The prenylated phenolic natural product isoglycycoumarin is a highly selective probe for human cytochrome P450 2A6

Accepted Manuscript The prenylated phenolic natural product isoglycycoumarin is a highly selective probe for human cytochrome P450 2A6

Qi Wang, Yi Kuang, Junbin He, Kai Li, Wei Song, Hongwei Jin, Xue Qiao, Min Ye PII: DOI: Reference:

S0928-0987(17)30491-8 doi: 10.1016/j.ejps.2017.08.035 PHASCI 4193

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

9 March 2017 7 July 2017 30 August 2017

Please cite this article as: Qi Wang, Yi Kuang, Junbin He, Kai Li, Wei Song, Hongwei Jin, Xue Qiao, Min Ye , The prenylated phenolic natural product isoglycycoumarin is a highly selective probe for human cytochrome P450 2A6, European Journal of Pharmaceutical Sciences (2017), doi: 10.1016/j.ejps.2017.08.035

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ACCEPTED MANUSCRIPT The prenylated phenolic natural product isoglycycoumarin is a highly selective probe for human cytochrome P450 2A6

Qi Wang,ab Yi Kuang,b Junbin He,b Kai Li,b Wei Song,b Hongwei Jin,b Xue Qiao,*b and

Department of Medicinal Chemistry and Natural Medicine Chemistry, College of

SC

a

RI

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Min Ye*b

Pharmacy, Harbin Medical University, Baojian Road 157, Nangang District, Harbin

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical

MA

b

NU

150081, China

Corresponding authors. Tel.: +86 10 82801516. Fax: +86 10 82802024. Email

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*

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Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, China

AC

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address: [email protected] (X. Qiao), [email protected] (M. Ye).

1

ACCEPTED MANUSCRIPT Abstract Prenylated phenolic compounds are an important class of bioactive natural products. One major in vivo metabolic pathway of these compounds is hydroxylation at terminal methyl of the isoprenyl group. This study aims to identify the P450

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isozyme catalyzing this metabolic reaction. In human liver microsomes, 16 out of 24

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screened compounds could be metabolized into their hydroxylated derivatives.

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Chemical inhibition assays using 11 isozyme specific inhibitors indicated the hydroxylation reactions of 12 compounds were primarily catalyzed by cytochrome

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P450 2A6 (CYP2A6). In particular, CYP2A6 was the major enzyme participating in

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the metabolism of isoglycycoumarin (IGCM). The product of IGCM was obtained and identified as licopyranocoumarin (4-hydroxyl isoglycycoumarin) using NMR

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spectroscopic analysis. The Km values for human liver microsomes and recombinant

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human CYP2A6 were 7.98 and 10.14 M, respectively. According to molecular docking analysis, the catalytic mechanism may involve cyclized isoprenyl group of

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IGCM entering the active cavity of CYP2A6. These results demonstrate that IGCM

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could serve as an ideal isozyme selective probe to evaluate CYP2A6 activities.

Keywords: Isoglycycoumarin; Cytochrome P450 2A6; Isozyme selective probe; Hydroxylation metabolism; Bioactive natural products; Prenylated phenolic compounds

2

ACCEPTED MANUSCRIPT 1. Introduction Prenylated phenolic compounds are important bioactive constituents of many medicinal plants. These natural products show significant biological activities, including antioxidant, antiviral, antitumor, antidiabetic, antibacterial, antifungal,

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estrogenic, and antiplasmodial activities [1,2]. Hydroxylation is one major in vivo

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metabolic reaction for prenylated phenolic compounds, and it mainly occurs

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regio-selectively at the terminal methyl of the isoprenyl group [3,4]. Our previous study indicated the hydroxylation reaction could be catalyzed by human liver

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microsomes, which mainly contain cytochrome P450 (CYP) enzymes [5-7]. CYP

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represents a big family of heme-containing enzymes belonging to the group of monooxygenases [8,9]. The human CYP superfamily contains 57 functional genes and

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58 pseudogenes, which are involved in the metabolism of many xenobiotic

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compounds such as drugs [10,11]. However, the specific CYP isoform catalyzing the hydroxylation of prenylated phenolics is still unknown. Thus far, only Guo et al.

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studied the P450 enzymes that metabolize isoxanthohumol and 8-prenylnaringenin,

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two prenylated flavonoids from hops [12]. Their hydroxylation reaction is catalyzed by CYP2C19 and CYP2C8, respectively.

CYP2A6 is an important hepatic phase I enzyme that metabolizes approximately 3% of therapeutic drugs, including pilocarpine, valproic acid, and tanshinone IIA [13-16]. It is responsible for 70-80% of the initial metabolism of nicotine and has been proposed to be a novel target for smoking cessation [17,18].

3

ACCEPTED MANUSCRIPT Environmental toxicants (e.g., gasoline additives) and many procarcinogens (e.g., nitrosamines and aflatoxin B1) are also substrates of CYP2A6 [14]. CYP2A6 is a highly

polymorphic

gene

with

at

least

45

allelic

variants

(http://www.cypalleles.ki.se/cyp2a6.htm). Many of these variants could remarkably

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modify the expression, activity, function and stability of the CYP2A6 enzyme (20-

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to >100-fold). Polymorphism of CYP2A6 may be associated with smoking behavior,

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drug clearance, and lung cancer risk [15]. Therefore, the discovery of probe substrates as a marker reaction for measuring catalytic activity of CYP2A6 is critically

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important. Coumarin has been used as a probe substrate for CYP2A6, and is

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metabolized to 7-hydroxycoumarin [14]. However, specificity of this reaction is

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compromised by the concurrent formation of 4-, 5-, 6- and 8-hydroxylated products.

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In the present work, we report that CYP2A6 is the predominant isozyme involved in the regio-selective hydroxylation of a series of prenylated phenolic

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compounds in human liver microsomes. Chemical inhibition assays were conducted to

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confirm the key role of CYP2A6. Furthermore, isoglycycoumarin (IGCM) as an isoform-specific probe for CYP2A6 was discovered, and the catalytic mechanism was interpreted via molecular docking.

2. Materials and methods 2.1. Chemicals and reagents Compounds

1-21,

isoglycycoumarin

4

(IGCM),

glycyrin

(GLN)

and

ACCEPTED MANUSCRIPT glycycoumarin (GCM) were isolated from Glycyrrhiza uralensis Fisch. by the authors. Their structures were fully characterized via NMR spectroscopy and mass spectrometry. The purities were above 98% as determined via HPLC/UV analysis. 1-Aminobenzotriazole (ABT) and pilocarpine were obtained from Santa Cruz

PT

Biotechnology (Santa Cruz, California, USA) and Abcam Biochemicals (Cambridge,

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United Kingdom), respectively. Montelukast was from Energy Chemical Ltd.

SC

(Shanghai, China). CYP3cide and clomethiazole were purchased from Toronto Research Chemical Inc. (Toronto, Ontario, Canada). Sulfaphenazole and furafylline

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were from Cayman Chemical Company (Ann Arbor, Michigan, USA). 8-Methoxsalen,

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omeprazole, quinidine, and ketoconazole were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade acetonitrile, methanol and formic acid were from

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Mallinkrodt Baker Inc. (Phillipsburg, NJ, USA). Ultra-pure water was prepared using

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a Milli-Q water purification system (Millipore, Billerica, MA, USA). The other reagents were of analytical grade. β-Nicotinamide adenine dinucleotide phosphate (β-NADP),

D-glucose

6-phosphate

sodium

salt

(G-6-P),

and

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hydrate

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glucose-6-phosphate dehydrogenase (G-6-P-DE) were purchased from Sigma-Aldrich (St. Louis, MO, USA). cDNA-expressed recombinant human CYP2A6 derived from baculovirus-infected insect cells (BTI-TN-5B1-4), co-expressing NADPH-CYP reductase and cytochrome b5, was obtained from BD Biosciences (MA, USA). Pooled human liver microsomes prepared from male Asians (20 donors) was obtained from iPhase Pharmaceutical Services (Beijing, China).

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ACCEPTED MANUSCRIPT 2.2. Human liver microsomes incubation The human liver microsome incubation experiments were conducted according to our optimized conditions (Fig. S1). The incubation mixture consisted of a nicotinamide adenine dinucleotide phosphate-oxidase (NADPH)-generating system (2

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mM NADP+, 8 mM glucose-6-phosphate, 3 U/ml of glucose-6-phosphate

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dehydrogenase, and 6 mM MgCl2), 100 mM potassium phosphate buffer (pH 7.4),

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and human liver microsomes or CYPs. All the analytes were dissolved in methanol and then diluted with PBS. The final concentration of the analytes in the 200 µL

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incubation mixture was 25 µM. The total amount of organic solvent was lower than 1%

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(v/v). PBS-containing methanol was used as the blank control. The reaction was initiated by adding the NADPH-generating system and further incubated at 37 °C in a

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shaking water bath. This reaction was terminated by 1000 μL of cold acetonitrile at

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4 °C after 1 h. For blank control samples, PBS was added instead of NADPH. The mixture was stored at 4 °C for 30 min, and the precipitated protein was removed via

AC

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centrifugation (10,000 g for 10 min at 4 °C).

2.3. Chemical inhibition assay Hydroxylation of the substrates in the pooled HLM in the absence or presence of selective inhibitors for different CYP isoforms was measured to identify the involved enzyme(s). In brief, the analyte (25 μM, relevant to the Km values) was incubated in HLM (0.4 mg protein/ml) using the NADPH-generating system in the absence (control) or presence of known CYP isoform-specific inhibitors/substrates. The

6

ACCEPTED MANUSCRIPT selective inhibitors and their concentrations (one, three, and ten times the IC50 values) were as follows: montelukast (0.03, 0.09, 0.3 µM) for CYP2C8 [19], sulfaphenazole (0.25, 0.75, 2.5 µM) for CYP2C9, quinidine (0.11, 0.33, 1.1 µM) for CYP2D6, ketoconazole (0.03, 0.09, 0.3 µM) for CYP3A, furafylline (5.6, 10, 56 µM) for

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CYP1A2 [20], omeprazole (5.4, 20, 54 µM) for CYP2C19 [21], clomethiazole (2, 29,

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50 µM) for CYP2E1 [22,23], CYP3cide (0.16, 0.48, 1 µM) for CYP3A4 [24],

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8-methoxsalen (0.34, 1.02, 2.5 µM) for CYP2A6 [25], pilocarpine (5.6, 10, 56 µM) for CYP2A6 [26,27], ticlopidine (0.32, 1, 3.2 µM) for CYP2B6 [23], and ABT (31, 93,

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500 µM) for broad CYPs [28], which were assayed via pre-incubation with the

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NADPH-generating system at 37 ºC for 30 min.

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2.4. Metabolism of IGCM by recombinant human CYP2A6

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The cDNA-expressed human CYP2A6 isoform co-expressing NADPH-P450 reductase was used. The incubations were carried out using the same procedure as

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described above for the HLMs. To generate adequate metabolites for detection, a

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relatively high substrate concentration (100 µM) was used and incubated with CYP2A6 (40 nM) at 37 ºC for 30 min. The sample was then treated with acetonitrile, and the supernatant was analyzed by LC/MS to characterize the metabolite of IGCM.

2.5. Enzyme kinetics analysis To characterize the isoform-specific biotransformation mediated by CYP2A6, kinetic studies were performed using different enzyme sources. The incubation

7

ACCEPTED MANUSCRIPT conditions were optimized to ensure that the formation rates of licopyranocoumarin was in the linear range in relation to incubation time and protein concentration at 37 °C. For kinetic analyses, IGCM (1, 2.5, 5, 10, 25, 50, 100 µM) was incubated with recombinant human P450 2A6 for 20 min or liver microsomes for 30 min at 37 °C in

PT

the presence of the NADPH-generating system as described above. All incubations

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were conducted in three independent experiments in duplicate. The apparent Km and

SC

Vmax values were calculated from a nonlinear regression analysis of the experimental data according to the following Michaelis-Menten equation.

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1/v = (Km/ (Vmax ×[S])) + 1/ Vmax

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where v is the rate of the reaction (nmol min-1 mg-1), [S] the substrate concentration (μM), Vmax the maximum velocity estimate, and Km the Michaelis-Menten constant

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(μM). Kinetic constants were estimated using Origin 8.0 (OriginLab Corporation,

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Northampton, MA). The results are graphically presented as Eadie-Hofstee plots,

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which are shown as insets in Figure 1.

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2.6. HPLC analysis

An Agilent 1260 Infinity HPLC system was employed (Agilent Technologies, Inc., USA). Samples were separated on an Agilent Eclipse XDB C18 column (4.6 × 250 mm, 5 µm) protected by a Zorbax Extend-C18 guard column (4.6 × 12.5 mm, 5 µm). The mobile phase consisted of acetonitrile (A) and water containing 0.1% (v/v) formic acid (B). A linear gradient elution program was used as follows: 0 min, 15% A; 6 min, 35% A; 16 min, 45% A; 25 min, 55% A; 33 min, 70% A; 40 min, 95% A; 45

8

ACCEPTED MANUSCRIPT min, 95% A. The flow rate was 1000 µL/min. The column temperature was 35 °C. An aliquot of 10 μL was injected for analysis.

2.7. LC/MS analysis

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The analysis was performed on an Agilent series 1100 HPLC instrument

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connected to a Finnigan LCQ Advantage ion trap mass spectrometer (Thermo Fisher

SC

Scientific, Waltham, MA) via an ESI ion source. The column and elution program were the same as described above for the HPLC analysis. The flow rate was 1.0

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mL/min, and the effluent was introduced into the ESI source of the mass spectrometer

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at 0.25 mL/min via a T-union splitter. UV spectra were obtained by scanning from 200 to 400 nm. The optimized parameters in the negative ion mode were as follows: ion

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spray voltage, 4.5 kV; sheath gas (nitrogen), 50 arbitrary units; auxiliary gas

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(nitrogen), 5 arbitrary units; capillary temperature, 340 °C; capillary voltage, -36 V; tube lens offset voltage, -56 V. Mass spectra were recorded in the range of m/z 100 to

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1000. MSn (n=2-4) was triggered by a data-dependent threshold. The collision energy

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for collision-induced dissociation was 20%, and the isolation width for precursor ions was 2.0 mass units. As an exception, GLN was detected in the positive ion mode, as we previously reported [6]. Data were processed using Xcalibur 2.0.7 software (Thermo Fisher).

2.8. Molecular docking and molecular dynamics simulation The crystal structures of human CYP 2A6 (PDB# 1Z11) was retrieved from the

9

ACCEPTED MANUSCRIPT PDB database (http://www.rcsb.org/pdb/home/home.do). The substrate recognition sites were indicated by the co-crystallized substrate, 8-methoxsalen. The protein structures were optimized via Discovery Studio v2.5 (Accelrys Software Inc., USA) using CHARMm-based algorithm following water removal and hydrogen addition.

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The substrates were prepared via Discovery Studio v2.5 to produce one to three

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optimized conformations. Docking simulations were performed using GOLD v5.1

SC

(CCDC Software Ltd., Cambridge, UK). The CYP-dedicated scoring function (chemscore.p450_pdb.params) was applied and the active site was defined as all

MA

NU

residues within 20 Å. Default parameters were used unless otherwise stated.

Molecular dynamics simulations (MDs) were performed using the AMBER 12

D

package [29]. The AMBER99 force field was used to describe the IGCM-2A6 and

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GCM-2A6 complexes. The force field for the heme moiety was applied from Cheatham et al. [30]. To obtain molecular mechanical parameters for IGCM and

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GCM, ab initio quantum chemical methods were employed using the Gaussian 09

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program [31]. The geometry was fully optimized, and the electrostatic potentials around them were determined at the HF/6-31G* level of theory. The RESP strategy [32] was used to obtain the partial atomic charges.

The starting structure of GCM-2A6 and IGCM-2A6 complexes obtained by docking was solvated in TIP3P water using an octahedral box, which extended 8 Å away from any solute atom. To neutralize the negative charges of the simulated

10

ACCEPTED MANUSCRIPT molecules, a Na+ counter ion was placed next to each phosphate group. MD simulations were carried out using the SANDER module. The calculations began with 500 steps of steepest descent followed by 500 steps of a conjugate gradient minimization with a large constraint of 500 kcal mol–1 Å–2 on the complexes. Then,

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1000 steps of steepest descent followed by 1500 steps of the conjugate gradient

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minimization with no restraints on the complexes were performed. Subsequently, after

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20 ps of MDs, during which the temperature was slowly raised from 0 to 300 K with weak (10 kcal mol–1 Å-2) restraints on the complex, the final unrestrained production

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simulations of 10.0 ns for the molecule were performed at a constant pressure (1 atm)

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and temperature (300 K). In the entire simulation, SHAKE was applied to all hydrogen atoms. Periodic boundary conditions with minimum image conventions

D

were applied to calculate the nonbonded interactions. A cutoff of 10 Å was used for

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the Lennard-Jones interactions. The final structure of IGCM-2A6 complex was produced from the 1,000 steps of the minimized averaged structure of the last 5.0 ns

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of MDs.

3. Results

3.1. Hydroxylation of 24 compounds in human liver microsomes (HLM) The metabolism of 24 prenylated phenolic natural products was investigated in human liver microsomes (Figure 2). These natural compounds were isolated from Glycyrrhiza uralensis, and many showed significant bioactivities [1,2,33,34]. Among them, compounds 1-9, glycyrin (GLN) and glycycoumarin (GCM) contain a chained

11

ACCEPTED MANUSCRIPT isoprenyl group, whereas 10-21 and IGCM contain a cyclized isoprenyl. HPLC analysis indicated 16 of them could form hydroxylated metabolites. These compounds included five coumarins (1, 8, GLN, GCM, IGCM), seven isoflavones (2, 3, 6, 7, 11, 15, 16), one isoflavan (10), one isoflavanone (14), and two flavonols (4, 5). The

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conversion rates were in the range of 2.60% to 90.00% (Table 1 and Figure 2).

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Structures of the metabolites were confirmed via HPLC/UV/MSn analysis. The

SC

tandem mass spectra for 1-8, 10, 11, 14-16 and their phase I metabolites are shown in Fig. S2. The metabolites of GLN and GCM were transformed via P450 enzymes

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catalysis, which had been reported [6]. Here, IGCM was taken as the example. After

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incubation in HLM, IGCM yielded one single metabolite, which showed an [M-H]ion at m/z 383. In accordance, the [M-H]- ion could readily eliminate CH3· from C-5

fragment

ions

at

m/z

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showed

D

to produce m/z 368. Upon collision-induced dissociation, its MS/MS spectrum 309

([M-H-CH3-C3H7O]-),

and

m/z

296

([M-H-CH3-C4H8O]-) (Fig. S2). These fragments indicated the hydroxylation reaction

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occurred at the terminal methyl of the isoprenyl group, which was consistent with our

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previous study [5-7].

3.2. Isozyme identification by chemical inhibition assays To further investigate the CYP isozymes involved in the hydroxylation of prenylated phenolic compounds, chemical inhibition assays were conducted using ABT (a broad spectrum CYP inactivator) [28] and 11 specific CYP isozyme inhibitors [19-27], including furafylline for CYP1A2, 8-methoxsalen and pilocarpine for

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ACCEPTED MANUSCRIPT CYP2A6, ticlopidine for CYP2B6, montelukast for CYP2C8, sulfaphenazole for CYP2C9, omeprazole for CYP2C19, quinidine for CYP2D6, clomethiazole for CYP2E1, ketoconazole for CYP3A, and CYP3cide for CYP3A4. The incubation

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conditions were optimized using IGCM as the substrate, as shown in Fig. S1.

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Among the 16 compounds which could be hydroxylated by HLM, 12 compounds

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were mainly catalyzed by CYP2A6, including IGCM, 1-5, 7, 8, 11, and 14-16. Their hydroxylation could be almost completely inhibited upon the addition of ABT,

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8-methoxsalen or pilocarpine, whereas it was hardly affected by the addition of other

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isozyme inhibitors (Table 1, Fig. S3). For compounds 6 and 10, their hydroxylation was only partially inhibited by CYP2A6 inhibitors and could also be inhibited by

D

other isozyme inhibitors. Thus, we could deduce that 6 and 10 were not metabolized

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by CYP2A6 alone. Other isozymes, such as CYP3A, 2C9, 2C19, 2D6 and 2B6, may also be involved in the hydroxylation of 6 and/or 10. However, GCM and GLN

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showed very different inhibition patterns from the other substrates. Their

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hydroxylation rates in HLM were 7.1% and 3.4%, respectively. These rates did not decrease or only decreased slightly upon the addition of different isozyme inhibitors. This result indicated that CYP2A6 did not participate in the hydroxylation of GCM and GLN. Their hydroxylation may not be catalyzed by one specific P450 isozyme.

3.3. Hydroxylation of IGCM by recombinant human CYP2A6 enzyme Based on the above results, IGCM could be hydroxylated by HLM at 46.2% to

13

ACCEPTED MANUSCRIPT produce one single product, and this metabolism was mainly catalyzed by CYP2A6. To further confirm the catalytic ability of CYP2A6 on IGCM, we conducted a chemical inhibition assay at three concentration levels, IC50, 3 times IC50, and 10 times IC50. As shown in Figure 3, the hydroxylation of IGCM could be completely

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inhibited by ABT or the specific 2A6 inhibitors, 8-methoxsalen and pilocarpine, at

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any of the three concentrations, but it was hardly inhibited by the inhibitors of other

SC

P450 isozymes. We further incubated IGCM with the cDNA-expressed human CYP2A6 enzyme, and found that IGCM could be specifically converted into one

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single hydroxylated metabolite, which was influenced by protein concentration

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(Figure 1-A). These results indicated that IGCM may be able to serve as a specific

metabolite

was

unambiguously

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The

D

probe substrate for the human CYP2A6 enzyme.

identified

as

licopyranocoumarin

(4-hydroxylated derivative of IGCM) by comparing with a reference standard

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isolated from Glycyrrhiza uralensis. Its 1H NMR spectrum was very similar to that of

AC

IGCM, and new methylene signals appeared at δH 3.41 and 3.39 (Figure 4). In addition, a new signal corresponding to a hydroxyl group was observed at δH 5.03. These data indicated one of the gem-methyl groups of IGCM was replaced by a hydroxymethyl group in the metabolite. In accordance, 5′′-CH3 (δC 21.6) (Table S1, Fig S7) shifted upfield by 4.8 ppm due to the γ-gauche effect of the hydroxy group. Combined with the previous report, the metabolite of IGCM was identified as 4-hydroxyl isoglycycoumarin (licopyranocoumarin) (Fig S6-S9) [35].

14

ACCEPTED MANUSCRIPT

3.4. Evaluation of IGCM as a probe substrate for CYP2A6 To further confirm the hydroxylation of IGCM was mediated by CYP2A6, the Michaelis-Menten kinetics were performed using pooled HLM and recombinant

PT

human CYP2A6, respectively (Figure 1-B and C). Furthermore, the Km value in the

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pooled HLM (7.98 μM) was close to that in recombinant human CYP2A6 (10.14 μM)

SC

(Table 2), supporting our speculation that CYP2A6 plays a predominant role in the

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hydroxylation metabolism of IGCM.

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3.5. Elucidation of catalytic mechanism of CYP2A6 by molecular docking CYP2A6 has the second smallest active site cavity among all known CYPs. Most

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of its substrates are small molecules with molecular weights below 200 Da [14].

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IGCM (368 Da) has a bigger size than the known substrates. Thus, we used molecular docking and molecular dynamics simulation to reveal the conformation of

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the IGCM-2A6 complex. According to the reported structures (PDB# 1Z10, 1Z11,

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2FDY, 3T3R, 4EJJ, 4RUI), substrates and inhibitors of CYP2A6 bind to the same site near the cysteinato-heme enzyme, and the electrons are transferred by the heme iron during the oxidation reaction [30]. Since the hydroxylation of IGCM could be inhibited by 8-methoxsalen, the co-crystallized structure of 8-methoxsalen and 2A6 (1Z10) was used [25]. The docking results showed that the cyclized isoprenyl group could enter the binding site, and the other side of the molecule stood alone from 8-methoxsalen, the initial substrate (Fig. S4). The smaller size of the initial substrate

15

ACCEPTED MANUSCRIPT partly explained the lower score of IGCM. Interestingly, in the case of GCM, the benzyl group, instead of the isoprenyl group was closer to the binding site.

The conformations were further supported by molecular dynamics simulation

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(Figure 5). The IGCM-2A6 complex maintained the docking results and was

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dynamically steady. The system became equilibrated after a 10.0-ns MD simulation,

SC

and the average RMSD value of IGCM was 2.05 Å. The distance between the terminal methyl group and the heme iron was 4.2 Å. Nonetheless, GCM exhibited a

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remarkably different orientation, where the distance was 6.6 Å from the terminal

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methyl group to the heme. According to a previous report, a close approach and optimal position of the carbon is critical to the oxidation that is catalyzed by CYP2A6

D

[36]. Therefore, geometry could be an important factor for the selectivity of CYP2A6

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on IGCM, but not on GCM. These reports are consistent with our finding that CYP2A6 does not contribute to the hydroxylation of GCM. Meanwhile, the isoprenyl

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group of other CYP2A6 substrates, compounds 1, 8, and 15, could also enter the

AC

binding site, as suggested by molecular docking (Fig. S4). These findings suggested IGCM is a good probe substrate, and that CYP2A6 may provide a metabolically favorable state in the metabolism of IGCM.

4. Discussion Prenylated phenolic compounds from medicinal plants have been reported to show numerous pharmacological activities. The hydroxylation on the terminal methyl

16

ACCEPTED MANUSCRIPT of the isoprenyl group is the primary metabolic pathway of most phenolic compounds [3,4]. Therefore, identification of the specific CYP isoform that can specifically catalyze the hydroxylation helps us to understand the drug metabolism of prenylated phenolic compounds. In the present work, we screened 24 prenylated phenolic

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compounds, and 16 of these compounds were metabolized by HLM to form

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hydroxylated metabolites. Moreover, the CYP2A6 was identified as the predominant

SC

isozyme in the hydroxylation of these prenylated phenolic compounds via chemical inhibition assays. Particularly, IGCM was found to be a novel isoform-specific probe

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substrate for CYP2A6 in our study. The hydroxylated metabolite was fully

MA

characterized as licopyranocoumarin (4''-hydroxyl isoglycycoumarin) via LC/MS and NMR analysis. Kinetic studies demonstrated that licopyranocoumarin in both HLM

D

and human recombinant CYP2A6 obeyed biphasic kinetics, displaying similar

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apparent kinetic parameters. The Km (10.14 μM) value of IGCM hydroxylation catalyzed by CYP2A6 was significantly lower than that for the hydroxylation of

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8-prenylnaringenin mediated by CYP2C19 (Km > 14.5 μM) [12], indicating that

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CYP2A6 displayed a high affinity with IGCM. Moreover, CYP2A6 showed good reactivity (kcat/Km = 496.06 L mol-1 s-1) for IGCM hydroxylation. With a high specificity and good reactivity, IGCM could serve as an ideal specific probe for the evaluation of CYP2A6 activity.

To confirm the key role of CYP2A6 in the hydroxylation of IGCM, potent inhibitors for different CYP isoforms were measured to explore the enzyme(s)

17

ACCEPTED MANUSCRIPT involved in this metabolism. The selective inhibitors and their concentrations were determined according to the previous reports [26, 37-39]. Finally, the enzymes involved in the formation of licopyranocoumarin were investigated using 11 selective chemical inhibitors of P450 enzymes at three concentrations (one, three, and ten times

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the IC50 values), and it was selectively catalysed by CYP2A6 in humans (Figure 3).

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Additionally, cDNA-expressed recombinant human CYP2A6 was used to

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determine the IC50 values of the known CYP2A6 inhibitors, 8-methoxsalen and pilocarpine, using IGCM as the probe substrate (Figure 6). The IC50 values of

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8-methoxsalen and pilocarpine for the hydroxylation of IGCM were 0.4 ± 0.01 µM

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and 5.6 ± 0.04 µM, respectively, which were similar to previous reports [26,40]. IGCM showed an advantage as a probe substrate over coumarin in that coumarin

(70-80%)

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7-hydroxycoumarin

D

produced four other hydroxylated metabolites aside from the major metabolite, [14],

whereas

IGCM

only

produced

licopyranocoumarin (Fig. S5). This finding suggested that IGCM is an ideal

AC

CE

isoform-specific probe for CYP2A6.

5. Conclusion

CYP2A6 is the key isozyme catalyzing the hydroxylation of the terminal isoprenyl methyl group of prenylated phenolic natural products. We discovered a novel isozyme-specific probe substrate for CYP2A6, isoglycycoumarin (IGCM). The hydroxylation reaction of IGCM to produce licopyranocoumarin can be used to determine the catalytic activity of CYP2A6.

18

ACCEPTED MANUSCRIPT

Conflict of interest There is no conflict of interest.

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Acknowledgements

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This work was supported by National Natural Science Foundation of China (No.

SC

81173644, No. 81222054), Science Foundation for Young Scholars of Peking

MA

Postdoctoral Fund (No. LBH-Z16161).

NU

University School of Pharmaceutical Sciences (YXQNF201507), and Heilongjiang

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PT E

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P450 2A6 (CYP2A6) allelic variants CYP2A6*15, CYP2A6*16, CYP2A6*21 and CYP2A6*22: differential susceptibility due to different sequence locations

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of the mutations. PLoS One 9, e86230.

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Glycyrrhiza uralensis and other Glycyrrhiza species, Microbiol. Immunol. 58,

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protein tyrosine phosphatase-1B inhibitory activities. Bioorg. Med. Chem. Lett.

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licopyranocoumarin, licoarylcoumarin and glisoflavone, and inhibitory effects of

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licorice phenolics on xanthine oxidase 2). Chem. Pharm. Bull. 37, 3005-3009. [36] Meunier, B., De Visser, S.P., Shaik, S., 2004. Mechanism of oxidation reactions catalyzed by cytochrome P450 enzyme. Chem. Rev. 104, 3947-3980. [37] Ma, X.C., et al. 2011. Comparative metabolism of cinobufagin in liver microsomes from Mouse, Rat, Dog, Minipig, Monkey, and Human. Drug Metab. Dispos. 39, 675-682. [38] Ning, J., et al. 2014. Characterization of the phase I metabolism of resibufogenin

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ACCEPTED MANUSCRIPT and evaluation of the metabolic effects on its antitumor activity and toxicity. Drug Metab. Dispos. 43, 299-308. [39] Ge, G.B., et al. 2013. A highly selective probe for human cytochrome P450 3A4: isoform selectivity, kinetic characterization and its applications. Chem.

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Commun., 49, 9779.

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[40] Lee, K.S., Kim, S.K., 2013. Direct and metabolism-dependent cytochrome P450

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inhibition assays for evaluating drug-drug interactions. J. Appl. Toxicol. 33,

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CE

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D

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100-108.

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ACCEPTED MANUSCRIPT Figures for review

A

B

CYP2A6

CYP2A6

6

DAD1 B, Sig=365,4 Ref=500,100 (D:\CHEM32\DATA\WQ\GC-39-P450KM-1 2015-11-21 22-12-19\2A6-1UM-3.D) mAU 50

licopyranocoumarin

1 µM

5

IGCM

25

30

35

min

20 0 5 10 15 DAD1 B, Sig=365,4 Ref=500,100 (D:\CHEM32\DATA\WQ\GC-39-P450KM-1 2015-11-21 22-12-19\2A6-5UM-3.D)

25

30

35

min

5 µM

20

3

2

25

30

35

min

0

0

10 µM

0 5 10 15 DAD1 B, Sig=365,4 Ref=500,100 (D:\CHEM32\DATA\WQ\GC-39-P450KM-1 2015-11-21 22-12-19\2A6-25UM-3.D)

20

25

30

35

min

C

25 µM

1.6 1.4

50 20

25

30

35

min

50 µM

0

mAU

20

25

30

100 µM

200 0 10

15

20

25

30

min

35

min

60

80

1.25

100

120

1

0.8 0.6 0.4

8

6 4 2 0

-0.5 -0.25

0.2

0

0.25

0.5

0.75

1

1.25

CIGCM-1 (µM-1)

0 0

20

40

60

80

100

Substrate concentration (µM)

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D

5

35

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5 10 15 DAD1 B, Sig=365,4 Ref=500,100 (D:\CHEM32\DATA\WQ\GC-39-P450KM-1 2015-11-21 22-12-19\2A6-100UM-3.D)

Formation Rate (nmol/min/mg protein)

5 10 15 DAD1 B, Sig=365,4 Ref=500,100 (D:\CHEM32\DATA\WQ\GC-39-P450KM-1 2015-11-21 22-12-19\2A6-50UM-3.D)

100

40

1

HLMs

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1.2

0

mAU

20

0.25 0.5 0.75 CIGCM-1 (µM-1)

Substrate concentration (µM)

20

mAU 100

1

0.5

SC

mAU

20

2

1.5

0 -0.5 -0.25 0

1

0 5 10 15 DAD1 B, Sig=365,4 Ref=500,100 (D:\CHEM32\DATA\WQ\GC-39-P450KM-1 2015-11-21 22-12-19\2A6-10UM-3.D)

2.5

Velocity-1 (nmol -1•min •— m g)

mAU 40

20

4

PT

25

Velocity-1 (nmol -1•min •— m g)

20

2.5 µM

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5 10 15 DAD1 B, Sig=365,4 Ref=500,100 (D:\CHEM32\DATA\WQ\GC-39-P450KM-1 2015-11-21 22-12-19\2A6-2-5UM-3.D) mAU

Formation Rate (nmol/min/mg protein)

0

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Figure 1. HPLC chromatograms for the catalytic products of different concentrations of IGCM by CYP2A6 (A), and Michaelis-menten kinetic plots for the formation of

AC

licopyranocoumarin from IGCM catalyzed by CYP2A6 (B) or pooled human liver microsomes (C). The corresponding Eadie-Hofstee plots are shown as insets.

25

120

ACCEPTED MANUSCRIPT

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

PT

90.0%

40.0%

15.0% 10.0%

RI

Conversion rate

65.0%

5.0%

NU

SC

0.0%

MA

Figure 2. Chemical structures of the 24 prenylated compounds and their incubation assay in human liver microsomes. The * means the compounds could be hydroxylated

AC

CE

PT E

D

by HLM.

26

120.00

10*IC50

100.00

3*IC50 IC50

80.00

PT

60.00 40.00

RI

20.00

N.D. N.D. N.D.

0.00

MA

NU

SC

% Control activity remaining

ACCEPTED MANUSCRIPT

Figure 3. Metabolism of IGCM into licopyranocoumarin by human liver microsomes

D

or CYP2A6, and the inhibition assay by different concentrations of selective CYP

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inhibitors in human liver microsomes. N.D. means not detected. Data represent the

AC

CE

mean±SD from three independent experiments.

27

ACCEPTED MANUSCRIPT 1H-NMR of

licopyranocoumarin

5-OCH3 5′′

2′-OH 4′-OH

4

8

1H-NMR of

3′

5′′-OH

5′

4′′A

4′′B

1′′

2′′B

2′′A

4′′, 5′′

PT

6′

IGCM

4

8 3′ 5′

1′′

2′′

MA

NU

6′

SC

2′-OH 4′-OH

RI

5-OCH3

AC

CE

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D

Figure 4. 1H NMR spectra (400 MHz, DMSO-d6) for IGCM and licopyranocoumarin.

28

ACCEPTED MANUSCRIPT

(A)

(B)

Leu212

6.6 Å

PT

4.2 Å

HEME

SC

RI

HEME

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Figure 5. Molecular dynamics simulation of IGCM (A) and GCM (B) into the

AC

CE

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D

MA

CYP2A6 protein.

29

ACCEPTED MANUSCRIPT D DoseResp Fit of D

B

A 100

100

80 80

B

50 50

25 25

40 40

20 00

IC50= 5.6 µM 0.0 0.0

60

0.6 0.6

IC50= 0.4 µM -1.2 -1.2

1.2 1.2

A

-0.6 -0.6

A

0.0 0.0

8-Methoxsalen (log µM)

NU

SC

RI

Pilocarpine (log µM)

PT

Control (%)

Control D(%)

75 75

00

B DoseResp Fit of B

100

Figure 6. CYP2A6 inhibition curves of pilocarpine and 8-methoxsalen using IGCM

AC

CE

PT E

D

MA

as probe.

30

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Graphical abstract

31

ACCEPTED MANUSCRIPT Table 1. Conversion rates for the hydroxylated metabolites of 16 prenylated compounds by adding three times of IC50 concentrations of selective CYP inhibitors in human liver microsomes (n = 3, %). Piloc 8-Methox ABT Anal

Clomet

CYP3c

Furafyl

Ketoco

Montel

Omepra

Sulfaph

Quinidi

Ticlopi

hiazole

ide

line

nazole

ukast

zole

enazole

ne

dine

(2E1)

(3A4)

(1A2)

(3A)

(2C8)

0.0±

43.1±2.

44.3±3.

43.5±4.

39.1±1.

45.5±2.

0.0

8

1

1

5

6.6±0.9

6.5±0.5

4.5±0.5

5.8±0.9

arpin ypsoralen

Contr

e (CYP

(2A6 (2A6)

s) IGC

46.3±

)

0.0±0. 0.0±0.0 0

GC

7.1±0

6.1±0.

6.5± 5.9±1.6

M

.4

8

GL

3.4±0

6.4±1.

1.6 1.3± 10.7±4.2

.6

6

20.0±

6.6±0.

1.9±0.8

0.0±

16.6±0.

18.9±2.

8

0

0.0±0.0 2

0.0

0.1±1.

0.8±

.5

0

22.7±

2.3±0.

0.0±0.0

2*

4.1±0.1 0.4 0.6±

22.6±3.

4.6

2

0.8

4

5.9±0

0.3±0. 2

8.7±0

0.4±0.

.5

2

34.6±

6.0±2.

6#

40.2±3

39.7±3.

.5

0

8

2

6.8±1.2

7.6±0.1

7.0±1.4

1.9±0.1

4.6±0.

2.1±0. 2.1±0.7

2.0±0.5

16.6±3.

15.8±6.

18.7±5.

3

1

2

3

2

2.5±0.2

0.5

2.2±0.1 1

15.8±5.

2.9±1.4

6.5±1.6 4

17.3±0

16.9±3.

.1

4

2.3±1. 2.9±1.1

2.3±1.2

2.4±1.0

2.6±0.5 9

20.6±3.

23.3±4.

21.1±3.

21.5±3.

20.5±3

20.6±3.

3

5

8

2

0

4

.8

0

4.8±1.6

4.2±0.4

4.0±2.4

4.5±2.2

3.5±1.3

4.2±1.4

7.0±0.1

8.1±1.4

5.5±1. 6.9±1.6

3.0±0.2 3

1.5±

0.0±0.0

5*

42.5±3.

21.3±4.

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.7

37.2±2.

0.4± 0.0±0.0

4*

(2B6)

22.0±4.

0.0±0.0

3*

2.4±0.1

(2D6)

12.9±0.

MA

1.0 5.3±0

1.9±0.9

(2C9)

D

1*

1.5±0.5

0.2

4.1±1.4

8.7±2.5

8.9±2.1

8.9±2.8

10.3±4

10.6±0.

.2

3

17.3±1

13.4±1.

.5

9

8.7±2.7

1.0

5.3±

24.9±3.

33.5±3.

33.9±4.

17.2±1.

35.6±4.

13.6±5.

16.4±2.

1.1

0

4

9

1

9

4

5

6.2±1.0 4.9

9

4.3±0

0.0±0.

.5

0

90.0±

2.3±0.

2.7±

CE

N

1.9±1.0

(2C19)

8

SC

1.5

NU

M*

PT

ol

RI

ytes

0.0±0.0

7*

4.4±1.0

3.2±0. 3.3±0.1

3.2±0.8

5.2±0.1

3.2±1.0

2.9±1.1

3.5±1.3

0.3

3.3±1.3 1

1.1±

90.6±9.

93.0±6.

71.5±3.

92.

93.5±7.

100.6±8

96.3±9.

90.4±5

97.1±7.

0.0

8

5

5

9±10.0

4

.7

2

.4

3

1.8±0.5

2.4±0.3

1.8±0.1

1.2±0.1

1.1±0.4

0.8±0.1

2.1±0.5

1.4±0.8

1.7

AC

8*

2.6±0 10#

7

0.7±0.

0.6±

1.0±0.5

.7

3.3±0

0

0.0±0.

0.1

.4

0

27.6±

3.3±0.

1.9±0.1 3

0.0± 0.0±0.0

11*

4.3±1.

3.0±1. 3.0±0.1

2.4±1.3

2.9±1.0

2.7±0.2

1.9±1.0

2.1±1.1

2.6±0.1

0.0

2.5±0.2 4

0.9±

27.0±4.

29.6±2.

17.9±1.

18.9±2.

17.3±5.

23.9±9.

22.2±2.

2

3

0

0

3

7

3

19.7±2

22.5±5.

.1

2

1.2±0.2

14* 3.6

2

0.1

6.2±1

1.2±0.

0.1±

.3

1

3.4±0

0.0±0.

0.0±0.0

15*

6.3±1.0

.7

0

6.7±1.1

5.6±1.3

5.5±3.4

4.5±2.0

4.9±3.0

5.3±1.7

0.0

5.9±2.2 2

0.0± 0.0±0.0

16*

4.4±0.

2.1±0. 2.7±0.5

2.7±0.9

1.9±0.1

0.0

2.0±1.0

2.6±0.3

2.5±1.7

3.1±0.2

2.2±0.1 9

32

ACCEPTED MANUSCRIPT *, compounds mainly catalyzed by CYP2A6; #, compounds catalyzed by CYP2A6 and other isozymes.

Table 2. Kinetic parameters for the formation of licopyranocoumarin from

pooled HLM

1.38±0.09

7.98±0.03

CYP2A6

5.03±0.02

10.14±0.08

Kcat/Km (L mol-1 s-1)

RI

Km (μM)

AC

CE

PT E

D

MA

NU

SC

Kcat (s-1)

PT

isoglycycoumarin catalyzed by CYP2A6 and pooled human liver microsomes (HLM).

33

172.93 496.06