Identification of the Metabolic Enzyme Involved Morusin Metabolism and Characterization of Its Metabolites by Ultraperformance Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (UPLC/Q-TOF-MS/MS)

Morusin, the important active component of a traditional Chinese medicine, Morus alba L., has been shown to exhibit many vital pharmacological activities. In this study, six recombinant CYP450 supersomes and liver microsomes were used to perform metabolic studies. Chemical inhibition studies and screening assays with recombinant human cytochrome P450s were also used to characterize the CYP450 isoforms involved in morusin metabolism. The morusin metabolites identified varied greatly among different species. Eight metabolites of morusin were detected in the liver microsomes from pigs (PLMs), rats (RLMs), and monkeys (MLMs) by LC-MS/MS and six metabolites were detected in the liver microsomes from humans (HLMs), rabbits (RAMs), and dogs (DLMs). Four metabolites (M1, M2, M5, and M7) were found in all species and hydroxylation was the major metabolic transformation. CYP1A2, CYP2C9, CYP2D6, CYP2E1, CYP3A4, and CYP2C19 contributed differently to the metabolism of morusin. Compared to other CYP450 isoforms, CYP3A4 played the most significant role in the metabolism of morusin in human liver microsomes. These results are significant to better understand the metabolic behaviors of morusin among various species.


Introduction
Flavonoids and their derivatives are important components of the human diet and exhibit many diverse pharmacological effects [1,2]. Morusin ( Figure 1) is a prenylated flavonoid that was first isolated from the root bark of Morus alba L. in 1978 and then later characterized in 2001 [3,4]. In previous studies, we discovered that morusin exhibits strong inhibitory activity against primary UGTs and CYP450s. Additionally, our findings revealed significant differences in its metabolism between species and found that dog models are the optimum model to study morusin metabolism [5].
Like most flavonoids, morusin has antitumor [6,7], antiinflammatory [8], and antibacterial [9] activity. However, it is unclear if morusin or its metabolites produce these biological activities. Additionally, the side effects and toxic reactions of morusin may also be related to its metabolites. Therefore, determination of only the parent drug may not fully reflect its true pharmacologic and toxic nature. Identification of metabolites of a potential drug candidate plays an important role in drug development and can provide essential information on drug efficacy and its toxicological profile. Recently, metabolic studies have been performed in the early stages of drug discovery to determine whether the potential drug candidate is worth further development [10].
A simple and effective analytical method to reveal the structures of possible active constituents is a significant and valuable tool. Mass spectrometry coupled to liquid chromatography (LC-MS) [11,12], mass spectrometry coupled to gas chromatography (GC-MS) [13,14], and nuclear magnetic resonance (NMR) [15,16] are most frequently used to identify the structures of drugs. Compared to GC-MS or NMR, LC-MS based techniques are often used for metabolite identification due to their superior selectivity, sensitivity, and rapid rate of analysis [12]. In this study, the morusin metabolite profiling was performed through ultraperformance liquid chromatography/ electrospray ionization quadruple time-of-flight/high-definition mass spectrometry (UPLC/ESI-Q-TOF/HDMS) combined with pattern recognition approaches and pathway analysis. The system has proven to have higher sensitivity than the traditional high-resolution MS system and has been used to identify many drug structures [17,18]. In addition, CYP450 isozymes involved in the metabolism of morusin were also confirmed by assays with recombinant CYP450 isoforms and chemical inhibition experiments. Finally, molecular docking was employed to further understand the interactions between morusin and CYP450s.
LC-MS/MS analysis was performed on Ultra-Performance Liquid Chromatograph (UPLC) equipped with a Q-TOF SYNAPT G2-Si High Definition Mass Spectrometry (Waters Corporation, Milford, MA). Each sample (2 L) was injected into a Welch C18 column (1.7 mm × 100 mm, 1.7 m, Milford, USA) using an Acquity H-class UPLC system (Waters Corporation, USA). The column oven was maintained at 40 ∘ C. The mobile phase consisted of LC grade water with 0.1% formic acid (A) and LC grade acetonitrile (B) with the following gradient profile: 0-12 min, 55% B; 12-13 min, 55-90% B; 13-19 min, 90% B; 19-20 min, 90-55% B; 20-25 min, 55% B. The mass spectrometer was operated in negative mode and the mass range was set from 100 / to 500 / . Optimum parameters were as follows: ESI collision gas was Argon, trap collision energies were 6 V (low energy) and 20-50 V (high energy), the capillary and cone voltages were 2 KV and 40 V, the source and desolvation temperatures were 120 and 600 ∘ C, and the cone and desolvation gas flows were 50 and 800 L⋅h −1 .

Incubation Systems with Liver Microsomes or Recombinant
CYP450 Supersomes. The optimum incubation conditions of microsomes have been reported [5]. In brief, the typical incubation system contains 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 MgCl 2 ), the appropriate concentration of liver microsomes or recombinant CYP450s supersomes, the corresponding probe substrate, and morusin (or positive inhibitor for different probe reactions) in a final volume of 200 L. According to preliminary experiments (data not shown), the final protein concentration of 0.3 mg/mL in liver microsomes or 15 nM in recombinant human supersomes and a 30 min reaction time were selected to ensure linear formation of metabolites during the incubations. There was a 3 min preincubation at 37 ∘ C before the reaction was initiated by adding the NADPH-generating system. The reaction was placed on ice and terminated by adding 200 L acetonitrile and an internal standard. The mixture was centrifuged at 20,000 ×g for 20 min and an Evidence-Based Complementary and Alternative Medicine 3 aliquot (10 L) of supernatant was transferred for HPLC or LC-MS/MS analysis.

Assay with Recombinant P450s.
Six cDNA-expressed human CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) were used to screen the involved isoform(s) for morusin metabolites. The incubations were carried out with each of the CYP450 isoforms using the protocol described above. Morusin (10 M) was incubated with each of the recombinant CYP450s (15 nM) at 37 ∘ C for 60 min and potential metabolites were monitored by HPLC.

Docking Studies of Morusin into the Reported Structures of CYP3A4.
In order to further assess morusin metabolism by CYP3A4, molecular docking analysis with Gold v5.2 was implemented. This docking method has been reported previously [19]. The crystal structures of human CYP3A4 (pdb: 4K9W) were obtained from RCSB Protein Databank (http://rcsb.org/). SYBYL X2.1 was used for protein and ligand preparation, and energy minimization was completed through the external Tripos force field. The protonation state and energy minimization of the protein and the ligands were calculated using the default settings in SYBYL X2.1. The docked poses were scored using CHEMPLP scoring function. The highest scored docked pose of the ligand was visualized using Pymol Molecular Graphics System v1.3.

Identifying CYP450s
Involved in the Metabolism of Morusin. cDNA-expressed human P450 isoforms were used to investigate the enzymes involved in the formation of M 1 . Quantification of M 1 in HLMs was normalized to 100% and other enzymes including CYP3A4, CYP2C19, CYP2D6, CYP2E1, CYP2C9, and CYP1A2 were compared with its counterpart from HLM incubation. As shown in Figure 2, the amount of M 1 generated by CYP3A4, CYP2C19, CYP2D6, CYP2E1, CYP2C9, and CYP1A2 was 22.6%, 6.4%, 0.6%, 1.7%, 5.9%, and 2.1% of the amount incubated in HLMs, respectively. In addition, we also used chemical inhibition assays to confirm the key role of CYP450s in metabolizing morusin. The formation of M 1 could be potently inhibited by ketoconazole (a potent inhibitor of CYP3A4) to less than 20% activity, while the selective inhibitors of other CYP450 isoforms had minor effects on the formation of M 1 (Figure 3).

Structure Characterization of Metabolites by LC-MS/MS.
In this study, the biotransformation of morusin was investigated by incubating morusin with different liver microsomes.  Table 1, and the structural skeleton of morusin and its potential metabolic pathways are shown in Figure 4. The parent compound, morusin, had a retention time of 9.17 min and was easily elucidated from the standard by comparison of retention time and MS data. Morusin ( /  were identified as the reduction and dihydroxylation products of morusin. Exact mass determination and the corresponding chemical composition verify that M 5 was produced through the addition of two hydroxyl groups to the parent molecule at C-12 and C-5 . M 6 also has two more hydroxyl groups than morusin: one hydroxyl group was added in phenyl and the other hydroxyl group was added at C-12. The molecular formula of M 7 was determined to be C 25 H 26 O 7 ( / 437.1389) based on MS data and its ionized form was 18 Da more than the parent morusin. According to its molecular formula and MS/MS spectra, M 7 was identified as the hydration product of morusin where the hydroxyl group was bonded to C-12. Structural formula and fragmentation pathway of M 7 are shown in Figure 5(  The ionized form of M 8 appeared at / 451.1393 and was 32 Da more than morusin. Additionally, corresponding main fragment ions were found at / 433.1366, 419.1509, 381.0969, and 375.0877. Therefore, M 8 was identified as a dihydroxylation product of morusin. According to the structures of fragment ions, the two new hydroxyl groups are potential bonded to C-14 and C-5 ( Figure 5(i)).

Analysis of Docking
Results. CYP3A4 is the major enzyme involved in the metabolism of morusin. Therefore, we used molecular docking to study the molecular mechanism of interactions between morusin and CYP3A4. As shown in Figure 6, morusin binds to the active cavity of CYP3A4 through hydrogen bonding and -stacking interactions. The hydrogen bonds occur with Arg106, MET371, Arg372, and Glu374, and -stacking interactions occur with Phe108.

Discussion
Morusin has many significant properties including antitumor, antibacterial, and anti-inflammatory properties and its pharmacological effects have been studied thoroughly. However, the metabolic pathway and behavior of morusin in human and experimental animals vary greatly and have not been examined. In this study, a comparison of metabolic profiles, enzymes involved, and catalytic efficiency of morusin metabolism in liver microsomes from different species was performed.
It is necessary during a toxicity assessment to identify the enzyme(s) involved in morusin biotransformation in order to predict potential metabolite-drug interactions [20]. We identified CYP450 isoforms involved in morusin metabolism through screening assays with commercially available cDNAexpressed CYP450 isoforms including CYP1A2, CYP3A4,