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Investigation of the interactions between flavonoids and human organic anion transporting polypeptide 1B1 using fluorescent substrate and 3D-QSAR analysis
BBA - Biomembranes 1862 (2020) 183210
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Investigation of the interactions between flavonoids and human organic anion transporting polypeptide 1B1 using fluorescent substrate and 3DQSAR analysis ⁎
T
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Yiqun Xiang1, Shuai Liu1, Jingjie Yang, Zhongmin Wang, Hongjian Zhang , Chunshan Gui College of Pharmaceutical Sciences, Soochow University, 199 Renai Road, Suzhou Industrial Park, Suzhou, Jiangsu 215123, China
A R T I C LE I N FO
A B S T R A C T
Keywords: OATP1B1 Transporter Flavonoid 3D-QSAR CoMFA CoMSIA
Organic anion transporting polypeptide 1B1 (OATP1B1) is a key hepatic uptake transporter whose inhibition could lead to adverse drug-drug and drug-food interactions. Flavonoids are widely distributed in food and beverages and thus our bodies are frequently exposed to them. Therefore, investigation of the interactions between OATP1B1 and flavonoids could be of great significance. In the present study, 25 common flavonoids were investigated for their interactions with OATP1B1 using the fluorescent substrate 2′,7′-dichlorofluorescein (DCF) and three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis. Kinetic study showed that OATP1B1-mediated DCF uptake exhibited a monophasic saturation kinetics with a Km value of 9.7 ± 2.4 μM. Inhibition assay for flavonoids on OATP1B1-mediated DCF uptake was performed and their IC50 values were determined upon which reliable and predictive CoMFA (q2 = 0.604, r2 = 0.841) and CoMSIA (q2 = 0.534, r2 = 0.807) models were developed. Our experimental and computational results showed that flavonoid aglycones interacted with OATP1B1 much stronger than their glycosides such as 3-O- and 7-O-glycosides as bulky hydrophilic and hydrogen-bond forming substituents at C-3 and C-7 positions on rings A and C were unfavorable for their binding. On the other hand, the presence of hydrogen-bond forming groups on ring B was beneficial as long as the number of hydroxyl groups was not > 2. Our results also indicated that flavones usually interacted with OATP1B1 much stronger than their 3-hydroxyflavone counterparts (flavonols). The obtained information and 3D-QSAR models could be useful for elucidating and predicting the interactions between flavonoids and human OATP1B1.
1. Introduction Organic anion transporting polypeptides (OATPs), which belong to the superfamily of solute carriers (SLCs) [1], are multispecific transporters that mediate the sodium-independent transport of a wide range of endo- and exo-genous amphipathic organic compounds [2,3]. Among the 11 known human OATPs, OATP1B1 is considered to be a liverspecific transporter under normal physiological conditions [4]. It is a key uptake transporter expressed on the sinusoidal membrane of hepatocytes and plays an important role in the hepatic uptake of many drugs [5]. Inhibition of OATP1B1-mediated hepatic uptake of its
substrate drugs by other drugs or natural products might lead to clinically relevant drug-drug and drug-food interactions [6–12]. Flavonoids are a diverse group of phytonutrients abundant in dietary plants and herbs. Chemically, flavonoids have a general structure of 15-carbon skeleton (C6-C3-C6), which consists of two phenyl rings (A and B) and a heterocyclic ring (C) [13]. Studies showed that flavonoids have diverse biological activities such as anti-cancer, antimicrobial, anti-diabetic, anti-inflammatory, and cardiovascular protective effects [14–18]. It has been reported that some flavonoids interact with OATPs [19–25]. Due to the wide distribution and frequent use of flavonoids and the importance of OATP1B1 for drug disposition,
Abbreviations: 3D-QSAR, three-dimensional quantitative structure-activity relationship; BSP, bromosulfophthalein; CHO, Chinese hamster ovary cells; CoMFA, comparative molecular field analysis; CoMSIA, comparative molecular similarity indices analysis; DCF, 2′,7′-dichlorofluorescein; EV, empty vector; E3S, estrone-3sulfate; EC, (−)-epicatechin; ECG, (−)-epicatechin gallate; EGC, (−)-epigallocatechin; EGCG, (−)-epigallocatechin gallate; FBS, fetal bovine serum; HTS, highthroughput screening; IC50, the half maximal inhibitory concentration; OATP, organic anion transporting polypeptide; PBS, phosphate buffered saline; PLS, partial least-squares regression; q2, crossvalidated r2; SLC, solute carrier ⁎ Corresponding authors. E-mail addresses: [email protected] (H. Zhang), [email protected] (C. Gui). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bbamem.2020.183210 Received 20 November 2019; Received in revised form 10 January 2020; Accepted 27 January 2020 0005-2736/ © 2020 Elsevier B.V. All rights reserved.
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Fig. 1. Chemical structures for the 25 tested flavonoids in this study.
fluorescein family, is a good substrate for OATP1B1 [26]. Due to its fluorescence, DCF can be conveniently detected and quantified. For these reasons, DCF should be an ideal probe substrate for OATP1B1 to be used in high-throughput screening (HTS) assay. Therefore, in the present study we used DCF as a substrate to investigate the interaction of flavonoids with OATP1B1. First, OATP1B1-mediated DCF uptake was characterized with Chinese hamster ovary (CHO) cells stably expressing human OATP1B1. Then, the effect of flavonoids on OATP1B1-mediated DCF uptake was investigated. After that, the half maximal inhibitory concentrations (IC50) for the flavonoids that showed significant
it is of great significance to extensively investigate the interaction between flavonoids and OATP1B1. In the present study, 25 common flavonoids were investigated for their interactions with OATP1B1, including 4 flavanols (epicatechin, epicatechin gallate, epigallocatechin, and epigallocatechin gallate), 11 flavonols (fisetin, galangin, gossypetin, kaempferol, myricetin, quercetin, hyperoside, isoquercitrin, myricitrin, quercitrin, and rutin), 5 flavones (apigenin, apigetrin, chrysin, luteolin, and oroxylin A), 2 flavanones (naringenin and prunin), and 3 isoflavones (daidzein, genistein, and puerarin) (Fig. 1). 2′,7′-Dichlorofluorescein (DCF), a commercially available dye of the 2
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lysed with 300 μL of 1% Triton X-100 in phosphate buffered saline (PBS) per well. After that, [3H]E3S was quantified by liquid scintillation counting and the fluorescence of DCF was measured by a Tecan Infinite M1000 PRO microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Total protein concentration was determined using the BCA assay with bovine serum albumin as a standard and uptake was normalized to total protein concentration. OATP1B1-specific uptake was calculated by subtracting the background uptake of CHO-EV cells from the uptake of CHO-OATP1B1 cells.
inhibition for OATP1B1 were measured. Finally, three-dimensional quantitative structure-activity relationship (3D-QSAR) studies were carried out on the investigated flavonoids using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) to elucidate their structure-activity relationships. 2. Materials and methods 2.1. Materials Radiolabeled [3H]estrone-3-sulfate ([3H]E3S) was from PerkinElmer (Waltham, MA). 2′,7′-Dichlorofluorescein (DCF) was purchased from Sigma-Aldrich (St. Louis, MO). The 25 flavonoid compounds were purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, Sichuan, China) and Nanjing Spring & Autumn Biological Engineering Co., Ltd. (Nanjing, Jiangsu, China) (purity 95–99%). Fetal bovine serum (FBS), Penicillin/Streptomycin, and trypsin were from Hyclone (Logan, UT). Ham's F12, L-glutamine, Lipofectamine 2000, Opti-MEM, and Flp-In system including Flp-In-CHO cell line, pcDNA5/ FRT vector, pOG44, zeocin, and hygromycin B were from Thermo Fisher Scientific (Carlsbad, CA). Sulfo-N-hydroxysuccinimide-SS-biotin, streptavidin-agarose beads, and the BCA protein assay kit were purchased from Pierce Chemical (Rockford, IL). Antibodies for detecting the 6-His tag and the Na+/K+-ATPase α subunit were purchased from Tiangen (Beijing, China) and Abcam (Boston, MA). Horseradish peroxidase-conjugated secondary antibodies were purchased from ProteinTech (Chicago, IL) and Sunshine Biotechnology (Nanjing, China). Immobilon Western blot detection kit was from Millipore (Billerica, MA).
2.3. Cell surface biotinylation and immunoblot analysis CHO-EV and CHO-OATP1B1 cells were plated in a 6-well plate at a density of 250,000 cells per well and induced with 5 mM sodium butyrate 48 h later. After 24 h of induction, cells were treated with 1 mL of sulfo-N-hydroxysuccinimide-SS-biotin (1 mg/mL in PBS) for 1 h at 4 °C. Then, cells were washed three times with 2 mL of ice-cold PBS containing 100 mM glycine and incubated for 10 min at 4 °C with the same buffer. After wash three times with ice-cold PBS, cells were lysed with 700 μL of lysis buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, and 1% Triton X-100, pH 7.4, containing protease inhibitors). Lysates were centrifuged at 10,000 ×g for 2 min. Supernatants were incubated with 140 μL of streptavidin-agarose beads for 1 h at room temperature. After wash three times with ice-cold lysis buffer, cell surface proteins were recovered from the resin by incubation of the beads with 2 × Laemmli buffer containing 100 mM dithiothreitol. Cell membrane proteins were then subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis. OATP1B1 was detected with a mouse anti-His antibody (Tiangen) (1:2000), followed by HRP-conjugated goat anti-mouse IgG (ProteinTech) (1:5000). Plasma membrane marker Na+/K+-ATPase was detected with a rabbit anti-Na+/K+ATPase α subunit antibody (Abcam) (1:5000), followed by HRP-conjugated goat anti-rabbit IgG (Sunshine) (1:10000). Immunoblots were developed with chemiluminescence method and scanned with ChemiDoc MP imaging system (Bio-Rad, Hercules, CA).
2.2. Cell culture, generation of stable cell lines, and uptake assay Flp-In-CHO cells were grown at 37 °C in a humidified 5% CO2 atmosphere in Ham's F12 medium supplemented with 10% FBS, 2 mM Lglutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 100 μg/ mL zeocin. Flp-In-CHO cells stably expressing OATP1B1 were generated as follows. First, a six His-tag was introduced at the C-terminal end of the open reading frame of human OATP1B1*1b [27] by PCR. The resulting construct was cloned into pcDNA5/FRT vector (Invitrogen, Carlsbad, CA) via NheI and NotI sites and verified by DNA sequencing. Then, Flp-In-CHO cells were transfected with pcDNA5/FRT empty vector (EV) or the constructed pcDNA5/FRT-OATP1B1 vector together with pOG44 using Lipofectamine 2000 following the manufacturer's instructions. Initially, the cell culture medium was supplemented with 600 μg/mL hygromycin B for transfectant selection. After single clones were isolated by limited dilution, the concentration of hygromycin B was lowered to 300 μg/mL. OATP1B1 clones were characterized by surface biotinylation and immunoblotting and functional assay, and one clone (CHO-OATP1B1) with highest transport function was employed for the following studies. One empty vector-transfected clone (CHO-EV) with hygromycin B resistance was used as negative control. Cells within ten passages were used for all experiments in the present study. For uptake assay, CHO-EV and CHO-OATP1B1 cells were plated at 40,000 cells per well on 24-well plates and 48-h later medium was replaced with medium containing 5 mM sodium butyrate, a histone deacetylase inhibitor, to induce nonspecific gene expression [28]. After another 24 h in culture, the cells were used for uptake experiments. Cells were washed three times with pre-warmed uptake buffer (116.4 mM NaCl, 5.3 mM KCl, 1 mM NaH2PO4, 0.8 mM MgSO4, 5.5 mM D-glucose and 20 mM Hepes, pH adjusted to 7.4 with Trizma base). Uptake was started by adding 200 μL of uptake buffer containing 0.4 μCi/mL of [3H]E3S or 5 μM DCF in the presence or absence of flavonoids. After incubation for a specific period of time, uptake was terminated by removing the uptake solution and washing the cells four times with 1 mL of ice-cold uptake buffer each time. Then cells were
2.4. Effect of flavonoids on OATP1B1-mediated DCF uptake and determination of their IC50 values First, inhibition screening assays were performed with 5 μM DCF in the absence and presence of 10 and 100 μM flavonoids. Flavonoids with significant inhibition effect were selected to determine their IC50 values by measuring the uptake of DCF in the absence and presence of increasing concentrations of each flavonoid. 2.5. Computational methods 2.5.1. Determination of the conformations of flavonoids and their structural alignment The molecular structures of flavonoids were sketched in Sybyl-X 2.0 program [29] and their initial conformations were generated by molecular mechanics optimization using the Tripos force field and Gasteiger-Hückel charges [30], with an energy gradient convergence criterion of 0.05 kcal/mol and a distance-dependent dielectric constant of 1. After that, conformational search with acyclic rotatable bonds was performed for each flavonoid by Confort program [31]. The conformation with lowest energy was selected and subjected to energy minimization until convergence. The resulting conformation was used for molecular alignment. Initial alignment was obtained by aligning the flavonoids according to their backbone structures. Initial CoMFA and CoMSIA models were developed and outliers (prediction error > 0.4 log unit) were subjected to conformational adjustment by rotating the rotatable bonds. However, all rotations were confined within 10 kcal/ mol of its lowest energy. 3
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significantly higher E3S transport activity than CHO-EV. Then, with the established cell lines we characterized OATP1B1-mediated DCF uptake. Time-dependent uptake assay showed that OATP1B1-mediated DCF uptake was linear up to at least 2 min (Fig. 3). Therefore, the uptake time for kinetic and inhibition assay in the present study was set to be 2 min. Kinetic study showed that OATP1B1-mediated DCF uptake exhibited a monophasic saturation kinetics (Fig. 4B) with a Km value of 9.7 ± 2.4 μM.
2.5.2. CoMFA and CoMSIA analyses The inhibition constants were expressed in pIC50 (−logIC50) values, which were used as the dependent variables in CoMFA and CoMSIA analyses. In CoMFA analysis, steric and electrostatic field energies were probed with an sp3 carbon atom having a charge of +1. Steric and electrostatic interactions were calculated using a Tripos force field with a distance-dependent dielectric constant. A cutoff value of 30 kcal/mol was applied on steric and electrostatic fields. Column filtering was set to be of 1.0 kcal/mol. In CoMSIA analysis, five different similarity fields, namely steric, electrostatic, hydrophobic, hydrogen-bond donor, and hydrogen-bond acceptor fields, were calculated with a probe atom having a radius of 1 Å, charge of +1, hydrophobicity of +1, and hydrogen bonding donor and acceptor properties of +1. These fields cover the major contributions to ligand binding [32]. The attenuation factor was set to be of 0.3. The crossvalidated r2 (q2) and optimum number of components were obtained by the partial least-squares (PLS) method with the leave-one-out option. With the obtained optimum numbers of components, the final non-crossvalidated CoMFA and CoMSIA models were developed.
3.2. Effect of flavonoids on OATP1B1-mediated DCF uptake As shown above, DCF is a good substrate for OATP1B1 and can be easily and conveniently detected and quantified due to its fluorescence. Therefore, DCF was used to investigate the interaction between flavonoids and OATP1B1 in this study. First, inhibition assay was carried out for the 25 common flavonoids on OATP1B1-mediated DCF uptake. Uptake of 5 μM DCF in the absence and presence of 10 and 100 μM flavonoids with CHO-EV and CHO-OATP1B1 cells was measured. The results were summarized in Fig. 5. Bromosulfophthalein (BSP), a known OATP1B1 inhibitor [33], was used as a positive control and it strongly inhibited OATP1B1-mediated DCF uptake as expected. At the concentration of 10 μM, luteolin, oroxylin A, quercetin, and apigenin showed the strongest inhibitory effect on OATP1B1 with inhibition rates > 70% and luteolin showing the greatest inhibition. Myricetin, fisetin, kaempferol, chrysin, genistein, and myricitrin showed moderate inhibitory effect with 40–70% inhibition. Isoquercitrin, naringenin, quercitrin, epigallocatechin gallate (EGCG), rutin, and apigetrin showed weak effect with 20–40% inhibition on OATP1B1-mediated DCF uptake (Fig. 5). However, daidzein, epicatechin (EC), epigallocatechin (EGC), galangin, gossypetin, hyperoside, prunin, and puerarin had no significant effect on DCF uptake. On the contrary, epicatechin gallate (ECG) showed a stimulating effect on OATP1B1-mediated DCF uptake. At the concentration of 100 μM, totally 19 flavonoids namely apigenin, apigetrin, chrysin, EGCG, fisetin, genistein, gossypetin, hyperoside, isoquercitrin, kaempferol, luteolin, myricetin, myricitrin, naringenin, oroxylin A, prunin, quercetin, quercitrin, and rutin showed inhibition rates > 60%. These 19 flavonoids were selected for further characterization by measuring their IC50 values on OATP1B1-mediated DCF uptake. At the concentration of 100 μM, ECG showed a very weak inhibitory effect on OATP1B1 instead of a stimulating effect as observed at the concentration of 10 μM.
2.6. Data analysis Data statistical analysis was performed with Prism 5 (GraphPad Software, San Diego, CA). Student's t-test was used when comparing two sets of data. One-way ANOVA followed by Dunnett's test was employed to analyze statistically significant difference for more than two groups. The p value for statistical significance was set to be < 0.05 at 95% confidence interval. The IC50 and Ki values were calculated by nonlinear least-squares data fitting. 3. Results 3.1. Characterization of OATP1B1-mediated 2′,7′-dichlorofluorescein (DCF) uptake Using Flp-In system, CHO cell lines stably transfected with empty vector (CHO-EV) and human OATP1B1 (CHO-OATP1B1) were constructed. The surface expression and function of OATP1B1 were confirmed by surface biotinylation and Western blot and uptake assay (Fig. 2). As shown in Fig. 2A, OATP1B1 had normal expression on cell surface. Estrone-3-sulfate (E3S), a model substrate for OATP1B1, was used to examine OATP1B1's function. As shown in Fig. 2B, the expressed OATP1B1 had normal function as CHO-OATP1B1 cells showed
Fig. 2. Surface expression and functional characterization of human OATP1B1 in Flp-In-CHO cells. (A) Surface expression of OATP1B1 in CHO cells. Plasma membrane proteins were isolated by surface biotinylation and OATP1B1 was detected with an anti-His antibody. The plasma membrane marker Na+/K+ATPase α subunit was used as protein loading control. (B) Functional characterization of OATP1B1 in CHO cells. Uptake of [3H]estrone-3-sulfate was measured at 37 °C for 1 min with CHO-EV and CHOOATP1B1 cells. Values were given as the mean ± SD of triplicate determinations; ⁎p < 0.05.
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Fig. 3. Time-dependent uptake of 2′,7′-dichlorofluorescein (DCF) mediated by OATP1B1. (A) Uptake mediated by CHO-EV and CHO-OATP1B1. (B) Net uptake mediated by OATP1B1. Uptake of 5 μM DCF was measured at 37 °C for indicated time points with CHO-EV and CHO-OATP1B1 cells. Net uptake was obtained by subtracting the uptake of CHO-EV cells from that of CHO-OATP1B1 cells. Data shown as mean ± SD of one representative of three independent experiments performed in triplicate.
Fig. 4. Concentration-dependent uptake of DCF mediated by OATP1B1. (A) Uptake mediated by CHO-EV and CHO-OATP1B1. (B) Net uptake mediated by OATP1B1. An Eadie-Hofstee plot of OATP1B1-mediated DCF uptake was included as a figure inset. Uptake of increasing concentrations of DCF was measured at 37 °C for 2 min with CHO-EV and CHO-OATP1B1 cells. Net uptake was obtained by subtracting the uptake of CHO-EV cells from that of CHO-OATP1B1 cells and fitted to the Michaelis–Menten equation to obtain Km and Vmax values. Data shown as mean ± SD of one representative of three independent experiments performed in triplicate. Fig. 5. Effect of flavonoids on OATP1B1-mediated DCF uptake. Uptake of 5 μM DCF was measured at 37 °C for 2 min with CHO-EV and CHO-OATP1B1 cells in the absence (control) and presence of 10 μM (open bars) and 100 μM (closed bars) BSP and indicated flavonoids. Values obtained with CHO-EV cells were subtracted from values obtained with CHO-OATP1B1 cells and were given as percent of control. Data shown as mean ± SD from three independent experiments done in triplicate; ⁎p < 0.05 vs control.
3.3. Concentration-dependent effect of flavonoids on OATP1B1-mediated DCF uptake
were listed in Table 1. Among them, luteolin had the strongest inhibitory effect with an IC50 value of 0.7 μM. Quercetin, fisetin, genistein, oroxylin A, and apigenin also had strong inhibitory effect on OATP1B1 with IC50 values smaller than 5 μM. EGCG showed the weakest inhibition among the selected 19 flavonoids with an IC50 value of 40.8 μM. Inhibition kinetic study was carried out for quercetin which is the
The concentration-dependent effect of flavonoids on OATP1B1mediated DCF uptake was investigated for the above-listed 19 flavonoids, which showed high OATP1B1 inhibition activity when tested at the concentration of 100 μM. The IC50 values of these 19 flavonoids 5
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Table 1 IC50 values for DCF and the selected 19 flavonoid compounds on OATP1B1mediated DCF uptake and the characteristic features affecting their inhibitory potency. Compounds
IC50 (μM)
Characteristic features affecting inhibitory potency
a
DCF Apigenin Apigetrin Chrysin EGCG Fisetin Genistein Gossypetin Hyperoside Isoquercitrin Kaempferol
14.7 4.9 ± 1.1 25.0 ± 1.3 7.4 ± 1.3 40.8 ± 1.8 3.3 ± 0.3 4.6 ± 1.0 18.4 ± 1.4 25.3 ± 1.3 20.8 ± 1.2 8.5 ± 0.4
Luteolin Myricetin
0.7 ± 0.2 5.8 ± 1.3
Myricitrin Naringenin Oroxylin A Prunin Quercetin
10.4 ± 1.2 15.6 ± 1.8 4.7 ± 0.1 36.6 ± 1.3 2.4 ± 1.1
Quercitrin Rutin
11.5 ± 0.9 20.5 ± 1.2
Sugar moiety on 7-OH reducing inhibitory potency
Sugar moiety on 3-OH reducing inhibitory potency Sugar moiety on 3-OH reducing inhibitory potency 3-OH reducing inhibitory potency 4′-OH enhancing inhibitory potency An additional –OH at 5′ reducing inhibitory potency Sugar moiety on 3-OH reducing inhibitory potency
Sugar moiety on 7-OH reducing inhibitory potency 3-OH reducing inhibitory potency 3′- and 4′-OH enhancing inhibitory potency Sugar moiety on 3-OH reducing inhibitory potency Sugar moiety on 3-OH reducing inhibitory potency
Fig. 7. Structural alignment of DCF and the 18 flavonoid compounds.
Concentration-dependent inhibition of 5 μM DCF by indicated flavonoids was measured at 37 °C for 2 min with CHO-EV and CHO-OATP1B1 cells. Values obtained with CHO-EV cells were subtracted from values obtained with CHOOATP1B1 cells and were used to calculate IC50 values by non-linear regression analysis. Values were given as the mean ± SE from two to four independent experiments. aThe IC50 of DCF was calculated with equation IC50 = Ki (1 + [S]/Km) where Ki was assumed to be equal to Km.
Activity changes of ligands are related to their molecular structural changes. To elucidate their structure-activity relationship in a quantitative manner, CoMFA and CoMSIA 3D-QSAR studies were carried out for the substrate DCF and 19 flavonoids with IC50 values measured.
3.4. CoMFA and CoMSIA analyses The conformations of DCF and the 19 flavonoids were generated with conformational search and aligned according to their backbone structures. Initial CoMFA and CoMSIA models were developed and improved by adjusting the conformations of some flavonoids that had high prediction error with visual inspection. However, gossypetin significantly decreased the crossvalidated r2 (q2) when included in CoMFA and CoMSIA model development and its conformation could be hardly adjusted to improve the models. Therefore, we excluded gossypetin and the remaining 18 flavonoids together with DCF were used for CoMFA and CoMSIA modeling. Fig. 7 showed the structural alignment of these 19 compounds. Among them, 17 compounds were used as training set to develop CoMFA and CoMSIA models. The remaining two compounds, namely apigenin (relatively strong inhibitor) and rutin (relatively weak inhibitor), were used as test set for model validation. CoMFA and CoMSIA models were developed with PLS analysis and their statistics were listed in Table 2. The q2 values for CoMFA and CoMSIA were 0.604 and 0.534, respectively. The optimal numbers of Fig. 6. Eadie-Hofstee plot of the inhibitory effect of quercetin on OATP1B1mediated DCF uptake. Uptake of increasing concentrations (3 to 50 μM) of DCF was measured at 37 °C for 2 min in the absence or presence of 1 and 3 μM quercetin. Data shown as mean ± SD of one representative of two independent experiments performed in triplicate.
Table 2 Statistical parameters of CoMFA and CoMSIA modeling.
PLS statistics q2 r2 Standard error of estimate Optimal number of components
most extensively studied flavonoid and one of the most abundant flavonoids in foods [34] and its inhibition type on OATP1B1-mediated DCF transport was determined. Fig. 6 showed the Eadie-Hofstee plot of OATP1B1-mediated uptake of DCF in the presence or absence of quercetin, indicating that quercetin inhibited OATP1B1-mediated DCF uptake in a competitive manner by affecting the apparent affinity but not the maximal transport rate. The apparent Ki value was determined to be 1.4 ± 0.1 μM.
Field contributions (%) Steric Electrostatic Hydrophobic Hydrogen-bond donor Hydrogen-bond acceptor
6
CoMFA
CoMSIA
0.604 0.841 0.203 3
0.534 0.807 0.216 2
50.3 49.7
10.5 26.0 13.8 23.9 25.8
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Fig. 8. CoMFA and CoMSIA predictions for DCF and the 18 flavonoid compounds. ●, training set (DCF and 16 flavonoids); , test set (apigenin and rutin).
models were developed to elucidate their structure-activity relationships. To the best of our knowledge, these were the first CoMFA and CoMSIA models developed for flavonoids as OATP1B1 inhibitors. Kinetic study showed that OATP1B1-mediated DCF transport exhibited a monophasic kinetic behavior (Fig. 4B, inset), indicating a single binding site for DCF in OATP1B1. However, several studies showed that the kinetics of OATP1B1-mediated E3S transport was biphasic and consisted of high-affinity low-capacity and low-affinity highcapacity components, indicating the presence of two binding sites for E3S in OATP1B1 [35–37]. Among the tested flavonoids in the present study, ECG exhibited a stimulating effect on OATP1B1-mediated DCF uptake (Fig. 5). This result suggested that ECG might allosterically alter OATP1B1-mediated DCF transport. Interestingly, another study showed that ECG had an inhibitory effect on OATP1B1-mediated E3S uptake [19]. These results indicated that DCF and ECG probably bind to different binding sites in OATP1B1. ECG might bind to the high-affinity binding site of E3S upon which it exerted an inhibitory effect on OATP1B1-meidated E3S uptake, while DCF binds to the other site. In addition, a study showed that E3S had an inhibitory effect on OATP1B1-mediated DCF uptake [26]. This is kind of expected as E3S can bind both binding sites. The effect of DCF on OATP1B1-mediated transport of E3S and other substrates and their mutual inhibition kinetics remain to be investigated. The only structural difference between luteolin and quercetin is that quercetin has an additional hydroxyl group at C-3 position (Fig. 1). This C-3 hydroxyl group decreased the inhibitory potential of the flavonoids 3-fold as the IC50 values for luteolin and quercetin were 0.7 and 2.4 μM, respectively (Table 1). A 2-fold decrease of activity was also observed for apigenin with this C-3 hydroxyl group as the IC50 values increasing from 4.9 μM (apigenin) to 8.5 μM (kaempferol) (Table 1). The C-3
components for CoMFA and CoMSIA were 3 and 2, respectively. Noncrossvalidation PLS analyses with the optimal numbers of components revealed conventional (non-crossvalidated) r2 of 0.841 and 0.807 for CoMFA and CoMSIA, respectively. The standard errors of estimate for CoMFA and CoMSIA were 0.203 and 0.216, respectively. In the CoMFA model, steric fields contributed 50.3% to the model's information, while electrostatic fields contributed the other 49.7%. In the CoMSIA model, the contributions of steric, electrostatic, hydrophobic, hydrogen-bond donor, and hydrogen-bond acceptor fields were 10.5, 26.0, 13.8, 23.9, and 25.8%, respectively. The reliability and predictive power of the CoMFA and CoMSIA models were verified with the compounds in training and test sets. The correlations between the predicted activities by CoMFA and CoMSIA and experimental activities for DCF and the 18 flavonoids were depicted in Fig. 8. The predicted activities by the CoMFA and CoMSIA models were in good agreement with the experimental data with R2 being of 0.837 and 0.808, respectively, indicating that our CoMFA and CoMSIA models were reliable. Figs. 9 and 10 showed the CoMFA and CoMSIA contour maps together with quercetin. 4. Discussion In the present study, a Flp-In-CHO cell line stably expressing human OATP1B1 was constructed and the interactions of 25 flavonoid compounds with OATP1B1 were investigated using a fluorescent substrate DCF which could be detected and quantified conveniently. Kinetic study showed that OATP1B1-mediated DCF uptake was saturable with a Km value of 9.7 ± 2.4 μM. Inhibition assay for the 25 flavonoids on OATP1B1-mediated DCF uptake was performed and 19 flavonoids were selected to determine their IC50 values upon which CoMFA and CoMSIA
Fig. 9. CoMFA contour maps in combination with quercetin. (A) Steric field. Green, steric bulk desirable regions; yellow, steric bulk undesirable regions. (B) Electrostatic field. Blue, positive charge desirable regions; red, negative charge desirable regions. 7
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Fig. 10. CoMSIA contour maps in combination with quercetin. (A) Steric field. Green, steric bulk desirable regions; yellow, steric bulk undesirable regions. (B) Electrostatic field. Blue, positive charge desirable regions; red, negative charge desirable regions. (C) Hydrophobic field. Yellow, hydrophobicity desirable regions; white, hydrophobicity undesirable regions. (D) Hydrogen-bond donor field. Cyan, donor desirable regions; purple, donor undesirable regions. (E) Hydrogen-bond acceptor field. Magenta, acceptor desirable regions; red, acceptor undesirable regions.
the inhibition of OATP1B1 were increased with increasing number of hydroxyl groups on ring B. The IC50 values of chrysin, apigenin, and luteolin were 7.4, 4.9, and 0.7 μM, respectively (Table 1). Similarly, galangin, kaemferol, and quercetin have zero, one, and two hydroxyl groups on ring B (Fig. 1) and their activities were also increased with increasing number of hydroxyl groups on ring B (Fig. 5 and Table 1). These results indicated that the hydroxyl groups at C-3′ and C-4′ positions on ring B of flavonoids could strengthen their interaction with OATP1B1. According to our CoMSIA model, hydrogen-bond forming groups at C-3′ and C-4′ positions of ring B were beneficial for flavonoid's binding (cyan and magenta contours in Fig. 10D and E). This could explain why chrysin, apigenin, and luteolin showed increasing order of activities (Fig. 1 and Table 1). The same order of activities was also observed for galangin, kaemferol, and quercetin for the same reason (Fig. 5 and Table 1). However, hydrogen-bond donor and acceptor unfavorable contours existed near C-5′ position of ring B (purple and red contours in Fig. 10D and E). Myricetin has an additional hydroxyl group at C-5′ position as compared to quercetin. According to our CoMSIA results, this addition hydroxyl group was not beneficial for myricetin's interaction with OATP1B1. In fact, our experimental data showed that myricetin had lower activity than quercetin (Table 1), indicating that our model had a good predictive ability. These results also indicated that the number of hydroxyl groups on ring B should not exceed two for flavonoids to have a strong inhibitory potency. Previously, we proposed an interaction model between OATP1B1 and nuclear receptor ligands, in which a negative charge center and a hydrophobic center of the ligands were important for their binding [38]. Flavonoids do not have an explicit negative charge center. However, the hydroxyl group on ring B was important for flavonoid's activity. It might play a somewhat similar role as the negative charge center in nuclear receptor ligands because a phenolic hydroxyl group can be partially negatively charged due to its proton dissociation. In the areas around rings A and C of flavonoids, bulky hydrophilic groups
hydroxyl group also decreased chrysin's activity as galangin showed lower inhibition rate than chrysin (Fig. 5). Therefore, for the flavonoids tested in the present study flavones had higher activity than their 3hydroxyflavone counterparts (flavonols). Attaching a sugar moiety to the C-3 hydroxyl group would further decrease the inhibitory activity of flavonoids on OATP1B1. Quercitrin (quercetin-3-O-rhamnoside), isoquercitrin (quercetin-3-O-glucoside), hyperoside (quercetin-3-O-galactoside), and rutin (quercetin-3-O-rutinoside) are 3-O-glycosides of quercetin (Fig. 1). Their IC50 values were 5–10 folds higher than that of quercetin (Table 1). Similarly, myricitrin (myricetin-3-O-rhamnoside) showed 2-fold decreased inhibitory activity on OATP1B1 as compare to myricetin whose IC50 values were 10.4 and 5.8 μM, respectively (Table 1). The presence of sugar moieties at positions C-7 and C-8 could also decrease flavonoid's inhibition activities. For example, apigenin showed 5-fold higher activity than apigetrin (apigenin-7-O-glucoside) and naringenin showed 2-fold higher activity than prunin (naringenin-7-Oglucoside) (Fig. 5 and Table 1). Similarly, daidzein had stronger inhibitory effect on OATP1B1 than puerarin (daidzein-8-C-glucoside) (Fig. 5). Therefore, it seems that flavonoid aglycones usually interact with OATP1B1 much stronger than their glycosides. Based on the steric and hydrophobic contour maps of our CoMFA and CoMSIA models, bulky and hydrophilic groups at C-3, C-4, C-7, and C-8 positions of rings A and C was detrimental to flavonoid's binding with OATP1B1 (yellow contours in Figs. 9A, 10A and C). In addition, hydrogen-bond forming groups in these areas were also detrimental to the interaction of flavonoids with OATP1B1 (purple and red contours in Fig. 10D and E). These could explain why quercetin, myricetin, apgenin, naringenin, and daidzein had stronger inhibition on OATP1B1 than their 3-, 7-, and 8-glycosides. The structural difference between chrysin, apigenin, and luteolin is the number of hydroxyl groups on ring B (Fig. 1). Chrysin, apigenin, and luteolin have zero, one (at C-4′ position), and two hydroxyl groups (at C-3′ and C-4′ positions) on ring B, respectively. Their activities for 8
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were unfavorable which might bear some resemblance to the hydrophobic center of nuclear receptor ligands. However, bulky substitution in the hydrophobic center area of nuclear receptor ligands was favorable, indicating that the binding sites for flavonoids and nuclear receptor ligands in OATP1B1 have some similarity but not identical. In conclusion, by using the fluorescent substrate DCF and 3D-QSAR analysis this study obtained some useful information on the interactions between flavonoids and OATP1B1. OATP1B1-mediated DCF uptake exhibited monophasic kinetics with a Km of 9.7 ± 2.4 μM. While most tested flavonoids had an inhibitory effect on OATP1B1-mediated DCF uptake, ECG showed a stimulating effect. Our experimental and computational results showed that flavonoid aglycones interacted with OATP1B1 much stronger than their glycosides such as 3-, 7-, and 8glycosides as bulky hydrophilic and hydrogen-bond forming groups at C-3, C-7, and C-8 positions on rings A and C of flavonoids were unfavorable for their binding. The presence of hydrogen-bond forming groups on ring B of flavonoids was beneficial for their interaction with OATP1B1 as long as the number of hydrogen-bond forming groups was not > 2. Our results also indicated that flavones usually had higher inhibitory activity on OATP1B1 than their 3-hydroxy derivatives (flavonols). The obtained information and 3D-QSAR models could be useful in elucidating and predicting the interactions between flavonoids and human OATP1B1.
[11] K. Mandery, B. Balk, K. Bujok, I. Schmidt, M.F. Fromm, H. Glaeser, Inhibition of hepatic uptake transporters by flavonoids, Eur. J. Pharm. Sci. 46 (2012) 79–85. [12] M. Wang, H. Qi, J. Li, Y. Xu, H. Zhang, Transmembrane transport of steviol glucuronide and its potential interaction with selected drugs and natural compounds, Food Chem. Toxicol. 86 (2015) 217–224. [13] F. Farhadi, B. Khameneh, M. Iranshahi, M. Iranshahy, Antibacterial activity of flavonoids and their structure-activity relationship: an update review, Phytother. Res. 33 (2019) 13–40. [14] T.P. Cushnie, A.J. Lamb, Antimicrobial activity of flavonoids, Int. J. Antimicrob. Agents 26 (2005) 343–356. [15] V.J. Jasmin, A review on molecular mechanism of flavonoids as antidiabetic agents, Mini Rev Med Chem. 19 (2019) 762–786. [16] D. Maaliki, A.A. Shaito, G. Pintus, A. El-Yazbi, A.H. Eid, Flavonoids in hypertension: a brief review of the underlying mechanisms, Curr. Opin. Pharmacol. 45 (2019) 57–65. [17] S.J. Maleki, J.F. Crespo, B. Cabanillas, Anti-inflammatory effects of flavonoids, Food Chem. 299 (2019) 125124. [18] D. Raffa, B. Maggio, M.V. Raimondi, F. Plescia, G. Daidone, Recent discoveries of anticancer flavonoids, Eur. J. Med. Chem. 142 (2017) 213–228. [19] M. Roth, B.N. Timmermann, B. Hagenbuch, Interactions of green tea catechins with organic anion-transporting polypeptides, Drug Metab. Dispos. 39 (2011) 920–926. [20] M. Roth, J.J. Araya, B.N. Timmermann, B. Hagenbuch, Isolation of modulators of the liver-specific organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 from Rollinia emarginata Schlecht (Annonaceae), J. Pharmacol. Exp. Ther. 339 (2011) 624–632. [21] Y. Zhang, A. Hays, A. Noblett, M. Thapa, D.H. Hua, B. Hagenbuch, Transport by OATP1B1 and OATP1B3 enhances the cytotoxicity of epigallocatechin 3-O-gallate and several quercetin derivatives, J. Nat. Prod. 76 (2013) 368–373. [22] F. Wen, M. Shi, J. Bian, H. Zhang, C. Gui, Identification of natural products as modulators of OATP2B1 using LC-MS/MS to quantify OATP-mediated uptake, Pharm. Biol. 54 (2016) 293–302. [23] D.G. Bailey, G.K. Dresser, B.F. Leake, R.B. Kim, Naringin is a major and selective clinical inhibitor of organic anion-transporting polypeptide 1A2 (OATP1A2) in grapefruit juice, Clin. Pharmacol. Ther. 81 (2007) 495–502. [24] H. Fuchikami, H. Satoh, M. Tsujimoto, S. Ohdo, H. Ohtani, Y. Sawada, Effects of herbal extracts on the function of human organic anion-transporting polypeptide OATP-B, Drug Metab. Dispos. 34 (2006) 577–582. [25] X. Wang, A.W. Wolkoff, M.E. Morris, Flavonoids as a novel class of human organic anion-transporting polypeptide OATP1B1 (OATP-C) modulators, Drug Metab. Dispos. 33 (2005) 1666–1672. [26] S. Izumi, Y. Nozaki, T. Komori, O. Takenaka, K. Maeda, H. Kusuhara, Y. Sugiyama, Investigation of fluorescein derivatives as substrates of organic anion transporting polypeptide (OATP) 1B1 to develop sensitive fluorescence-based OATP1B1 inhibition assays, Mol. Pharm. 13 (2016) 438–448. [27] R.G. Tirona, B.F. Leake, G. Merino, R.B. Kim, Polymorphisms in OATP-C: identification of multiple allelic variants associated with altered transport activity among European- and African-Americans, J. Biol. Chem. 276 (2001) 35669–35675. [28] D.P. Palermo, M.E. DeGraaf, K.R. Marotti, E. Rehberg, L.E. Post, Production of analytical quantities of recombinant proteins in Chinese hamster ovary cells using sodium butyrate to elevate gene expression, J. Biotechnol. 19 (1991) 35–47. [29] Sybyl-X 2.0 Tripos International, St, USA, Louis, MO, 2012. [30] J. Gasteiger, M. Marsili, Iterative partial equalization of orbital electronegativity—a rapid access to atomic charges, Tetrahedron 36 (1980) 3219–3228. [31] R.S. Pearlman, R. Balducci, Confort: A Novel Algorithm for Conformational Analysis, National Meeting of the American Chemical Society, (1998). [32] G. Klebe, U. Abraham, Comparative molecular similarity index analysis (CoMSIA) to study hydrogen-bonding properties and to score combinatorial libraries, J. Comput. Aided Mol. Des. 13 (1999) 1–10. [33] Y. Nie, J. Yang, S. Liu, R. Sun, H. Chen, N. Long, R. Jiang, C. Gui, Genetic polymorphisms of human hepatic OATPs: functional consequences and effect on drug pharmacokinetics, Xenobiotica 50 (2020) 297–317. [34] D.B. Haytowitz, X. Wu, S. Bhagwat, USDA Database for the Flavonoid Content of Selected Foods, Release 3.3., U.S. Department of Agriculture, Agricultural Research Service. Nutrient Data Laboratory Home Page: http://www.ars.usda.gov/ nutrientdata/flav (2018). [35] I. Tamai, T. Nozawa, M. Koshida, J. Nezu, Y. Sai, A. 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Declaration of competing interest All authors declare no conflicts of interest. Acknowledgement This work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References [1] M.A. Hediger, M.F. Romero, J.B. Peng, A. Rolfs, H. Takanaga, E.A. Bruford, The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteins. Introduction, Pflugers Arch. 447 (2004) 465–468. [2] B. Hagenbuch, C. Gui, Xenobiotic transporters of the human organic anion transporting polypeptides (OATP) family, Xenobiotica 38 (2008) 778–801. [3] M. Roth, A. Obaidat, B. Hagenbuch, OATPs, OATs and OCTs: the organic anion and cation transporters of the SLCO and SLC22A gene superfamilies, Br. J. Pharmacol. 165 (2012) 1260–1287. [4] A. Obaidat, M. Roth, B. Hagenbuch, The expression and function of organic anion transporting polypeptides in normal tissues and in cancer, Annu. Rev. Pharmacol. Toxicol. 52 (2012) 135–151. [5] U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), In Vitro Metabolism- and Transporter- Mediated Drug-Drug Interaction Studies Guidance for Industry, 2017. [6] Y. Shitara, M. Hirano, H. Sato, Y. Sugiyama, Gemfibrozil and its glucuronide inhibit the organic anion transporting polypeptide 2 (OATP2/OATP1B1:SLC21A6)-mediated hepatic uptake and CYP2C8-mediated metabolism of cerivastatin: analysis of the mechanism of the clinically relevant drug-drug interaction between cerivastatin and gemfibrozil, J. Pharmacol. Exp. Ther. 311 (2004) 228–236. [7] Y. Shitara, T. Itoh, H. Sato, A.P. Li, Y. Sugiyama, Inhibition of transporter-mediated hepatic uptake as a mechanism for drug-drug interaction between cerivastatin and cyclosporin A, J. Pharmacol. Exp. Ther. 304 (2003) 610–616. [8] S.G. Simonson, A. Raza, P.D. Martin, P.D. Mitchell, J.A. Jarcho, C.D. Brown, A.S. Windass, D.W. Schneck, Rosuvastatin pharmacokinetics in heart transplant recipients administered an antirejection regimen including cyclosporine, Clin. Pharmacol. Ther. 76 (2004) 167–177. [9] A. Treiber, R. Schneiter, S. Hausler, B. Stieger, Bosentan is a substrate of human OATP1B1 and OATP1B3: inhibition of hepatic uptake as the common mechanism of its interactions with cyclosporin A, rifampicin, and sildenafil, Drug Metab. Dispos. 35 (2007) 1400–1407. [10] M. Hirano, K. Maeda, Y. Shitara, Y. Sugiyama, Drug-drug interaction between pitavastatin and various drugs via OATP1B1, Drug Metab. Dispos. 34 (2006) 1229–1236.
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BBA - Biomembranes journal homepage: www.elsevier.com/locate/bbamem
Investigation of the interactions between flavonoids and human organic anion transporting polypeptide 1B1 using fluorescent substrate and 3DQSAR analysis ⁎
T
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Yiqun Xiang1, Shuai Liu1, Jingjie Yang, Zhongmin Wang, Hongjian Zhang , Chunshan Gui College of Pharmaceutical Sciences, Soochow University, 199 Renai Road, Suzhou Industrial Park, Suzhou, Jiangsu 215123, China
A R T I C LE I N FO
A B S T R A C T
Keywords: OATP1B1 Transporter Flavonoid 3D-QSAR CoMFA CoMSIA
Organic anion transporting polypeptide 1B1 (OATP1B1) is a key hepatic uptake transporter whose inhibition could lead to adverse drug-drug and drug-food interactions. Flavonoids are widely distributed in food and beverages and thus our bodies are frequently exposed to them. Therefore, investigation of the interactions between OATP1B1 and flavonoids could be of great significance. In the present study, 25 common flavonoids were investigated for their interactions with OATP1B1 using the fluorescent substrate 2′,7′-dichlorofluorescein (DCF) and three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis. Kinetic study showed that OATP1B1-mediated DCF uptake exhibited a monophasic saturation kinetics with a Km value of 9.7 ± 2.4 μM. Inhibition assay for flavonoids on OATP1B1-mediated DCF uptake was performed and their IC50 values were determined upon which reliable and predictive CoMFA (q2 = 0.604, r2 = 0.841) and CoMSIA (q2 = 0.534, r2 = 0.807) models were developed. Our experimental and computational results showed that flavonoid aglycones interacted with OATP1B1 much stronger than their glycosides such as 3-O- and 7-O-glycosides as bulky hydrophilic and hydrogen-bond forming substituents at C-3 and C-7 positions on rings A and C were unfavorable for their binding. On the other hand, the presence of hydrogen-bond forming groups on ring B was beneficial as long as the number of hydroxyl groups was not > 2. Our results also indicated that flavones usually interacted with OATP1B1 much stronger than their 3-hydroxyflavone counterparts (flavonols). The obtained information and 3D-QSAR models could be useful for elucidating and predicting the interactions between flavonoids and human OATP1B1.
1. Introduction Organic anion transporting polypeptides (OATPs), which belong to the superfamily of solute carriers (SLCs) [1], are multispecific transporters that mediate the sodium-independent transport of a wide range of endo- and exo-genous amphipathic organic compounds [2,3]. Among the 11 known human OATPs, OATP1B1 is considered to be a liverspecific transporter under normal physiological conditions [4]. It is a key uptake transporter expressed on the sinusoidal membrane of hepatocytes and plays an important role in the hepatic uptake of many drugs [5]. Inhibition of OATP1B1-mediated hepatic uptake of its
substrate drugs by other drugs or natural products might lead to clinically relevant drug-drug and drug-food interactions [6–12]. Flavonoids are a diverse group of phytonutrients abundant in dietary plants and herbs. Chemically, flavonoids have a general structure of 15-carbon skeleton (C6-C3-C6), which consists of two phenyl rings (A and B) and a heterocyclic ring (C) [13]. Studies showed that flavonoids have diverse biological activities such as anti-cancer, antimicrobial, anti-diabetic, anti-inflammatory, and cardiovascular protective effects [14–18]. It has been reported that some flavonoids interact with OATPs [19–25]. Due to the wide distribution and frequent use of flavonoids and the importance of OATP1B1 for drug disposition,
Abbreviations: 3D-QSAR, three-dimensional quantitative structure-activity relationship; BSP, bromosulfophthalein; CHO, Chinese hamster ovary cells; CoMFA, comparative molecular field analysis; CoMSIA, comparative molecular similarity indices analysis; DCF, 2′,7′-dichlorofluorescein; EV, empty vector; E3S, estrone-3sulfate; EC, (−)-epicatechin; ECG, (−)-epicatechin gallate; EGC, (−)-epigallocatechin; EGCG, (−)-epigallocatechin gallate; FBS, fetal bovine serum; HTS, highthroughput screening; IC50, the half maximal inhibitory concentration; OATP, organic anion transporting polypeptide; PBS, phosphate buffered saline; PLS, partial least-squares regression; q2, crossvalidated r2; SLC, solute carrier ⁎ Corresponding authors. E-mail addresses: [email protected] (H. Zhang), [email protected] (C. Gui). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bbamem.2020.183210 Received 20 November 2019; Received in revised form 10 January 2020; Accepted 27 January 2020 0005-2736/ © 2020 Elsevier B.V. All rights reserved.
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Fig. 1. Chemical structures for the 25 tested flavonoids in this study.
fluorescein family, is a good substrate for OATP1B1 [26]. Due to its fluorescence, DCF can be conveniently detected and quantified. For these reasons, DCF should be an ideal probe substrate for OATP1B1 to be used in high-throughput screening (HTS) assay. Therefore, in the present study we used DCF as a substrate to investigate the interaction of flavonoids with OATP1B1. First, OATP1B1-mediated DCF uptake was characterized with Chinese hamster ovary (CHO) cells stably expressing human OATP1B1. Then, the effect of flavonoids on OATP1B1-mediated DCF uptake was investigated. After that, the half maximal inhibitory concentrations (IC50) for the flavonoids that showed significant
it is of great significance to extensively investigate the interaction between flavonoids and OATP1B1. In the present study, 25 common flavonoids were investigated for their interactions with OATP1B1, including 4 flavanols (epicatechin, epicatechin gallate, epigallocatechin, and epigallocatechin gallate), 11 flavonols (fisetin, galangin, gossypetin, kaempferol, myricetin, quercetin, hyperoside, isoquercitrin, myricitrin, quercitrin, and rutin), 5 flavones (apigenin, apigetrin, chrysin, luteolin, and oroxylin A), 2 flavanones (naringenin and prunin), and 3 isoflavones (daidzein, genistein, and puerarin) (Fig. 1). 2′,7′-Dichlorofluorescein (DCF), a commercially available dye of the 2
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lysed with 300 μL of 1% Triton X-100 in phosphate buffered saline (PBS) per well. After that, [3H]E3S was quantified by liquid scintillation counting and the fluorescence of DCF was measured by a Tecan Infinite M1000 PRO microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Total protein concentration was determined using the BCA assay with bovine serum albumin as a standard and uptake was normalized to total protein concentration. OATP1B1-specific uptake was calculated by subtracting the background uptake of CHO-EV cells from the uptake of CHO-OATP1B1 cells.
inhibition for OATP1B1 were measured. Finally, three-dimensional quantitative structure-activity relationship (3D-QSAR) studies were carried out on the investigated flavonoids using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) to elucidate their structure-activity relationships. 2. Materials and methods 2.1. Materials Radiolabeled [3H]estrone-3-sulfate ([3H]E3S) was from PerkinElmer (Waltham, MA). 2′,7′-Dichlorofluorescein (DCF) was purchased from Sigma-Aldrich (St. Louis, MO). The 25 flavonoid compounds were purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, Sichuan, China) and Nanjing Spring & Autumn Biological Engineering Co., Ltd. (Nanjing, Jiangsu, China) (purity 95–99%). Fetal bovine serum (FBS), Penicillin/Streptomycin, and trypsin were from Hyclone (Logan, UT). Ham's F12, L-glutamine, Lipofectamine 2000, Opti-MEM, and Flp-In system including Flp-In-CHO cell line, pcDNA5/ FRT vector, pOG44, zeocin, and hygromycin B were from Thermo Fisher Scientific (Carlsbad, CA). Sulfo-N-hydroxysuccinimide-SS-biotin, streptavidin-agarose beads, and the BCA protein assay kit were purchased from Pierce Chemical (Rockford, IL). Antibodies for detecting the 6-His tag and the Na+/K+-ATPase α subunit were purchased from Tiangen (Beijing, China) and Abcam (Boston, MA). Horseradish peroxidase-conjugated secondary antibodies were purchased from ProteinTech (Chicago, IL) and Sunshine Biotechnology (Nanjing, China). Immobilon Western blot detection kit was from Millipore (Billerica, MA).
2.3. Cell surface biotinylation and immunoblot analysis CHO-EV and CHO-OATP1B1 cells were plated in a 6-well plate at a density of 250,000 cells per well and induced with 5 mM sodium butyrate 48 h later. After 24 h of induction, cells were treated with 1 mL of sulfo-N-hydroxysuccinimide-SS-biotin (1 mg/mL in PBS) for 1 h at 4 °C. Then, cells were washed three times with 2 mL of ice-cold PBS containing 100 mM glycine and incubated for 10 min at 4 °C with the same buffer. After wash three times with ice-cold PBS, cells were lysed with 700 μL of lysis buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, and 1% Triton X-100, pH 7.4, containing protease inhibitors). Lysates were centrifuged at 10,000 ×g for 2 min. Supernatants were incubated with 140 μL of streptavidin-agarose beads for 1 h at room temperature. After wash three times with ice-cold lysis buffer, cell surface proteins were recovered from the resin by incubation of the beads with 2 × Laemmli buffer containing 100 mM dithiothreitol. Cell membrane proteins were then subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis. OATP1B1 was detected with a mouse anti-His antibody (Tiangen) (1:2000), followed by HRP-conjugated goat anti-mouse IgG (ProteinTech) (1:5000). Plasma membrane marker Na+/K+-ATPase was detected with a rabbit anti-Na+/K+ATPase α subunit antibody (Abcam) (1:5000), followed by HRP-conjugated goat anti-rabbit IgG (Sunshine) (1:10000). Immunoblots were developed with chemiluminescence method and scanned with ChemiDoc MP imaging system (Bio-Rad, Hercules, CA).
2.2. Cell culture, generation of stable cell lines, and uptake assay Flp-In-CHO cells were grown at 37 °C in a humidified 5% CO2 atmosphere in Ham's F12 medium supplemented with 10% FBS, 2 mM Lglutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 100 μg/ mL zeocin. Flp-In-CHO cells stably expressing OATP1B1 were generated as follows. First, a six His-tag was introduced at the C-terminal end of the open reading frame of human OATP1B1*1b [27] by PCR. The resulting construct was cloned into pcDNA5/FRT vector (Invitrogen, Carlsbad, CA) via NheI and NotI sites and verified by DNA sequencing. Then, Flp-In-CHO cells were transfected with pcDNA5/FRT empty vector (EV) or the constructed pcDNA5/FRT-OATP1B1 vector together with pOG44 using Lipofectamine 2000 following the manufacturer's instructions. Initially, the cell culture medium was supplemented with 600 μg/mL hygromycin B for transfectant selection. After single clones were isolated by limited dilution, the concentration of hygromycin B was lowered to 300 μg/mL. OATP1B1 clones were characterized by surface biotinylation and immunoblotting and functional assay, and one clone (CHO-OATP1B1) with highest transport function was employed for the following studies. One empty vector-transfected clone (CHO-EV) with hygromycin B resistance was used as negative control. Cells within ten passages were used for all experiments in the present study. For uptake assay, CHO-EV and CHO-OATP1B1 cells were plated at 40,000 cells per well on 24-well plates and 48-h later medium was replaced with medium containing 5 mM sodium butyrate, a histone deacetylase inhibitor, to induce nonspecific gene expression [28]. After another 24 h in culture, the cells were used for uptake experiments. Cells were washed three times with pre-warmed uptake buffer (116.4 mM NaCl, 5.3 mM KCl, 1 mM NaH2PO4, 0.8 mM MgSO4, 5.5 mM D-glucose and 20 mM Hepes, pH adjusted to 7.4 with Trizma base). Uptake was started by adding 200 μL of uptake buffer containing 0.4 μCi/mL of [3H]E3S or 5 μM DCF in the presence or absence of flavonoids. After incubation for a specific period of time, uptake was terminated by removing the uptake solution and washing the cells four times with 1 mL of ice-cold uptake buffer each time. Then cells were
2.4. Effect of flavonoids on OATP1B1-mediated DCF uptake and determination of their IC50 values First, inhibition screening assays were performed with 5 μM DCF in the absence and presence of 10 and 100 μM flavonoids. Flavonoids with significant inhibition effect were selected to determine their IC50 values by measuring the uptake of DCF in the absence and presence of increasing concentrations of each flavonoid. 2.5. Computational methods 2.5.1. Determination of the conformations of flavonoids and their structural alignment The molecular structures of flavonoids were sketched in Sybyl-X 2.0 program [29] and their initial conformations were generated by molecular mechanics optimization using the Tripos force field and Gasteiger-Hückel charges [30], with an energy gradient convergence criterion of 0.05 kcal/mol and a distance-dependent dielectric constant of 1. After that, conformational search with acyclic rotatable bonds was performed for each flavonoid by Confort program [31]. The conformation with lowest energy was selected and subjected to energy minimization until convergence. The resulting conformation was used for molecular alignment. Initial alignment was obtained by aligning the flavonoids according to their backbone structures. Initial CoMFA and CoMSIA models were developed and outliers (prediction error > 0.4 log unit) were subjected to conformational adjustment by rotating the rotatable bonds. However, all rotations were confined within 10 kcal/ mol of its lowest energy. 3
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significantly higher E3S transport activity than CHO-EV. Then, with the established cell lines we characterized OATP1B1-mediated DCF uptake. Time-dependent uptake assay showed that OATP1B1-mediated DCF uptake was linear up to at least 2 min (Fig. 3). Therefore, the uptake time for kinetic and inhibition assay in the present study was set to be 2 min. Kinetic study showed that OATP1B1-mediated DCF uptake exhibited a monophasic saturation kinetics (Fig. 4B) with a Km value of 9.7 ± 2.4 μM.
2.5.2. CoMFA and CoMSIA analyses The inhibition constants were expressed in pIC50 (−logIC50) values, which were used as the dependent variables in CoMFA and CoMSIA analyses. In CoMFA analysis, steric and electrostatic field energies were probed with an sp3 carbon atom having a charge of +1. Steric and electrostatic interactions were calculated using a Tripos force field with a distance-dependent dielectric constant. A cutoff value of 30 kcal/mol was applied on steric and electrostatic fields. Column filtering was set to be of 1.0 kcal/mol. In CoMSIA analysis, five different similarity fields, namely steric, electrostatic, hydrophobic, hydrogen-bond donor, and hydrogen-bond acceptor fields, were calculated with a probe atom having a radius of 1 Å, charge of +1, hydrophobicity of +1, and hydrogen bonding donor and acceptor properties of +1. These fields cover the major contributions to ligand binding [32]. The attenuation factor was set to be of 0.3. The crossvalidated r2 (q2) and optimum number of components were obtained by the partial least-squares (PLS) method with the leave-one-out option. With the obtained optimum numbers of components, the final non-crossvalidated CoMFA and CoMSIA models were developed.
3.2. Effect of flavonoids on OATP1B1-mediated DCF uptake As shown above, DCF is a good substrate for OATP1B1 and can be easily and conveniently detected and quantified due to its fluorescence. Therefore, DCF was used to investigate the interaction between flavonoids and OATP1B1 in this study. First, inhibition assay was carried out for the 25 common flavonoids on OATP1B1-mediated DCF uptake. Uptake of 5 μM DCF in the absence and presence of 10 and 100 μM flavonoids with CHO-EV and CHO-OATP1B1 cells was measured. The results were summarized in Fig. 5. Bromosulfophthalein (BSP), a known OATP1B1 inhibitor [33], was used as a positive control and it strongly inhibited OATP1B1-mediated DCF uptake as expected. At the concentration of 10 μM, luteolin, oroxylin A, quercetin, and apigenin showed the strongest inhibitory effect on OATP1B1 with inhibition rates > 70% and luteolin showing the greatest inhibition. Myricetin, fisetin, kaempferol, chrysin, genistein, and myricitrin showed moderate inhibitory effect with 40–70% inhibition. Isoquercitrin, naringenin, quercitrin, epigallocatechin gallate (EGCG), rutin, and apigetrin showed weak effect with 20–40% inhibition on OATP1B1-mediated DCF uptake (Fig. 5). However, daidzein, epicatechin (EC), epigallocatechin (EGC), galangin, gossypetin, hyperoside, prunin, and puerarin had no significant effect on DCF uptake. On the contrary, epicatechin gallate (ECG) showed a stimulating effect on OATP1B1-mediated DCF uptake. At the concentration of 100 μM, totally 19 flavonoids namely apigenin, apigetrin, chrysin, EGCG, fisetin, genistein, gossypetin, hyperoside, isoquercitrin, kaempferol, luteolin, myricetin, myricitrin, naringenin, oroxylin A, prunin, quercetin, quercitrin, and rutin showed inhibition rates > 60%. These 19 flavonoids were selected for further characterization by measuring their IC50 values on OATP1B1-mediated DCF uptake. At the concentration of 100 μM, ECG showed a very weak inhibitory effect on OATP1B1 instead of a stimulating effect as observed at the concentration of 10 μM.
2.6. Data analysis Data statistical analysis was performed with Prism 5 (GraphPad Software, San Diego, CA). Student's t-test was used when comparing two sets of data. One-way ANOVA followed by Dunnett's test was employed to analyze statistically significant difference for more than two groups. The p value for statistical significance was set to be < 0.05 at 95% confidence interval. The IC50 and Ki values were calculated by nonlinear least-squares data fitting. 3. Results 3.1. Characterization of OATP1B1-mediated 2′,7′-dichlorofluorescein (DCF) uptake Using Flp-In system, CHO cell lines stably transfected with empty vector (CHO-EV) and human OATP1B1 (CHO-OATP1B1) were constructed. The surface expression and function of OATP1B1 were confirmed by surface biotinylation and Western blot and uptake assay (Fig. 2). As shown in Fig. 2A, OATP1B1 had normal expression on cell surface. Estrone-3-sulfate (E3S), a model substrate for OATP1B1, was used to examine OATP1B1's function. As shown in Fig. 2B, the expressed OATP1B1 had normal function as CHO-OATP1B1 cells showed
Fig. 2. Surface expression and functional characterization of human OATP1B1 in Flp-In-CHO cells. (A) Surface expression of OATP1B1 in CHO cells. Plasma membrane proteins were isolated by surface biotinylation and OATP1B1 was detected with an anti-His antibody. The plasma membrane marker Na+/K+ATPase α subunit was used as protein loading control. (B) Functional characterization of OATP1B1 in CHO cells. Uptake of [3H]estrone-3-sulfate was measured at 37 °C for 1 min with CHO-EV and CHOOATP1B1 cells. Values were given as the mean ± SD of triplicate determinations; ⁎p < 0.05.
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Fig. 3. Time-dependent uptake of 2′,7′-dichlorofluorescein (DCF) mediated by OATP1B1. (A) Uptake mediated by CHO-EV and CHO-OATP1B1. (B) Net uptake mediated by OATP1B1. Uptake of 5 μM DCF was measured at 37 °C for indicated time points with CHO-EV and CHO-OATP1B1 cells. Net uptake was obtained by subtracting the uptake of CHO-EV cells from that of CHO-OATP1B1 cells. Data shown as mean ± SD of one representative of three independent experiments performed in triplicate.
Fig. 4. Concentration-dependent uptake of DCF mediated by OATP1B1. (A) Uptake mediated by CHO-EV and CHO-OATP1B1. (B) Net uptake mediated by OATP1B1. An Eadie-Hofstee plot of OATP1B1-mediated DCF uptake was included as a figure inset. Uptake of increasing concentrations of DCF was measured at 37 °C for 2 min with CHO-EV and CHO-OATP1B1 cells. Net uptake was obtained by subtracting the uptake of CHO-EV cells from that of CHO-OATP1B1 cells and fitted to the Michaelis–Menten equation to obtain Km and Vmax values. Data shown as mean ± SD of one representative of three independent experiments performed in triplicate. Fig. 5. Effect of flavonoids on OATP1B1-mediated DCF uptake. Uptake of 5 μM DCF was measured at 37 °C for 2 min with CHO-EV and CHO-OATP1B1 cells in the absence (control) and presence of 10 μM (open bars) and 100 μM (closed bars) BSP and indicated flavonoids. Values obtained with CHO-EV cells were subtracted from values obtained with CHO-OATP1B1 cells and were given as percent of control. Data shown as mean ± SD from three independent experiments done in triplicate; ⁎p < 0.05 vs control.
3.3. Concentration-dependent effect of flavonoids on OATP1B1-mediated DCF uptake
were listed in Table 1. Among them, luteolin had the strongest inhibitory effect with an IC50 value of 0.7 μM. Quercetin, fisetin, genistein, oroxylin A, and apigenin also had strong inhibitory effect on OATP1B1 with IC50 values smaller than 5 μM. EGCG showed the weakest inhibition among the selected 19 flavonoids with an IC50 value of 40.8 μM. Inhibition kinetic study was carried out for quercetin which is the
The concentration-dependent effect of flavonoids on OATP1B1mediated DCF uptake was investigated for the above-listed 19 flavonoids, which showed high OATP1B1 inhibition activity when tested at the concentration of 100 μM. The IC50 values of these 19 flavonoids 5
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Table 1 IC50 values for DCF and the selected 19 flavonoid compounds on OATP1B1mediated DCF uptake and the characteristic features affecting their inhibitory potency. Compounds
IC50 (μM)
Characteristic features affecting inhibitory potency
a
DCF Apigenin Apigetrin Chrysin EGCG Fisetin Genistein Gossypetin Hyperoside Isoquercitrin Kaempferol
14.7 4.9 ± 1.1 25.0 ± 1.3 7.4 ± 1.3 40.8 ± 1.8 3.3 ± 0.3 4.6 ± 1.0 18.4 ± 1.4 25.3 ± 1.3 20.8 ± 1.2 8.5 ± 0.4
Luteolin Myricetin
0.7 ± 0.2 5.8 ± 1.3
Myricitrin Naringenin Oroxylin A Prunin Quercetin
10.4 ± 1.2 15.6 ± 1.8 4.7 ± 0.1 36.6 ± 1.3 2.4 ± 1.1
Quercitrin Rutin
11.5 ± 0.9 20.5 ± 1.2
Sugar moiety on 7-OH reducing inhibitory potency
Sugar moiety on 3-OH reducing inhibitory potency Sugar moiety on 3-OH reducing inhibitory potency 3-OH reducing inhibitory potency 4′-OH enhancing inhibitory potency An additional –OH at 5′ reducing inhibitory potency Sugar moiety on 3-OH reducing inhibitory potency
Sugar moiety on 7-OH reducing inhibitory potency 3-OH reducing inhibitory potency 3′- and 4′-OH enhancing inhibitory potency Sugar moiety on 3-OH reducing inhibitory potency Sugar moiety on 3-OH reducing inhibitory potency
Fig. 7. Structural alignment of DCF and the 18 flavonoid compounds.
Concentration-dependent inhibition of 5 μM DCF by indicated flavonoids was measured at 37 °C for 2 min with CHO-EV and CHO-OATP1B1 cells. Values obtained with CHO-EV cells were subtracted from values obtained with CHOOATP1B1 cells and were used to calculate IC50 values by non-linear regression analysis. Values were given as the mean ± SE from two to four independent experiments. aThe IC50 of DCF was calculated with equation IC50 = Ki (1 + [S]/Km) where Ki was assumed to be equal to Km.
Activity changes of ligands are related to their molecular structural changes. To elucidate their structure-activity relationship in a quantitative manner, CoMFA and CoMSIA 3D-QSAR studies were carried out for the substrate DCF and 19 flavonoids with IC50 values measured.
3.4. CoMFA and CoMSIA analyses The conformations of DCF and the 19 flavonoids were generated with conformational search and aligned according to their backbone structures. Initial CoMFA and CoMSIA models were developed and improved by adjusting the conformations of some flavonoids that had high prediction error with visual inspection. However, gossypetin significantly decreased the crossvalidated r2 (q2) when included in CoMFA and CoMSIA model development and its conformation could be hardly adjusted to improve the models. Therefore, we excluded gossypetin and the remaining 18 flavonoids together with DCF were used for CoMFA and CoMSIA modeling. Fig. 7 showed the structural alignment of these 19 compounds. Among them, 17 compounds were used as training set to develop CoMFA and CoMSIA models. The remaining two compounds, namely apigenin (relatively strong inhibitor) and rutin (relatively weak inhibitor), were used as test set for model validation. CoMFA and CoMSIA models were developed with PLS analysis and their statistics were listed in Table 2. The q2 values for CoMFA and CoMSIA were 0.604 and 0.534, respectively. The optimal numbers of Fig. 6. Eadie-Hofstee plot of the inhibitory effect of quercetin on OATP1B1mediated DCF uptake. Uptake of increasing concentrations (3 to 50 μM) of DCF was measured at 37 °C for 2 min in the absence or presence of 1 and 3 μM quercetin. Data shown as mean ± SD of one representative of two independent experiments performed in triplicate.
Table 2 Statistical parameters of CoMFA and CoMSIA modeling.
PLS statistics q2 r2 Standard error of estimate Optimal number of components
most extensively studied flavonoid and one of the most abundant flavonoids in foods [34] and its inhibition type on OATP1B1-mediated DCF transport was determined. Fig. 6 showed the Eadie-Hofstee plot of OATP1B1-mediated uptake of DCF in the presence or absence of quercetin, indicating that quercetin inhibited OATP1B1-mediated DCF uptake in a competitive manner by affecting the apparent affinity but not the maximal transport rate. The apparent Ki value was determined to be 1.4 ± 0.1 μM.
Field contributions (%) Steric Electrostatic Hydrophobic Hydrogen-bond donor Hydrogen-bond acceptor
6
CoMFA
CoMSIA
0.604 0.841 0.203 3
0.534 0.807 0.216 2
50.3 49.7
10.5 26.0 13.8 23.9 25.8
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Fig. 8. CoMFA and CoMSIA predictions for DCF and the 18 flavonoid compounds. ●, training set (DCF and 16 flavonoids); , test set (apigenin and rutin).
models were developed to elucidate their structure-activity relationships. To the best of our knowledge, these were the first CoMFA and CoMSIA models developed for flavonoids as OATP1B1 inhibitors. Kinetic study showed that OATP1B1-mediated DCF transport exhibited a monophasic kinetic behavior (Fig. 4B, inset), indicating a single binding site for DCF in OATP1B1. However, several studies showed that the kinetics of OATP1B1-mediated E3S transport was biphasic and consisted of high-affinity low-capacity and low-affinity highcapacity components, indicating the presence of two binding sites for E3S in OATP1B1 [35–37]. Among the tested flavonoids in the present study, ECG exhibited a stimulating effect on OATP1B1-mediated DCF uptake (Fig. 5). This result suggested that ECG might allosterically alter OATP1B1-mediated DCF transport. Interestingly, another study showed that ECG had an inhibitory effect on OATP1B1-mediated E3S uptake [19]. These results indicated that DCF and ECG probably bind to different binding sites in OATP1B1. ECG might bind to the high-affinity binding site of E3S upon which it exerted an inhibitory effect on OATP1B1-meidated E3S uptake, while DCF binds to the other site. In addition, a study showed that E3S had an inhibitory effect on OATP1B1-mediated DCF uptake [26]. This is kind of expected as E3S can bind both binding sites. The effect of DCF on OATP1B1-mediated transport of E3S and other substrates and their mutual inhibition kinetics remain to be investigated. The only structural difference between luteolin and quercetin is that quercetin has an additional hydroxyl group at C-3 position (Fig. 1). This C-3 hydroxyl group decreased the inhibitory potential of the flavonoids 3-fold as the IC50 values for luteolin and quercetin were 0.7 and 2.4 μM, respectively (Table 1). A 2-fold decrease of activity was also observed for apigenin with this C-3 hydroxyl group as the IC50 values increasing from 4.9 μM (apigenin) to 8.5 μM (kaempferol) (Table 1). The C-3
components for CoMFA and CoMSIA were 3 and 2, respectively. Noncrossvalidation PLS analyses with the optimal numbers of components revealed conventional (non-crossvalidated) r2 of 0.841 and 0.807 for CoMFA and CoMSIA, respectively. The standard errors of estimate for CoMFA and CoMSIA were 0.203 and 0.216, respectively. In the CoMFA model, steric fields contributed 50.3% to the model's information, while electrostatic fields contributed the other 49.7%. In the CoMSIA model, the contributions of steric, electrostatic, hydrophobic, hydrogen-bond donor, and hydrogen-bond acceptor fields were 10.5, 26.0, 13.8, 23.9, and 25.8%, respectively. The reliability and predictive power of the CoMFA and CoMSIA models were verified with the compounds in training and test sets. The correlations between the predicted activities by CoMFA and CoMSIA and experimental activities for DCF and the 18 flavonoids were depicted in Fig. 8. The predicted activities by the CoMFA and CoMSIA models were in good agreement with the experimental data with R2 being of 0.837 and 0.808, respectively, indicating that our CoMFA and CoMSIA models were reliable. Figs. 9 and 10 showed the CoMFA and CoMSIA contour maps together with quercetin. 4. Discussion In the present study, a Flp-In-CHO cell line stably expressing human OATP1B1 was constructed and the interactions of 25 flavonoid compounds with OATP1B1 were investigated using a fluorescent substrate DCF which could be detected and quantified conveniently. Kinetic study showed that OATP1B1-mediated DCF uptake was saturable with a Km value of 9.7 ± 2.4 μM. Inhibition assay for the 25 flavonoids on OATP1B1-mediated DCF uptake was performed and 19 flavonoids were selected to determine their IC50 values upon which CoMFA and CoMSIA
Fig. 9. CoMFA contour maps in combination with quercetin. (A) Steric field. Green, steric bulk desirable regions; yellow, steric bulk undesirable regions. (B) Electrostatic field. Blue, positive charge desirable regions; red, negative charge desirable regions. 7
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Fig. 10. CoMSIA contour maps in combination with quercetin. (A) Steric field. Green, steric bulk desirable regions; yellow, steric bulk undesirable regions. (B) Electrostatic field. Blue, positive charge desirable regions; red, negative charge desirable regions. (C) Hydrophobic field. Yellow, hydrophobicity desirable regions; white, hydrophobicity undesirable regions. (D) Hydrogen-bond donor field. Cyan, donor desirable regions; purple, donor undesirable regions. (E) Hydrogen-bond acceptor field. Magenta, acceptor desirable regions; red, acceptor undesirable regions.
the inhibition of OATP1B1 were increased with increasing number of hydroxyl groups on ring B. The IC50 values of chrysin, apigenin, and luteolin were 7.4, 4.9, and 0.7 μM, respectively (Table 1). Similarly, galangin, kaemferol, and quercetin have zero, one, and two hydroxyl groups on ring B (Fig. 1) and their activities were also increased with increasing number of hydroxyl groups on ring B (Fig. 5 and Table 1). These results indicated that the hydroxyl groups at C-3′ and C-4′ positions on ring B of flavonoids could strengthen their interaction with OATP1B1. According to our CoMSIA model, hydrogen-bond forming groups at C-3′ and C-4′ positions of ring B were beneficial for flavonoid's binding (cyan and magenta contours in Fig. 10D and E). This could explain why chrysin, apigenin, and luteolin showed increasing order of activities (Fig. 1 and Table 1). The same order of activities was also observed for galangin, kaemferol, and quercetin for the same reason (Fig. 5 and Table 1). However, hydrogen-bond donor and acceptor unfavorable contours existed near C-5′ position of ring B (purple and red contours in Fig. 10D and E). Myricetin has an additional hydroxyl group at C-5′ position as compared to quercetin. According to our CoMSIA results, this addition hydroxyl group was not beneficial for myricetin's interaction with OATP1B1. In fact, our experimental data showed that myricetin had lower activity than quercetin (Table 1), indicating that our model had a good predictive ability. These results also indicated that the number of hydroxyl groups on ring B should not exceed two for flavonoids to have a strong inhibitory potency. Previously, we proposed an interaction model between OATP1B1 and nuclear receptor ligands, in which a negative charge center and a hydrophobic center of the ligands were important for their binding [38]. Flavonoids do not have an explicit negative charge center. However, the hydroxyl group on ring B was important for flavonoid's activity. It might play a somewhat similar role as the negative charge center in nuclear receptor ligands because a phenolic hydroxyl group can be partially negatively charged due to its proton dissociation. In the areas around rings A and C of flavonoids, bulky hydrophilic groups
hydroxyl group also decreased chrysin's activity as galangin showed lower inhibition rate than chrysin (Fig. 5). Therefore, for the flavonoids tested in the present study flavones had higher activity than their 3hydroxyflavone counterparts (flavonols). Attaching a sugar moiety to the C-3 hydroxyl group would further decrease the inhibitory activity of flavonoids on OATP1B1. Quercitrin (quercetin-3-O-rhamnoside), isoquercitrin (quercetin-3-O-glucoside), hyperoside (quercetin-3-O-galactoside), and rutin (quercetin-3-O-rutinoside) are 3-O-glycosides of quercetin (Fig. 1). Their IC50 values were 5–10 folds higher than that of quercetin (Table 1). Similarly, myricitrin (myricetin-3-O-rhamnoside) showed 2-fold decreased inhibitory activity on OATP1B1 as compare to myricetin whose IC50 values were 10.4 and 5.8 μM, respectively (Table 1). The presence of sugar moieties at positions C-7 and C-8 could also decrease flavonoid's inhibition activities. For example, apigenin showed 5-fold higher activity than apigetrin (apigenin-7-O-glucoside) and naringenin showed 2-fold higher activity than prunin (naringenin-7-Oglucoside) (Fig. 5 and Table 1). Similarly, daidzein had stronger inhibitory effect on OATP1B1 than puerarin (daidzein-8-C-glucoside) (Fig. 5). Therefore, it seems that flavonoid aglycones usually interact with OATP1B1 much stronger than their glycosides. Based on the steric and hydrophobic contour maps of our CoMFA and CoMSIA models, bulky and hydrophilic groups at C-3, C-4, C-7, and C-8 positions of rings A and C was detrimental to flavonoid's binding with OATP1B1 (yellow contours in Figs. 9A, 10A and C). In addition, hydrogen-bond forming groups in these areas were also detrimental to the interaction of flavonoids with OATP1B1 (purple and red contours in Fig. 10D and E). These could explain why quercetin, myricetin, apgenin, naringenin, and daidzein had stronger inhibition on OATP1B1 than their 3-, 7-, and 8-glycosides. The structural difference between chrysin, apigenin, and luteolin is the number of hydroxyl groups on ring B (Fig. 1). Chrysin, apigenin, and luteolin have zero, one (at C-4′ position), and two hydroxyl groups (at C-3′ and C-4′ positions) on ring B, respectively. Their activities for 8
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were unfavorable which might bear some resemblance to the hydrophobic center of nuclear receptor ligands. However, bulky substitution in the hydrophobic center area of nuclear receptor ligands was favorable, indicating that the binding sites for flavonoids and nuclear receptor ligands in OATP1B1 have some similarity but not identical. In conclusion, by using the fluorescent substrate DCF and 3D-QSAR analysis this study obtained some useful information on the interactions between flavonoids and OATP1B1. OATP1B1-mediated DCF uptake exhibited monophasic kinetics with a Km of 9.7 ± 2.4 μM. While most tested flavonoids had an inhibitory effect on OATP1B1-mediated DCF uptake, ECG showed a stimulating effect. Our experimental and computational results showed that flavonoid aglycones interacted with OATP1B1 much stronger than their glycosides such as 3-, 7-, and 8glycosides as bulky hydrophilic and hydrogen-bond forming groups at C-3, C-7, and C-8 positions on rings A and C of flavonoids were unfavorable for their binding. The presence of hydrogen-bond forming groups on ring B of flavonoids was beneficial for their interaction with OATP1B1 as long as the number of hydrogen-bond forming groups was not > 2. Our results also indicated that flavones usually had higher inhibitory activity on OATP1B1 than their 3-hydroxy derivatives (flavonols). The obtained information and 3D-QSAR models could be useful in elucidating and predicting the interactions between flavonoids and human OATP1B1.
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