Food Flavonoid Ligand Structure/Estrogen Receptor-Α Affinity
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Food and Chemical Toxicology 129 (2019) 328–336 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox Food flavonoid ligand structure/estrogen receptor-α affinity relationships – toxicity or food functionality? T ∗ Hui Ye, Ian C. Shaw Human Toxicology Research Group, School of Physical and Chemical Sciences, University of Canterbury, Private Bag 4800, Christchurch, 8140, New Zealand ARTICLE INFO ABSTRACT Keywords: In silico molecular modelling is used to study interactions between flavonoid phytoestrogens and estrogen re- Food functionality and toxicity ceptor (ER)α. Twenty flavonoids from foods were studied; e.g., genistein from soy, naringenin from grapefruit, estrogen receptor α phloretin from pears, chrysin from oyster mushrooms. These potential ligands' molecular attributes and their dietary phytoestrogen spatial arrangements that favour binding to the ligand binding cleft (LBC) of ERα are identified, and Docking binding affinity Scores calculated. The Docking Score order is the same as the estrogenicity order for 8 of the flavonoids studied structural features in detail. The number and position of flavonoid ring hydroxyls influence the Docking Scores which might relate flavonoids to ERα′s bio-activity. Hydrophobic interactions between ligands and ERα are also important; the number of rotatable CeC bonds in ligands likely affects the magnitude of hydrophobic interactions and ligand fit. Our findings suggest that flavonoids with diverse structural features could have different binding energies and binding affinities with ERα, which might confer different functionalities and toxicities. 1. Introduction separated by 9.6 Å of hydrophobicity (Brzozowski et al., 1997). Mole- cules with similar molecular attributes (i.e. estrogen mimics) activate A better understanding of the molecular mechanisms underlying ERs, but to a lesser extent than E2. For example, the estrogen mimic food functionality is crucial to our knowledge of potential health risks genistein (a phytoestrogen; Fig. 1) from soy has a hydroxyl separation − and benefits. However, such studies are often time consuming and ex- of 11.6 Å: genistein has a relative estrogenicity of 3.9 × 10 5 (where pensive. Therefore, the application of in silico modelling studies to in- E2 = 1) (Berckmans et al., 2007) because its ER binding characteristics vestigate receptor-mediated mechanisms might be useful in exploring are not ideal. food component functionality. Flavonoids are important food components; they include subclasses In this paper, we will focus on estrogen receptor (ER)-mediated based on their chemical structures; for example, flavone, flavonol, and mechanisms of food functionality and the use of in silico molecular isoflavone (Table 1). Flavonoids are present in many commonly con- modelling (Schrödinger platform) to give insights into the potential sumed fruits, vegetables and herbs (e.g., apigenin in parsley, quercetin benefits and risks of estrogen-mimicking (xenoestrogen) food compo- in red onion, genistein in soy, bergamottin in grapefruit) (Bailey et al., nents. 2003), and can mimic E2 (Fig. 1) and thus activate ERs (Harris et al., ERs occur as two isoforms (ERα and ERβ) and are present in many 2005). The binding affinities of both E2 and estrogen mimics to ER cell types (Heldring et al., 2007); e.g. ERα predominates in mammary isoforms are different this means that they will have differential tissue- cells and ERβ in colon cells (Kuiper et al., 1997). ERs are important in based biological activity which is dependent on the distribution of ER growth and development (Mosselman et al., 1996) with functions far isoforms in the tissue (Kuiper et al., 1997). Interestingly, ERβ (pre- exceeding the conventional concepts of sex hormone receptors (Ye dominates in colon cells) (Kuiper et al., 1997) provides a more suitable et al., 2018). The most potent natural ligand of ERs is 17β-estradiol (E2, binding environment for flavonoids and so might play an important role Fig. 1) and when bound to the receptor it initiates a sequence of events in flavonoid-containing colon-mediated food functionality. that leads to regulation of genes involved in growth and development The intimate interactions between flavonoids and ERα have been and expression of sexual characteristics (Reid et al., 2002). In order to more extensively studied than their ERβ interactions because of the activate ERs the ligand ideally has two hydroxyl groups, which hy- dominant role of ERα in some important diseases (Ye et al., 2018) in- drogen bond to amino acid residues in the ligand binding cleft (LBC), cluding breast cancer (more than 80% of human breast cancers are ERα ∗ Corresponding author. E-mail address: [email protected] (I.C. Shaw). https://doi.org/10.1016/j.fct.2019.04.008 Received 23 January 2019; Received in revised form 27 March 2019; Accepted 8 April 2019 Available online 12 April 2019 0278-6915/ © 2019 Published by Elsevier Ltd. H. Ye and I.C. Shaw Food and Chemical Toxicology 129 (2019) 328–336 based ERα cell assays. They concluded that a better fit to the ERα LBC and a greater estrogenicity was associated with longer molecules with hydroxyl groups that could interact with the LBC by forming hydrogen bonds. In this paper, we report the structure-affinity relationship between ERα and flavonoid ligands in silico (using the Schrödinger platform). We use these findings to better understand flavonoid estrogenicity and, importantly, the estrogenicity changes that might result following fla- vonoid metabolism (e.g., by the gut microbiome); we discuss our findings in the context of food functionality and toxicity. 2. Experimental 2.1. In silico modelling studies The molecular docking studies were carried out using Schrödinger Small-Molecule Drug Discovery Suit 2017–1 (Schrödinger LLC, New York, USA) to determine the likelihood that the studied molecule (li- gand) would bind to the LBC of ERα. Maestro (Schrödinger Release, 2017–1: Maestro, Schrödinger LLC, New York, NY, 2017) was used as the interface for preparing ERα and ligands for study. 2.2. Protein preparation The X−ray crystallographic coordinate of ERα were taken from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) (http://www.rcsb.org), the X-ray crystal structures of ERα complexed with ligands (e.g., genistein in PDB entry 1 × 7R) were used. Protein fragment crystals are derived from different polypeptides of the ERα protein according to the particular preparative methodolo- gies. This means that they might have different polypeptide chains and Fig. 1. Top: Two-dimensional structures of E2 (A) and genistein (B) showing each chain has a docked ligand (e.g. GEN). One of these chains was that E2’s two hydroxyl have approximately the same hydrophobic separations arbitrarily used as the ligand–receptor subunit for these docking stu- ’ as 2 of genistein s hydroxyls. Bottom: three-dimensional structures of E2 (C) dies, others were deleted. Missing amino acid residues (Ser 305, Tyr and genistein (D) to show the spatial alignment of their hydroxyl groups. These 331, Asp 332, Pro 333, Thr 334, Arg 335, Pro 336, Phe 337, Arg 548, commonalities of special arrangement of hydroxyls explain why genistein is an Leu 549) based on ERα′s primary sequence were added to complete the estrogen mimic. protein structure using the Schrödinger ‘Prime’ command. Besides the ligand, a single water molecule hydrogen bonded to Arg 394 and Glu positive) (Turner et al., 2015), atherosclerosis (Evangelopoulos et al., 353 was kept because it is important in ligand binding)− other water α′ 2003), ovarian cancer (Lu et al., 2006), and ER s potential role in molecules (e.g., solvent water) were removed (Manas et al., 2004). diabetes because of its role in regulation of glucose homeostasis Restrained Minimization was run to provide controls for optimizing the (Bryzgalova et al., 2006). corrected structure, relieving any strain and fine−tuning the placement Since there is more structural information (e.g., X-ray crystal- of specific groups (e.g., hydroxyls). Hydrogen atoms were always op- α β lography) (Brzozowski et al., 1997); for ER than ER , we are able to timized fully, which allows relaxation of the H-bond network and al- α – construct a good ER in silico model for this reason we have focused leviates potential steric clashes by user–selected root–mean–square α our food in silico functionality molecular mechanism studies on ER / deviations (RMSD) with a tolerance of 0.3 Å (Ignatowicz and Baer- fl food avonoid interactions. Dubowska, 2001). In the context of receptor (e.g., ERα)-mediated biological activity, ffi the structure-a nity relationship is the interdependency between li- 2.3. Receptor Grid Generation gand structures and receptor binding affinity (i.e. ligand/receptor fit) ffi (Ye et al., 2018). Structure-a nity relationships are good predictors of The receptor grid for docking studies was set up and generated from biological activity (including toxicity) and are useful screening methods the Receptor Grid Generation panel of Glide (Schrödinger Release, to select potential new receptor-mediated compound for pharmaceu- 2017–1: Glide, Schrödinger LLC, New York, NY, 2017). The natural LBC tical development (Hall et al., 2003). This has led to in silico studies was used for these docking studies, but the original bound ligand (e.g., becoming a key tool in drug discovery and development (Ekins et al., GEN) was excluded; this determines the position and size of the active 2007) - our studies extend this thinking to food functionality. site (i.e. the LBC) for ligands docking. Flavonoids are important food components that impart significant biological activity including estrogenicity, which is a key factor in food 2.4. Preparation of ligands functionality (e.g., isoflavone-rich bread is used as partial ‘hormone’ replacements in peri- and postmenopausal women (Simons et al., In this study, 20 flavonoids from different subclasses were selected α 2000)). Further to this, Zhang et al. (2018) studied both ER interac- as potential ligands for ERα.