A molecular dynamics perspective into estrogen receptor inhibition by selective flavonoids as alternative therapeutic options

Figures & data

Figure 1. Chemical structures of the ERα ligands. (A) Estradiol, the endogenous stimulatory ERα-ligand (ChemSpider ID: 5554). (B) ZEA, the stimulatory mycoestrogen (ChemSpider ID: 4444897). (C) 4-Hydroxytamoxifen, the synthetic selective estrogen-receptor modulator (ChemSpider ID: 395987).

Figure 2. Basic chemical structures of several different flavonoid classes (Panche et al., 2016).

Figure 3. X-ray crystallography structures of the ERα ligand binding domain (LBD). (A) The LBD encompasses helices: H1-H12. The ligand binding pocket is comprised of H3, H6, H8, H11 and H2, PDB ID: 3ert. (B) ERα LBD complexed with zearalenone in the active/closed conformation, PDB ID: 5krc. (C) ERα LBD complexed with 4-hydroxytamoxifen in the inactive/open conformation, PDB ID: 3ert. (D) The apo form of ERα LBD complexed with 17β-estradiol, PDB ID: IA52.

Table 1. Flavonoid compounds obtained from the Biological Magnetic Resonance Data Bank.

Compound	Abbreviation	Pubchem compound ID	Classification	Source	References
4-Hydroxytamoxifen (Afimoxifen)	OHT	449459	Selective estrogen receptor modifier	Synthetic hormone.	(Ahmad, 2018; Neven et al., 2018; Sanchez-Spitman et al., 2019)
Quercetrin (Quercetin 3-rhamnoside)	QRM	5280459	Flavonol	star anise, grapes, cocoa, pepper.	(Dabeek & Marra, 2019; Ulusoy & Sanlier, 2020)
Hesperidin (Hesperitin-7-rhamnoglucoside)	HPN	53477767	Flavanone	Citrus fruits	(Binkowska, 2020; Ganeshpurkar & Saluja, 2019; Selmi et al., 2017)
Epigallocatechin 3-gallate	EGCG	65064	Flavan-3-ols	Green tea extract	Vázquez Cisneros et al., 2017)
Kaempferol 7-O-glucoside	K7G	10095180	flavonol	Delphinium, Witch-hazel, grapefruit.	(Cid-Ortega & Monroy-Rivera, 2018; Huynh et al., 2018)
Naringenin-7-O-glucoside (Prunin)	NGN	92794	flavanone	Apigenin metabolite.	(Karim et al., 2018; Venkateswara Rao et al., 2017)
Apigenin 7-O-glucoside	APN	5385553	flavone	Chamomile.	(Cvetanović et al., 2017; Wang et al., 2019)
Daidzein	DZN	5281708	Isoflavone	Soybeans	(Sumardi et al., 2017)
Phloretin	PRT	4788	Chalcone	Fruits	(Behzad et al., 2017; Mariadoss et al., 2019)
Baicalein 6-glucoside	BCN	44258404	Flavone	Fruits, vegetables, medicinal plants	(de Oliveira et al., 2015; Dinda et al., 2017)
Luteolin	LTN	5280445	flavone	Flowering food plants.	(Somerset & Johannot, 2008)
Fisetin	FST	5281614	flavonol	Berries, grapes.	(Grynkiewicz & Demchuk, 2019; Khan et al., 2013; Somerset & Johannot, 2008)
Myricetin	MCT	5281672	flavonol	Vegetables. fruits and beverages.	(Gupta et al., 2020)
Tangeretin	TGN	68077	Flavone	Apples, tangerines, Fortunella, japonica	(Ashrafizadeh et al., 2020)
Cyanidin	CYN	128861	Anthocyanidin	Fruits and vegetables.	(Somerset & Johannot, 2008)

Figure 4. 2 D structures of the flavonoids.

Figure 5. Lateral view of ERα LBD showing the validation of the docking protocol. The original crystal ERα LBD structure (PDB: 3ert) in green was superimposed with the newly docked complex (in blue and OHT in brown). RMSD = 0.004A˚.

Table 2. Thermodynamic binding free energy components (Kcal/mol) for all the flavonoids compared to OHT (reference drug).

	Energy components (kcal.mol^-1)
	ΔEvdw	ΔEelec	ΔGgas	ΔGsolv	ΔGbind
OHT	−50.744 ± 3.170	−170.383 ± 24.083	−221.127 ± 24.936	176.402 ± 22.526	−44.724 ± 4.306
HPN	−69.495 ± 4.825	−41.980 ± 15.293	−111.476 ± 16.009	55.410 ± 11.203	−56.066 ± 6.696
QRM	−40.415 ± 5.505	−74.587 ± 22.181	−115.002 ± 18.785	59.369 ± 10.954	−55.633 ± 9.099
EGCG	−39.509 ± 5.004	−77.166 ± 11.204	−116.675 ± 11.998	68.5883 ± 8.165	−48.087 ± 6.098
K7G	−45.012 ± 3.806	−54.586 ± 12.723	−99.598 ± 11.091	53.089 ± 8.014	−46.509 ± 4.428
NGN	−37.498 ± 2.903	−30.468 ± 4.055	−67.967 ± 3.623	25.023 ± 2.066	−42.943 ± 2.688
APN	−39.777 ± 5.352	−52.169 ± 11.325	−91.947 ± 12.127	49.196 ± 8.647	−42.750 ± 5.105
PRT	−39.837 ± 2.908	−29.380 ± 6.617	−69.217 ± 6.343	27.201 ± 3.352	−42.016 ± 4.224
DZN	−35.808 ± 2.811	−33.620 ± 3.661	−69.429 ± 3.213	27.440 ± 1.868	−41.989 ± 2.514
BCN	−50.919 ± 3.978	−19.380 ± 9.151	−70.300 ± 9.561	27.861 ± 6.837	−42.438 ± 4.751
LTN	−29.569 ± 3.948	−52.599 ± 8.135	−82.168 ± 7.714	41.990 ± 5.680	−40.178 ± 4.209
FST	−39.759 ± 3.657	−28.113 ± 7.935	−67.873 ± 6.668	28.618 ± 4.392	−39.254 ± 3.568
MCT	−30.390 ± 3.710	−57.558 ± 8.732	−87.948 ± 7.334	51.083 ± 6.281	−36.865 ± 3.158
TGN	−42.146 ± 6.023	−18.402 ± 7.392	−60.549 ± 10.457	25.905 ± 6.648	−34.644 ± 5.854
CYN	−38.289 ± 5.012	−156.49 ± 12.523	−194.78 ± 12.216	160.238 ± 9.477	−34.548 ± 4.567

ΔEelec: electrostatic, ΔEvdW: van der Waals, ΔGbind: calculated total free binding energy, ΔGgas: gas phase interaction, and ΔGsolv: solvation free energy.

Figure 6. (A) The RMSD plots of Cα for the apo (black), OHT (red), HPN (cyan) and QRM (purple). (B) The RMSD plots of Cα apo (black), OHT (red), K7G (green) and QRM (blue).

Figure 7. Radii of gyration (RoG)of apo and bound systems were measured over a 200 ns simulation. Data obtained were presented as a plot exhibiting the differences arising in radius deviation between the apo and bound systems. (A) The RoG plots for apo(black), OHT (red), HPN (cyan) and QRM (purple). (B) The RoG plots for apo (black), OHT (red), K7G (green) and QRM (blue).

Figure 8. (A) The RMSF plots for the apo system (black), OHT complex (red), HPN (cyan) complex and QRM (purple) complex. (B) The RMSF plots for the apo system (black), OHT (red) complex, K7G (green) complex and QRM (blue) complex.

Table 3. The average Cα RMSF values of specific regions of the ER LBC.

Helices	Residues	Apo	OHT	HPN	QRM	EGCG	K7G
H3	R333-R341	2.8	2.6	4.4	3.8	5.4	4.7
H9	R454-R471	2.2	3.3	3.6	2.6	4.7	3.3
H12	R525-R535	4.8	4.2	2.8	3.2	3.6	3.6
H12	R545-R551	8.9	6.0	2.7	4.8	3.3	6.0

The units of measurements are in Angstrom.

Figure 9. The graphical representation of HPN, QRM, EGCG and K7G residue interactions within the LBD of ERα.

Table 4. The flavonoid- ERα interaction details. The number of interactions and the interacting residues.

Compound	Total interactions (Hydrogen bonds)	Types of hydrogen bonds	Interacting residues (hydrogen bonds)
OHT	27 (9)	Conventional:2 Carbon hydrogen: 7	HIE524, CYS530. THR 347, THR347, ASP351, GLY521, CYS530, CYS530, ASN532.
HPN	26 (7)	Conventional:2 Carbon hydrogen:5	GLU339, THR347 GLU339, LEU346, THR347, TRP383, CYS530
QRM	17 (9)	Conventional: 6 Carbon hydrogen:3	GLU323, GLU353, GLU353, HIE356, ARG394, HIE356 Arg394, PHE445, HIE356
EGCG	17 (8)	Conventional: 7 Carbon hydrogen: 1	ASP351, ASP351, MET522, ASN532, ASN532, VAL534, VAL534 TYR526
K7G	18 (6)	Conventional: 4 Carbon hydrogen: 2	LEU327, GLU353, GLU353, ILE386 GLY390, LYS444

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