Minggu, 02 Desember 2018

Quantitative structure-activity relationship for estrogenic flavonoidsfrom Psoralea corylifolia

Quantitative structure-activity relationship for estrogenic flavonoidsfrom Psoralea corylifolia

a b s t r a c t
A combination of in vitro and in silico approaches was employed to investigate the estrogenic activities of flavonoid compounds from Psoralea corylifolia. In order to develop fluorescence polarization (FP) assay for flavonoids, a soluble recombinant protein human estrogen receptor ligand binding domain(hER-LBD) was produced in Escherichia coli strain. The competition binding experiment was performedby using coumestrol (CS) as a tracer. The result of FP assay suggested that the tested flavonoids can bindto hER-LBD as affinity ligands, except for corylin. Then, molecular modeling was conducted to explorethe binding modes between hER-LBD and flavonoids. All the tested compounds fit into the hydrophobicbinding pocket of hER-LBD. The hydrophobic and hydrogen-bonding interactions are dominant forcesto stabilize the flavonoids-hER-LBD binding. It can be speculated from molecular docking study that thehydroxyl groups and prenyl group are essential for flavonoid compounds to possess estrogenic activities. Both methylation of hydroxyl group and cyclization of prenyl group significantly diminish the estrogenicpotency of flavonoids. Furthermore, quantitative structure-activity relationship (QSAR) analysis was performed by the calculated binding energies of flavonoids coupled with their determined binding affinities.Comparison between the docking scores and the pIC50values yields an R-squared value of 0.9722, indicating that the estrogenic potency of flavonoids is structure-dependent. In conclusion, molecular docking canpotentially be applied for predicting the receptor-binding properties of undescribed compounds basedon their molecular structure.


1. IntroductionAs an erect annual herb, Psoralea corylifolia (Leguminosae)has been used in traditional practices of Ayurvedic and Chinesemedicine [1–5]. It is widely distributed and considered as a naturalalternative remedy due to its diverse beneficial effects, including hepatoprotective, estrogenic, antidepressant, antimicrobial,antioxidant, and antitumor activities [6,7]. The flavonoid com-pounds derived from the fruits of Psoralea corylifolia can be dividedinto isoflavones, flavanones and chalcones [8]. Multiple biologicalactivities of these components have been confirmed, demonstrating their potential for treating diseases [6,9]. As phytoestrogens,flavonoids share similar structure with endogenous estrogens(such as 17 -estradiol). They can interfere with endocrine regulations in the human body through binding to estrogen receptors(ERs) [10–14]. Estrogen receptors are the transcriptional factors playing important roles by binding and activating estrogen response elements(EREs) on target genes, subsequently controlling cell proliferation and survival in normal mammary tissue [15]. They belong tothe nuclear receptor (NR) superfamily [16] and participate in theregulation of reproduction, development, metabolism, and homeostasis [17,18]. Two isoforms of estrogen receptors (ER and ER )have been identified and share the similar crystallographic structure [19–22]. Current available endocrine therapies for ER-positivebreast cancers mainly focus on the selective estrogen receptormodulators (SERMs) [23]. They exert dual agonistic or antagonistic effect on ER transcription and have been applied for treatinghormone responsive breast cancers for decades [24,25]. Estrogen mimetics including both natural and synthetic chemicals have been reported to selectively activate ERs [26–29].Phytoestrogens, a group of plant-derived compounds with estrogenic properties [30], can structurally or functionally mimicmammalian estrogens [31,32]. They are considered as a naturalsource of SERMs eliminating the side-effects of hormone replacement therapy. Furthermore, they are also known to exert widelybenefits to human health, especially against cancer, osteoporosis,irregular menopause syndrome, cardiovascular disease, etc. [33,34]. Genistein is a typical phytoestrogen isolated from soybeans andbelongs to isoflavonoids [35], a class of secondary metabolites thatmainly occur in Leguminosae [36]. It has been confirmed to bindto the human estrogen receptors and disrupt normal estrogenicsignaling. However, the estrogenic potential of many other naturally occurring flavonoid compounds and the underlying molecularmechanism of their pharmacological activities are still unclear.Hence, the present work focuses on the estrogenicity of flavonoidsisolated from the fruits of Psoralea corylifolia.A combination of in vitro and in silico approaches was employedto investigate the estrogenic activities of flavonoid compounds,including two isoflavones corylin and neobavaisoflavone, three flavanones bavachin, isobavachin and bavachinin, three chalconesbavachalcone, isobavachalcone, and 4 -O-methylbavachalcone. Inorder to develop fluorescence polarization assay for flavonoid compounds, a soluble recombinant protein human estrogen receptor ligand binding domain (hER-LBD) was produced first. The competition binding experiment was performed by using coumestrol (CS)as a tracer. Based on the determined binding affinities of hER-LBDwith flavonoids, molecular docking was conducted to explore theirbinding modes, in an attempt to establish a quantitative structure-activity relationship (QSAR) model for evaluating and predictingthe estrogenic potential of flavonoid compounds.


2. Materials and methods
2.1. Materials and chemicals
Isopropyl -D-1-thiogalactopyranoside (IPTG), dimethylsulfoxide (DMSO), and coumestrol (CS) were purchased fromSigma-Aldrich (St. Louis, MO, USA) and TCI (Tokyo, Japan). Corylin Corylin(≥98%), neobavaisoflavone (≥98%), bavachin (≥98%), isobavachin(≥98%), bavachinin (≥99%), bavachalcone (≥98%), isobavachalcone(≥98%), and 4 -O-methylbavachalcone (≥98%) were purchasedfrom Yuanye Biotechnology Co., Ltd. (Shanghai, China). The structures of these flavonoid compounds are shown in Fig. 1. All otherreagents used were of analytical grade.


2.2. Expression and purification of hER˛-LBD

The coding sequences of human estrogen receptor ligandbinding domain (hER-LBD) and glutathione S-transferase (GST)were inserted into the pGEX-4T-1 vector at restriction sites BamHIand XhoI. The expression plasmid pGEX-4T-1-hER-LBD was introduced into Escherichia coli strain BL21(DE3)pLysS. Cells weretreated with 0.5 mM IPTG overnight at 20◦C to induce the expression of hER-LBD. A 0.22 m membrane filter (Millipore, Bedford,MA, USA) was used to remove all the bacterial cells from suspension. Afterward, the supernatant was loaded onto an IDA-Ni2+column (Novagen, Madison, WI, USA) to purify the target protein.


2.3. Fluorescence polarization assay

In this work, an autofluorescent exogenous estrogen coumestrol(CS) was employed as a probe. The protein hER-LBD (250 nM)and the probe (10 nM) were mixed in a total volume of 290 Land titrated with various concentrations of flavonoids (10 L). Themicroplate was subjected to FlexStation 3 (Molecular Devices, Sunnyvale, CA, USA) after being incubated at room temperature for 2 h.The excitation and emission wavelengths were 355 and 405 nm,respectively. The IC50value (concentration of flavonoid for 50%inhibition of binding between CS and hER -LBD) was calculatedaccording to a four parameter logistic equation Y = (A − D) / [1 + (X/ IC50)B] + D, where Y and X correspond to the polarization value andthe tanshinone concentration, A and D are the polarization valuesat zero and an infinite concentration respectively, and B is the slope parameter. Data analysis was performed using GraphPad Prism 5(GraphPad Software, USA).


2.4. Molecular docking

The crystal structure of hER-LBD complexed with diethylstilbestrol (DES) was obtained from the Protein Data Bank (PDBID: 3ERD) [37]. The initial structures of flavonoid compoundswere constructed with GaussView 5.0.9 and optimized using theB3LYP/6-31G(d) method with Gaussian 09W. The molecular lengthand Connolly solvent-excluded volume (CSEV) of flavonoids werecalculated by using AutoDockTools-1.5.6 and Chem3D Ultra 8.0,respectively. Then automated docking with grid-based energy evaluation was carried out by AutoDockTools-1.5.6 to explore theflavonoids-hER-LBD binding modes. The center of the grid boxwas identified by the location of the synthetic nonsteroidal agonist DES with its size adjusted so as to enclose all the key residues.We built the box around hER-LBD with 38 points cube coverage and set a spacing of 0.375 Å between the grid points. Thetested compounds were docked into rigid receptor structure usinga Lamarckian genetic algorithm (GA) provided by AutoDockTools-1.5.6. All other docking parameters, such as number of GA runs(10), population size (150), maximum number of evals (medium),maximum number of generations (27,000), rate of gene mutation(0.02), and rate of crossover (0.8), were set to defaults. The predicted binding energies (kcal mol−1) were calculated based on thescoring function. For each flavonoid, 10 independent docking runswere conducted and the first-ranked conformation with the lowest binding energy was chosen to provide insights into its bindingmodes toward hER-LBD. The intermolecular interactions werevisualized by using the program PyMol.



3. Results and discussion

3.1. Determination of binding potency between hER˛-LBD andflavonoids
For competitive fluorescence polarization assay, a soluble protein hER-LBD and an exogenous estrogen coumestrol (CS) wereemployed as recognition element and fluorescent tracer, respectively. The recombinant protein was expressed in Escherichia coliand purified with immobilized metal affinity chromatography(IMAC). At the beginning of the determination, the receptor andthe tracer form a CS-hER-LBD complex. With a large molecular volume, CS-hER -LBD rotates slowly and produces a high FPvalue. Then, if the added flavonoid can compete for the bindingsite of receptor, CS is displaced from the complex. The unboundtracer molecule, with decreasing size, rotates remarkably fast andcauses a low FP value. Therefore, the FP signal can be monitored todifferentiate the bound and unbound tracer.
As can be observed in Fig. 2, all the tested flavonoids (exceptfor corylin) exhibited dose-dependent binding to hER-LBD andtheir IC50values obtained from the competition curves werelisted in Table 1. The flavonoid compounds from Psoralea corylifolia demonstrate distinct binding potency toward hER-LBDwith IC50values ranging from 5.6 nM to 949.6 nM, except forcorylin. Their binding affinities with hER -LBD is in the order ofneobavaisoflavone > isobavachin > bavachalcone > isobavachalcone > 4'-O-methylbavachalcone > bavachin > bavachinin > corylin. For theisoflavones derivatives investigated herein, neobavaisoflavonewith a prenyl group exhibits remarkably greater estrogenicpotency than corylin, whose prenyl group is cyclized. It has beenreported that the presence of prenyl group appears to be crucialfor estrogenic effects [38], which is confirmed in this work. In summary, the flavonoid compounds from Psoralea corylifolia canbind to hER-LBD as phytoestrogens.


3.2. Structural basis for estrogenicity of flavonoids

It is generally assumed that phytoestrogens and xenoestrogens exert their stimulatory effects on ERs through binding atthe same site as that occupied by endogenous estrogens such as17-estradiol [39,40]. Previous studies have revealed the crystal structures of hER-LBD complexed with several flavonoid-likecompounds, such as genistein (PDB ID: 1 × 7R and 2QA8) andbiochanin A (PDB ID: 5JMM) [41,42]. However, the protein structures co-crystallized with the flavonoid compounds from Psoraleacorylifolia have not been reported yet. In this work, moleculardocking was conducted to explore the binding modes betweenhER -LBD and flavonoids.
The hydrophobic binding pocket of hER -LBD possesses a probeaccessible volume of 450 Å3, nearly twice as large compared todiethylstilbestrol (DES), a synthetic nonsteroidal agonist ligand[43]. The molecular length and Connolly solvent-excluded volume(CSEV) of flavonoid compounds were calculated and compared tothat of DES (Table 2). The sizes of flavonoids are close to that ofDES, making the hydrophobic pocket large enough to accommodate these compounds. As shown in Fig. 3, flavonoids completely fitinto the cavity without disruption of the coactivator binding site,known as a transcriptional activation function 2 (AF-2). Furthermore, it can be observed that all these flavonoids fit into the bindingsite similar to that of DES. As the core helix of AF-2, H12 is stabilized by flavonoids to pack against H3 and H11, constraining theirmovement (Fig. 4). As a result, the receptor seals flavonoids in thecavity and forms a hydrophobic groove to facilitate co-activator(CoA) recruitment.

Molecular docking results suggest that hydrophobic andhydrogen-bonding interactions are the dominant forces to stabilize the flavonoids-hER -LBD binding. As shown in Fig. 4, withregard to isoflavones, neobavaisoflavone makes significant morehydrogen-bonding interactions than that of corylin. The two phenolic hydroxyl groups of neobavaisoflavone form hydrogen bondswith polar residues located at the two ends of the cavity, whichsignificantly help to improve the stability of neobavaisoflavone-hER-LBD binding. However, only a hydrogen bond can beobserved between corylin and hER-LBD, which may be attributedto the cyclization between hydroxyl and prenyl group. For three fla-vanones investigated in this work, both bavachin and isobavachinform hydrogen bonds at two active sites, namely H3 and H11.Unfortunately, the phenolic hydroxyl group at A-ring of bavachininis methylated, resulting in the loss of hydrogen-bonding interaction with Gly521 in H11. Similarly, for the tested chalcones,4 -O-methylbavachalcone loses the hydrogen bond toward Gly521(H11) compared to bavachalcone and isobavachalcone, due to themethylation of one of its phenolic hydroxyl group at A-ring. Hence,it can be speculated that the hydroxyl groups and prenyl group are essential for flavonoid compounds to possess estrogenic activities. Furthermore, as shown in Fig. 4 and Table 2, all the testedcompounds form a hydrogen bond with a water molecule, exceptfor corylin. Loss of hydrogen bonds with the water molecule andthe key residues in H3 greatly diminish the estrogenic potencyof corylin, which is consistent with aforementioned result in thefluorescence polarization assay.


3.3. Quantitative structure-activity relationship (QSAR) model

For each docked ligand, the binding energy (score) were calculated by AutoDock. Among these flavonoids, neobavaisoflavoneseems to be the most potent ER ligand with maximum binding energy (−9.92 kcal mol−1), as shown in Table 1. Due tothe cyclization between hydroxyl and prenyl group, corylindisplays a minimum binding energy (−3.53 kcal mol−1).The order of the calculated binding energies is as follow:neobavaisoflavone > isobavachin > bavachalcone > isobavachalcone> 4 -O-methylbavachalcone > bavachin > bavachinin > corylin.Interestingly, the predicted binding potency for flavonoids withhER -LBD is in agreement with their experimentally determinedbinding affinities. As shown in Fig. 5, comparison of the docking scores with the pIC50values, namely -log10(IC50) values[44], yields an R-squared value of 0.9722, indicating that theestrogenic potency of flavonoids is structure-dependent. Thena quantitative structure-activity relationship (QSAR) model was established for evaluating and predicting the estrogenic potentialof flavonoid compounds. Based on the structures of undescribedcompounds, molecular docking may be helpful for predicting theirreceptor-binding properties.



4. Conclusion

The present work aims to investigate the estrogenic activitiesof flavonoid compounds from Psoralea corylifolia by a combinationof fluorescence polarization and molecular docking approaches.Both in vitro and in silico studies indicate that the tested flavonoidcompounds from Psoralea corylifolia can bind to hER-LBD asaffinity ligands, except for corylin. The hydrophobic and hydrogen-bonding interactions are the dominant forces to stabilize theflavonoids-hER-LBD binding. Structure-activity relationship analysis of estrogenic flavonoids suggests that both methylation ofhydroxyl group and cyclization of prenyl group significantly diminish their estrogenic potency. Therefore, it can be speculated thatthe hydroxyl groups and prenyl group are essential for flavonoidcompounds to possess estrogenic activities. Additionally, the correlation analysis between the docking scores and the pIC50valuesindicates that the estrogenic potency of flavonoids is structure-dependent. This work may be helpful for in silico screeningof selective estrogen receptor modulators (SERMs) from naturalbioactive compounds based on their molecular structures.


Acknowledgements

This work was supported by the National Natural ScienceFoundation of China (31871717, 31601534 and 31701349), theNational Key Research and Development Program of China(2018YFD0300200 and 2016YFD0300103), the China PostdoctoralScience Foundation (2018T110249 and 2017M621213), and theScience and Technology Development Project Foundation of JilinProvince (20180520102JH, 20180201062SF, and 20150519010JH).



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