Structure-based design of eugenol analogs as potential estrogen receptor antagonists

Background

Eugenol (compound 1) is an essential oil mainly found in thebuds and leaves of clove (Syzygium aromaticum (L.) Merrill andPerry [1]. This essential oil has some biological activities, e.g.,antiinfective (i.e., antibacterial, anthelmintic, antifungal,antiplasmodial and antiviral), anti-inflammatory, analgesic,antioxidant, antimutagenic, antigenotoxic, modulatory andanticancer [1,2]. As an anticancer, eugenol inhibits cellproliferation and induces apoptosis in human MCF-7 breastcancer cells [2,3]. This type of cancer is the most common formamong women [4]. Therefore, drug discovery efforts byexploring the potency of eugenol in order to develop novel andpotent pharmaceuticals for breast cancer therapy are ofconsiderable interest.

The biological activities of eugenol in human breast cancer cellscan be correlated to its potential activity as an estrogen receptorα (ER) antagonist [1–3]. Interestingly, the standard adjuvant forpostmenopausal women with hormone-receptor–positive earlybreast cancer Tamoxifen (Astra Zeneca) is an ER antagonist [5].Tamoxifen itself is a prodrug that is metabolized in the liverresults in some active metabolites (e.g., 4-hydroxytamoxifenand N-desmethyl-4-hydroxy-tamoxifen), with 30-100 foldactivity in the binding to ER compared to its original form [6].On the other hand, compared to tamoxifen and its metabolites,eugenol can be considered as a small fragment that has apotency to be developed further in a direction guided by acomputer-aided structure-based design [7,8] to havecompounds that have similar or even better affinities to ER thantamoxifen and its metabolites.

We described previously the construction and validation of thestructure-based virtual screening (SBVS) protocols to discovercyclooxygenase-2 (COX-2) inhibitors [9]. In this article, similarapproaches were employed to construct and validate SBVSprotocols to discover potent ER antagonist. Fortunately, similarto the retrospective validation of the SBVS to discover COX-2inhibitors, the dataset to retrospectively validate SBVS protocolsto discover potent ER antagonist has been made publiclyavailable in the directory of useful decoys (DUD;http://dud.docking.org/r2/er_antagonist.tar.gz) [10]. Thevalidated protocol has a better enrichment factor in 1% falsepositive (EF_1%) compared to the first SBVS campaign usingDUD to retrospectively identify ER antagonists [10]. Moreover,the EF_1% value of the validated protocol constructed here issignificantly higher than the average value (17.3) resulted fromthe first SBVS campaign of 40 targets employing DUD and cantherefore be considered as acceptable [10]. The validatedprotocol was subsequently employed to virtually screeneugenol (compound 1), its analogues (compounds 2-7) and theirdimers (8-14). None of the compounds show better dockingscore as compared to the threshold compound of the EF_1%value. However, instead of being considered as drug-likecompounds, the screened compounds are considered asfragments that can be developed further [7,11]. Therefore, byemploying docking score ligand efficiency (DSLE; the absolutevalue of docking score divided by number of heavy atoms)value these initial results guide us to select dimer 11 (4-[4-hydroxy-3-(prop-2-en-1-yl)phenyl]-2-(prop-2-en-1-yl)phenol) asthe most potential fragment to be developed further in order todiscover novel and potent ER antagonists.

Methodology

Results and Discussion

The aim of this research was to construct a validated SBVSprotocol to discover potent ER antagonist and subsequently usethe protocol to virtually screen small fragments eugenol and itsanalogs in order to develop novel and potent ER antagonists.Potential small fragments with low potency but high ligandefficiency recognized in a SBVS campaign can successfully leadto high affinity ligand after structure-based optimization [7,8,17].

The crystal structure with PDB code of 3ERT, which was usedin the reference SBVS protocol using DUD, was selected as thereference target [10,12]. This crystal structure has an acceptableresolution (1.90 Å) and the ER in this crystal structure was cocrystalizedwith ligand 4-hydroxytamoxifen, a high affinity ERantagonist with binding affinity (Ki) value in the range ofnanomolar concentrations [12]. By visual inspection of thecrystal structure 3ERT, the optimal protein-ligand interactionscan be studied. There were 70 residues recognized as thebinding pocket residues: LEU327, TYR328, SER329, GLU330,SER341, MET342, MET343, GLY344, LEU345, LEU346, THR347,ASN348, LEU349, ALA350, ASP351, ARG352, GLU353, LEU354,VAL355, MET357, LEU379, GLU380, CYS381, ALA382, TRP383,LEU384, GLU385, ILE386, LEU387, MET388, ILE389, GLY390,LEU391, VAL392, ARG394, SER395, LEU402, LEU403, PHE404,ALA405, LEU408, LEU410, GLY415, VAL418, GLU419, GLY420,MET421, VAL422, GLU423, ILE424, PHE425, LEU428, ILE514,HIS516, MET517, SER518, ASN519, LYS520, GLY521, MET522,GLU523, HIS524, LEU525, TYR526, SER527, MET528, LYS529,CYS530, LEU536, and LEU539. Interestingly, one watermolecule was observed in the binding pocket and this watermolecule can be considered as conserved [18]. Two hydrogenbonds networks were observed during the visual inspection:

• the 4-hydroxy moiety of the 4-hydroxytamoxifen with the conserved water molecule and residues GLU353 and ARG394, and
• the (2-hydroxyethyl) dimethylazanium moiety of the 4- hydroxytamoxifen with residues THR347 and ASP351.

The internal validation was aimed to examine whether thedocking simulation used by the SBVS protocols can reproducethe pose of the co-crystal ligand [15]. The objective functionused in the internal validation was the root mean squaredistance (RMSD) value between the heavy atoms of the dockedpose and the crystal structure pose. The default configurationresulted in the RMSD value of 1.670 Å, while the configurationthat took into account the conserved water molecule resulted inthe RMSD value of 1.403 Å. Although the configurationconsidering the conserved water molecule gave a slightly betterRMSD value, since a protocol is acceptable if the RMSD value isless than 2.0 [15], both protocols can be considered asacceptable. Interestingly, the docked poses resulted from bothprotocols still maintain the hydrogen bonds networks withresidues THR347, ASP351, GLU353, and ARG394, though thedefault protocol did not involve the conserved water molecule.

The reference retrospective SBVS campaign using DUD showedEF_1% value of 12.7 [10]. Moreover, the most recent retrospectiveSBVS campaign using enhanced DUD showed EF_1% value of 15[19]. Remarkably, the independent retrospective SBVScampaigns using DUD dataset employing PLANTS1.2described here showed that the default protocol resulted inEF_1% value of 15.9 and the protocol that involved the conservedwater molecule resulted in EF_1%value of 21.2. Both values givebetter EF_1% value compared to the reference SBVS campaigns.Notably, the SBVS protocol that involved the conserved watermolecule gave significantly higher EF_1%value compared toothers. This indicates that the conserved water molecule playsan important role in the SBVS campaigns to identify ERantagonists. The EF_1%value of the validated protocolconstructed here (21.2) is above the average value (17.3)resulted from the first SBVS campaign of 40 targets employingDUD [10]. Thus, the SBVS protocol that involved the conservedwater molecule is therefore acceptable and selected for furtherSBVS campaign in subsequent prospective efforts. Using theEF_1%value, a reference compound that can be used as thethreshold compound in the prospective SBVS was recognized.The compound is ZINC01914469 (compound 15), an ERantagonist with IC₅₀ value of 69.23 nM [20].

The prospective screening results of eugenol (1), its analogues(2-7) and their dimers (8-14) together with compound 15 as thereference compound are presented in Table 1 (seesupplementary material). None of the screened compoundshows a better ChemPLP score as compared to compound 15.However, in order to rank the small fragments 1-14 to bedeveloped further, another objective value named DSLE isintroduced. This value is a modified ligand efficiency [17]which uses docking score instead of the observed affinity.

According to Table 1, eugenol and its analogs in this researchresulted in higher DSLE values than the reference compound15. This indicates that compounds 1-7 can serve as good startingpoints in the development of novel and potent ER antagonists.In order to narrow the degree of freedom in the furtherdevelopment, initial design by dimerization (compounds 8-14)was proposed. The prospective SBVS campaign showed that thesuccess of the strategy was monomer dependent sincecompounds 8, 10, 13 and 14 were shown significant decrease inthe DSLE values, which were lower than the DSLE value of thereference compound. Notably, the dimer 11 4-[4-hydroxy-3-(prop-2-en-1-yl) phenyl]-2-(prop-2-en-1-yl) phenol showed thehighest DSLE value among the dimers and therefore has beensuggested to be developed further. The superposition of thedocked poses of compounds 11 and 15 is presented in (Figure 1). Based on (Figure 1), the phenolic moieties nearest to theconserved water molecule of both compound 11 and 15 arelocated very similar. This creates the hydrogen bonds networkto residues GLU353 and ARG394.However, compound 11 lacksof basic moiety that can bind to residue ASP351. Therefore therecommended design strategy to develop compound 11 is toadd at least a basic moiety in the similar position to basicmoiety of compound 15. Subsequently, another phenol moietyto fulfill the hydrophobic pocket possessed by compound 15can be added to increase the affinity (Figure 1).

Conclusion

The construction and the retrospective validation of SBVSprotocols to identify ER antagonists have successfully provideda valid tool to screen potential ER antagonists virtually. Thevalidated protocol has an EF_1% of 21.2, which is considered asacceptable. The validation processes have also revealed that theconserved water molecule in the binding pocket of the crystalstructure 3ERT plays an important role in the quality of theSBVS protocol. Subsequent prospective screen on eugenol, itsanalogs and their dimers has suggested dimer 11 4-[4-hydroxy-3-(prop-2-en-1-yl)phenyl]-2-(prop-2-en-1-yl)phenol to bedeveloped further in order to discover novel and potent ERantagonists.

Supplementary material