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Molecular Docking Fevicordin on Human Estrogen α Receptor

Using MOE Software

Aulia Rachman1, Muchtaridi1, Abdul Mutalib2,3

1Faculty of Pharmacy Padjadjaran University Bandung

2Department of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University Bandung 3Center for Radioisotopes and Radiopharmaceuticals, BATAN, PUSPIPTEK, Serpong

Abstract

Fevicordin have similar structure to estradiol and cytotoxic in nature, thus can be

developed as cancer drug potentially. Docking method can be applied to simulate it interactions with human estrogen α receptor which known involve in growth of breast cancer. Information from simulation can be used to optimize it activity on target. The purpose of this research was to analyze interaction between fevicordin and human estrogen α receptor (hERα) LBD and to predict its affinity with MOE. Docking method was validated with redocking x-ray estradiol and modeled estradiol into hERα LBD. Fevicordin 3D structure was modeled using molecular mechanics in MMFF94s force field and fevicordin conformer given by stochastic conformational search.. Each conformer was docked into x-ray estradiol binding pocket (SP). Validation of docking method proved that MOE could predict the binding mode of both x-ray estradiol and modeled estradiol. Docking with Affinity dG scoring function show result that fevicordin have less binding affinity to LBD (ligand binding domain) human estrogen-α- receptor than estradiol. Fevicordin has predict binding to alternative pocket LBD (ligand binding domain) human estrogen-α- receptor so that fevicordin has predict not competitive with estradiol. Binding free energy fevicordin has predicted -12,4645 kkal/mol. Keywords: Fevicordin, estrogen receptor, docking, estradiol

Introduction

Compound fevicordin isolated from mahkota dewa seed (Phaleria marcocarpa (Scheff) Boerl.) be a steroid compound with framework looking like estradiol. This toxic compound to cancer cell (Diantini et al., 2007). Existence of hydroxy bunch at C3 aromatic ring of fevicordin indicates ability for binding with estrogen receptor. This thing haves occasion to because one of farmakofor required for binding with estrogen receptor is existence of aromatic ring substitution with hydroxy bunch (Duax et al.,1985).

2

3

4

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10

1

6

7

8

9 14

13

12

11

15

16

17

HO

HO

O

20

21

22

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24

2527

26

O

28

O

OOH

OH

29

18

3019

Figure 1 fevicordin structure

CH3

HO

OH

Figure 2 estradiol structure

Resemblance of main frame fevicordin with estradiol is estimated render affinity to estrogen α receptor through interaction of van der waals with ligand binding area estrogen α receptor ( Anstead, Carlson, Katzenellenbogen, 1997) Cytotoxic characteristic by fevicordin and possibility that interaction with estrogen α receptor potency in therapy of breast cancer. Human estrogen α receptor known stands in growth of breast cancer cells -yang-dipengaruhi-hormon ( Fujita et al., 2003) so that ligand which inhibition of activity estrogen α receptor can prevent growth of cancer cells. Research before all about the relation of estradiol analogue struktur-aktivity indicates that conformation of structure, position of subtituen and character fisikokimia subtituen-subtituen influences estradiol analogue binding affinity to Human estrogen α receptor (Anstead, Carlson, Katzenellenbogen, 1997). Subtituen-subtituen at fevicordin is differing from estradiol is anticipated to influences affinity fevicordin to human estrogen α receptor. Affinity fevicordin to human estrogen α receptor can be predicted with simulation docking. Principle docking is location technique of ligand into active side of receptor continued with evaluation of molecule based on conformity of form and nature of like electrostatic ( Kroemer, 2003). Information docking about relative position of ligand in receptor, involving chemistry binding and the role of subtituen-

ISBN 978-979-18962-0-7

A. Rachman et al.

Proceeding of The International Seminar on Chemistry 2008 (pp. 325-331)

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subtituen ligand in interaction ligan-reseptor serve the purpose of information to predict strength of binding fevicordin to human estrogen α receptor.

Materials and Method

Structure 3D LBD ( ligand binding domain) human estrogen α receptor wild type which is kristal together estradiol with resolution 2,9 Å obtained from online data base Protein Data Bank ( www.rcsb.org) with access code PDB: 1G50. Structural two dimensions fevicordin obtained from Diantini (Diantini et al., 2007). Hardware applied for calculation, molecule modelling, and docking molecule for example personal computer ( personal computer) what equiped processor Mobile Intel® Pentium™ 4-M CPU (Central Processing Unit) 18GHz (www.intel.com),harddisk 80 GB, graph ATI Mobility Radeon 16 MB and memory RAM 512 MB. Software applied is program package stochastic conformation search to model estradìol and fevicordin , program package MOE Sequence editor applied to present, and dissociates ligand structure from macromolecules structure. The licence MOE 2007.09 (Chemical Computing Group www.chemcomp.com) from the Department of Chemistry Faculty of Mathematics and Natural Science Padjadjaran University applied to draw up protein structure and ligand structure. While program package MOE dock applied for and draws up parameter docking and simulation of process docking . Program package Alpha Site Finder which applicable to predict location and binding pocket volume place of the happening of interaction of ligand with macromolecules.

Docking validation

The goal of validation is to get evidence that is believable that method and also technique docking applied will give result of docking matching with the one which actually or comes near actually. Validation is started with docking estradiol x-ray at LBD (ligand binding domain) human estrogen α receptor with aim to observe and performance test MOE 2007.09 reproducing orientation and interactions macromolekul-ligan result of crystallography. Validation is continued with docking estradiol result of modelling at LBD human estrogen α receptor with aim to observe and performance test MOE 2007.09 reproducing orientation and interactions makromolekul-ligan result of modelling. Paramater-parameter result docking evaluated covers orientation of ligand, deviation value position of ligand space result of docking compared to position of ligand result of crystallography (RMSD), hydrophobic interaction, electrostatic, hydrogen bond happened between proteins and ligand, binding free energy.

Then the parameters is compared to the same parameters observed in complex LBD human estrogen α receptor-estradiol. Docking with MOE 2007.09 assumed to be valid if when: a).RMSD value between x-ray estradiol or model estradiol result of docking with estradiol x-ray is less or equal to 2 Å; b).Can reproduce orientation of ligand in binding pocket and also binding site initialy through equality in hydrophobic interaction, electrostatic, hydrogen bond and amino acid residue standing in process of the interaction; and c)Contiguity of free energy value of binding result of docking with free energy value result of experiment. Validation stages;steps is as follows: 1. Preparation of Macromolecules

Structure 3D of Human estrogen α receptor wild type is containing estradiol molecule as ligand (access code PDB:1G50) presented through MOE 2007.09 files open. Pass program package MOE 2007.09 Sequence editor of all water molecule, chain B and C is vanished except chain A is let like existence. With the same program package of estradiol locked out of chain A, then each structure result of dissociation kept in separate file in the form of moe file format. Chain A structure which has separated then is given hydrogen atom, while parsial charge every atom is calculated by using compute partial charges with amber forcefield 99 have been available in program package MOE 2007.09. 2. Preparation of Ligand

Preparation of ligand done for x-ray estradiol hereinafter for model estradiol. X-ray estradiol structure obtained through dissociation from chain A LBD human estrogen α receptor then is equiped with hydrogen. Partial charge for every molecule atom is calculated to applies partial charges which have been available in program package MOE 2007.09. 3. Estradiol modelling

Estradiol three dimensions structure is built with program package Chem Office 8.0 and kept by in moe file format. Then file with the moe format opened in program package MOE and kept in file format moe. Minimization of Model structure total energy is done with MOE current forcefield that is with calculation molecular mechanics forcefield MMFF94s in program package MOE Energy Minimization. Then model estradiol minimum energy structure kept in file format moe.

Model estradiol minimum energy structure is searched by best conformation with program package MOE Stochastic Conformational Search. Forcefield is arranged with potentially setup MMFF94s. Then conformation of model estradiol is obtained by arranging the parameter at Compute Conformations Stochastic Search. Result of best conformation with low energy hereinafter applied in process of docking model estradiol at LBD human estrogen α receptor

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Proceeding of The International Seminar on Chemistry 2008 (pp. 325-331)

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4. Re-docking x-ray estradiol and model estradiol

to LBD human estrogen α receptor in binding

pocket initially

Docking parameter is a given by location of binding pocket between chain A from LBD human estrogen α receptor as macromolecules with estradiol as ligand. Process docking is done to applies two methods scoring that is method Affinity dG scoring Function and London dG scoring Function in program package dock MOE 2007.09. Other docking parameter as receptor is selected receptor and solvent, site is selected dummy atoms, placement method is applied Alpha Triangle, refinement or attenuation is selected gridmin, and rescoring applies method Affinity dG scoring Function or London dG scoring Function. Result of process docking then kept in file is having format moe. Data result of docking presented by data base viewer and kept automatically in file format mdb. 5. Docking Evaluation

Evaluation is done to confomer with topscore ranking result of various method scoring. Evaluation parameter in the form of orientation x-ray estadiol and also model estradiol, hydrophobic interaction, electrostatic, hydrogen bond formed, free energy value result of docking compared to the same parameters observed at result of experiment of crystallography estradiol with LBD human estrogen α receptor.

Modelling Fevicordin

Fevicordin three dimensions structure is modeled with stages;steps as follows : Structural three dimensions fevicordin is built with program package Chem Office 8.0 and save as in moe file format. Then file with the moe format opened in program package MOE and kept in file format moe. Minimization of model structure total energy is done with MOE current forcefield that is with calculation molecular mechanics forcefield with forcefield MMFF94s in program package MOE Energy Minimization. Then minimum energy model structure saved in moe file format. Model fevicordin minimum energy structure is searched by determine the best conformation with program package MOE Stochastic Conformational Search. Force field is arranged potentially setup MMFF94s. Then conformation model fevicordin is obtained by arranging its the parameter at Compute Conformations Stochastic Search, hereinafter conformation fevicordin is obtained by implementing Conformations Stochastic Search. Result of best conformation with low energy hereinafter applied in process of docking fevicordin model at LBD human estrogen α receptor

Docking Fevicordin model at Steroid Pocket (SP)

Fevicordin model and macromolecules in the form of LBD human estrogen α receptor prepared in the same way like at preparation procedure of ligand and macromolecules for validation of method docking. Parameter docking is determine by location of binding pocket between chain A from LBD human estrogen α receptor as macromolecules with fevicordin as ligand. Docking process is done to applies two methods scoring that is method Affinity dG scoring Function and London dG scoring Function in program package dock MOE 2007.09. Other docking parameter as receptor is selected receptor and solvent, site is selected is dummy atoms, placement method is applied by Alpha Triangle, refinement or attenuation is selected by gridmin, and rescoring applies method Affinity dG scoring function or London dG scoring function. Result of docking process then kept in file is having format moe. Data result of docking presented by data base viewer and kept automatically in file format mdb. Result of docking in the form of orientation of ligand, interactions happened between ligands with binding pocket amino acid residue, binding energy value from conformation of topscore rank in population compared to data result of experiment of complex LBD human estrogen α receptor estradiol initialy.

Results and Discussion Docking validation

Result of docking estradiol x-ray with scoring affinity dG and london dG shows equality by three amino acid residues that is successfully embosomed that is Arg394, Glu353, and His524. At method affinity dG contribution to the biggest free energy value with score 69.2% is yielded as result of interaction of hydrogen bond between atoms H Arg394 with atom O 4009 estradiols x-ray with ligand as proton acceptor. While at method london dG contribution to the biggest free energy value with score 69.2% is yielded as result of interaction of hydrogen bond between atoms H Arg394 with atom O 4157 estradiols x-ray with ligand as proton acceptor. Both methods scoring shows residue Glu353 to form hydrogen bond that is between atoms O Glu353 with atom H estradiol x-ray with contribution to free energy value equal to 43.1% for method affinity dG and london dG. Both methods skoring shows residue His524 to form hydrogen bond that is between atoms N His524 with atom O estradiol x-ray with contribution to free energy value each of 26.4%

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(a) (b)

Figure 3 Visualisation of interaction of estradiol x-ray with residue closest. (a)Visualisation of interaction of

estradiol x-ray with residue closest with scoring affinity dG method. (b)Visualisation of interaction of estradiol x-ray with residue closest with scoring London dG method.

(a) (b) (c)

Figure 4 Conformer-Conformer On Unique Fevicordin.Description: Conformer K1 ( a), conformer K2 ( b), conformer K3 ( c); ring B at K1, K2 and K3 in the form of boat conformation is distortion, ring C K2 and K3 in the form of chair conformation; all conformer is showed [by] without hydrogen atom for clarity.

Table.1 Free energy value result of re-docking

No. Docking Free energy(s)

1. Estradiol x-ray with affinity dG -12,8608

2. Estradiol x-ray with London dG

-12,0734

Modelling estradiol

RMSD Value is calculated by summing up estradiol binding length difference square x-ray with model estradiol who divided with many binding length data, under square root label. At tables is upper, value RMSD shows model estradiol binding length comes near estradiol binding length x-ray although mean is experiencing curtailment. Single binding length between carbon atoms is 1,40 Å- 1,54 Å while single binding length between carbon atoms with oxygen atom is 1,4 Å- 1,51 Å (Fessenden and Fessenden, 1982) so that modelling of estradiol with style field MMFF94s and Stochastic Conformational Search at program package MOE 200709 giving result that is believable.

Modelling of estradiol with forcefield MMFF94s and Stochastic Conformational Search at program package MOE 200709 showing model who obtained as according to original structure, thereby modelling of new structure with forcefield MMFF94s and Stochastic Conformational Search at program package MOE 2007.09 can be done with level of high trust MOE 2007.09 valid in docking model estradiol to LBD human estrogen α receptor with result of RMSD 0.033 angstrom and residue having interaction approximant to look like at docking estradiol x ray.

Modelling fevicordin

Simulation of Dynamics molecular to model fevicordin yields three structure conformations given code K1, K2 and K3 in total 17 conformer Conformation K1 is in the form of letter similar "U" (Figure 4(a)) while conformation K2 and K3 in the form of conformation of letter similar " L" ( Figure 4(b) and 4(c)).

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Figure 5 Binding Modes fevicordin K1 with method scoring affinity dG

(a) (b) (c)

Figure 6 visualisation of surface of molecular steroid pocket result of docking with scoring Affinity dG method K1(a), K2(b), K3(c) fevicordin with MOE. Green (hydrophobic), purple (hydrogen bond) blue (mild polar).

Table 2 Simulation Data Docking Conformer-conformer Fevicordin at Steroid Pocket ( Scoring Affinity dG )

Ka EBb Nearest closest Residu Hydrogen bonding

(donor→akseptor) K1 -12,4645

Gly521, Pro324, Ile386, Ile424, Leu387, Lys449, Met357, Leu384, Leu525, Met421, Glu353, His524, Phe404, Leu391, Arg394, Met388, Leu428

OH3→Glu353 (1,71 Å) Lys449→OH16 (2,82Å) Arg394→O20(2,75 Å) Arg394→O22(2,63 Å)

K2 -12,2077

Leu384, Leu387, Leu391, Phe402, Leu525, Gly521, Ile386, Arg394, Glu353, Trp383, Met388, Leu346, Met522, Lys449, Gly390, Pro324, Ala350

O4251→His524 (2,81 Å) H4287→Gly521 (3,25 Å) His524→O4251 (2,81 Å) Leu525→O4251( 2,65Å)

K3 -11,7361

Arg394, Leu387, Leu391, Met357, Thr347, Gly521, Leu346, Trp383, Ile386, Leu384, Phe404, Gly390, Met522, Glu353, Leu540, Leu525, Lys449, Ala350.

Arg394→O4251 (3,05Å) H4287→Leu387 (3,22Å) H4307→Gly521(1,73Å) O4268→His524 (2,93Å) His524→O4268 (2,93Å)

a Conformer fevicordin b Binding free energy prediction (kkal/mol)

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Docking fevicordin

Result of docking fevicordin K1 with method affinity dG shows to residue Arg394 happened hydrogen bond with atom O 4260 and O4268 at ring side chain D fevicordin with contribution to interaction free energy 12,7 % and 47,4 %. Residue Glu353 forms hydrogen bond with atom H4307 as proton donor ( 1,71 Å). Lys449 forms binding hydrogen with atom O4265 as proton acceptor ( 2,82Å).Docking fevicordin K1 shows four hydrogen bonds with free energy value equal - 12,4645 kkal/mol., so that fevicordin K1 has good enough affinity compared to fevicordin K2 and K3. Table 2 showing free energy value , near residues closest, and hydrogen bond formed result of docking fevicordin K1,K2, and K3 applies method scoring affinity dG From table 2 knowable that fevicordin K1 has free energy value of result docking which is lowest compared to K2 and K3, besides K1 also has hydrogen bond looking like estradiol estradiol x-ray that is Glu353,Arg449, and Lys449 so that inferential KI has better affinity from at K2 and K3. Table 3 showing free energy value ambit result of docking estradiol x-ray, model estradiol , and fevicordin K1 applies two methods scoring.

Table 3 Free Energy Values Result Of Docking

No. Docking Free Energy

(s)

1. Estradiol x-ray with affinity dG

-12,8608

2. Estradiol x-ray with London dG

-12,0734

3. Estradiol model with affinity dG

-10.5497

4. Estradiol model with London dG

-11,7911

5. Fevicordin model K1 with affinity dG

-12,4645

6. Fevicordin model K1 with London dG

-14.8896

From tables 3 show that scoring london dG method is less suitable for ligand having bulky structure like fevicordin, this thing indicated from free energy value yielded by scoring london dG method shows difference that significant with free energy value of other method. This thing is assured with binding mode formed applies scoring london dG method differs from estradiol binding mode x-ray. Conclusions

MOE 2007.09 applicable to predict estradiol binding mode x-ray and model estradiol in accurate figure.

Simulation of Molecular dynamics with Stochastic Conformational Search yields three best conformer of structure fevicordin. Result of docking with scoring London dG method is less suitable for ligand that is bulky enough like fevicordin Result of docking with scoring Affinity dG method indicates that fevicordin to have feebler binding to LBD (ligand binding domain) human estrogen α receptor from at estradiol. Fevicordin is predicted bound at alternative pocket of LBD human estrogen α receptor. Free energy value predicted equal -12,4645 kkal/mol

Acknowledgements

We thanks to Muchtaridi and Abdul Mutalib for guidance and assistance in finalizing this paper.

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