In-Silico study of Flavonoid Compounds And its Modifications for the Medications against Breast Canc

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In-Silico study of Flavonoid Compounds And its Modifications for the Medications against Breast Cancer Growth.

Department of Biotechnology, School of Bio Science and Technology, Vellore Institute of Technology (VIT), Vellore, Tamil Nadu, India. ***

Abstract - Breast cancer is one of the greatest global issues causing to increase in death rates in females due to uncontrolled growth of a tumor that can metastasize to different parts of the human body. The binding of Estrogen receptor (ER) and Human Epidermal Growth Factor Receptor 2 (HER-2) with breast cancer cells is mainly responsible to cause malignant tumor that regulates the transcription of various genes and act as a transcription factor. Maximum of the drugs used to cure breast cancer have a negative effect on body so, we focused on natural flavonoids such as Chrysin, Equol, Hesperetin and Naringenin and modified these flavonoids which will interact with breast cancer cell receptors, such as Human Estrogen Receptor Alpha (HERα) and Human Epidermal Growth Factor Receptor 2 (HER-2), showing less toxic effect to human body. The new way to deal with the immune system studies that combine techniques of systems biology with data provided by information-driven prediction methods. The structure information of these flavonoids were collected from PubChem and the structural data of HERα and HER-2 receptor were collected from Protein Data Bank (PDB). The four ligands were modified using the Corina classic tool and drug-likeness and molecular properties of all the flavonoids’ original structure and modified structures were examined using the Molinspiration server to check if all the flavonoids satisfy “Lipinski’s Rule of Five.” Computational docking allows to study of the biomolecular interactions and ligand interactions with thousands of compounds and this virtual screening method is done using the Auto Dock tool. This study focused on the molecular docking of modified flavonoids (ligands) with breast cancer cell receptors and the bestmodified ligand against each receptor were analysed and evaluated to cure breast cancer. The best-modified flavonoid of Hesperetin is (2S)-8-amino-2-(3-hydroxyphenyl)-6-iodo-3,4dihydro-2H-1benzopyran-4-one whose highest interaction energy is6.3 Kcal/mol and -7.0 Kcal/mol respectively, with HERα and HER2 receptor of breast cancer. All the best-modified structures of Naringenin, Chrysin, and Equol flavonoid did not show potential significant difference between the original and modified structure activation energy.

Keywords: Flavonoids,activationenergy,breastcancer,autodock,naringenin,chrysin,equol,andhesperetin.

1. Introduction:

Breastcancerisoneofthemajormalignantgrowthsaffectingadultwomenbetweentheagesof20and59andtheunderlying cause of the highest mortality rate among women in India. Over the nation, the highest rates of illiteracy were found among femaleswhoareengagedinhouseholdactivitieswheretheilliteracyrateis8%to46%.Theexplanationincorporatestheneed forvalueinstruction,individuallifestyletendencies,andreproductiveage[Guptaetal.,2015;Torreetal.,2017]

Estrogenreceptorshave beendevelopedto bethe best objectiveandeffectivefor breastcancertreatmentsinceendogenous estrogens are assumed to have a significant role in the advancement of breast cancer [Debeb et al.,2015]. ER fundamentally existsin2types:ERbetaandERalpha. Estrogenreceptorα(ER)isthemajordriverof∼75%ofbreastcancers andplaysan important role in controlling the transcription of nuclear DNA necessary for mammary [Siersbæk et al., 2018].HER2 amplificationwasfirstnotedinhumanbreastcancerandwassubsequentlyidentifiedinovariancancer [Berchucketal.1990; Slamonetal.1987,1989;Zhangetal.1989].HER2proteinexpressionismainlyincreasedduetotheHER2geneamplification mechanism, which prompts overexpression of the receptor and disturbs typical control mechanism, possibly prompting the development of aggressive tumor cells [Salmon et al., 1989; Hynes et al., 1994; Hung et al., 1986]. This situation along these lines requests the advancement of better HER2-TK inhibitors with minimal toxicity issues to be presented as focused anticancertherapeutics[Chandrikaetal.,2016].

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Correct cancer treatments are those that are specifically tailored to the individual's needs in order to provide the most effectivetreatment.ThefirstPARPinhibitorhasbeenapprovedforclinicaluse[Bryantetal.,2005].Thetamoxifencitratedrug has a widespread role in treating the metastatic breast cancer (Manni et al., 1982). The use of an LH-RH agonist (zoladex) it was used with the combination with Tamoxifen as the observation was not good and there were unpredictable endocrinological responses [Klijn et al., 1984] The serious side effects of these artificial drugs used in breast cancer are uterinecancerwhichcausesasmallincreasedrisk,strokevisionproblems,thedrugsareanticancerdrugsinbreastcancerbut areacarcinogenicagentintheendometriumwhichaffectsthefemalegenitaltract,andahighriskofendometrialmalignancy [Fleming et al., 2018]. Paclitaxel and carboplatin are also used in the treatment of breast cancer and have some urinary symptomsinfemales,therewereworseningofexistingurinarycontinenceandanoverflow[Stemmleretal.,2008]

Cancercellswillingeneraldodgeapoptosis,aprocessbywhichcellsundergothroughtheprogrammedcelldeathbyharming DNA,whichthusrestrictsthecellproliferation[Evanetal.,1998;Hanahanetal.,2011].Inthisway,apoptosisinductionisone ofthebesttechniquestostopcancer,whichisusedinmostanticancertechniques,forexample,irradiationandchemotherapy treatment [Fulda et al.,2006]. But due to side effects and constraints related to these therapies, for example, cardiovascular toxicityandneuropathy, the need to discovernew chemopreventive agentswith theleastside effectsis growingin demand [Monsuezetal.,2010;Riveraetal.,2015].

So, Kawaii (1999) and Pouget (2001) discovered an alternative approach for the treatment of breast cancer by the use of traditional herbal drugs called flavonoids that have anti-breast cancer activity and show fewer side effects against human normal cells. Naturally, occurring flavonoid products are derived from plants and are found in various parts of plants [Havsteen et al., 2002]. Flavonoids are classified as flavones, flavonols, anthocyanidins, flavanols, flavanines, flavanonols, aurones,furanchromones,isoflavones,isoflavanones,biflavones,xanthones,chaoconesanddihydrochalcones(fig1) [Wanget al.,2018]

Figure1:-Flavonoidsclassifications(Wangetal.,2018)

Flavonoids are well known for their antioxidant properties [Mishra et al., 2013], anti-bacterial & anti-viral activities [Wu et al.,2008; Wang et al., 1998], anti-inflammatory and anti-thrombogenic properties [Alcaraz et al., 1987], anticancer activity [Sheikh et al., 2020] In cancer therapy, apoptosis is the known target pathway of chrysin and it affects different molecular pathways [Moghadam et al., 2020]. Hence chrysin can be used as an anti-tumor agent [Yadav et al., 2018; Wang et al., 2020; Zhangetal.,2020].Prakashetal(2020)demonstratedtheanticanceractivityofhesperetinforcervicalcancer.Naringeninisa potentialagentforanti-tumorasdemonstratedbyHughesetal(2008),Erdogduetal(2009),andVerbeeketal(2004).Shiet al(2011)Thisstudydemonstratedthatequolcanbeusedtoinhibittheexpressionofnuclearfactor-kappaBinhumanbreast

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cancer cells. Many studies have found that high intakes of flavonoids may reduce the risk of cancer in humans. [Hui et al., 2013].

FlavonoidsusedinthisprojectareChrysin,Equol,Hesperetin,andNaringeninagainsttwobreastcancercellreceptors,Human Estrogen Receptor Alpha (HERα) and Human Epidermal Growth Factor Receptor 2(HER-2). In silico study of Chrysin and Equol flavonoid is done by Suganya et al (2014) against receptor Human Estrogen Receptor Alpha (HERα), also the In silico studyofHesperetinandNaringeninflavonoidagainstHumanEpidermalGrowthFactorReceptor2(HER-2)wasconductedby Chandrikaetal(2016).So,wemodifiedallthefourflavonoids10timeseachandallmodifiedflavonoidssuccessfullysatisfied Lipinski’s ruleoffive.Allthemodifiedstructuresofeachflavonoidsweredockedagainstthereceptors HERαandHER-2and successfully the best-modified flavonoid results were obtained by comparing the activation energy of original structure of flavonoids and modified structure of flavonoids with HERα and HER2 receptors using the Auto Dock tool, and the bestmodified flavonoid was selected having more activation energy than original flavonoid, which can be used to cure breast cancerwithnosideeffects.

2. MATERIALS AND METHOD:

2.1) Protein preparation

The 3D crystallographic structure of the receptor Human Estrogen Alpha (HERα) having Protein ID:2IOG and Human Epidermal Growth Factor Receptor 2 (HER-2) having Protein ID: 3PP0 was retrieved from Protein Data Bank (PDB). All the hydrogen atoms present in the proteins were merged and all the non-essential water molecules and heteroatoms were removedduringdockinganalysisusingtheAutoDocktool.Hencethesetwowerethetargetproteinsthatweredockedagainst thefourflavonoids.

2.2) Ligand preparation

PDB format, two- and three-dimensional structures of flavonoids: Chrysin, Equol, Hesperetin, and Naringenin were obtained fromPubChemfordockinganalysisagainstHERαandHER-2receptors [Suganyaetal.,2014;Chandrikaetal.,2016].

2.3) Ligand modifications

All fourflavonoids,Chrysin, Equol,Hesperetin,andNaringenin,weremodifiedusinga Corina classictool.Three-dimensional structuresofeachmodifiedflavonoidweregeneratedusingthistool.Eachflavonoidwasmodified10times,eachofwhichwas testedagainstthetworeceptors.NamingmodifiedflavonoidsusingtheKingDrawtool.

2.4) Lipinski rule of five of ligand molecule

The molecular propertiesand drug-likenessof all the flavonoids’ original structures and modified structures were examined usingtheMolinspirationserver.ChristopherALipinskiformulatedathumbruletoassessthepropertyof drug-likeness,ithas criteriasuchasapartitionco-efficientlogPwhichislessthan5andnotmorethan5hydrogenbonddonors,andthemolecular weightshouldbebelow500Daltons.

2.5)

Molecular docking using the Auto Dock tool

The active site residues were determined after the protein and the modified ligand were prepared, providing information about the protein’s secondary structure and the protein-ligand interaction, and preserving the protein and ligand in PDBQT format. The X, Y,andZcoordinates were recorded whenthegrid box wascreated usingtheAutoDock tool, whichidentifies theresiduesimplicatedintheactivesite.Theupdatedligandandtheproteinwerestoredastheconfigurationfileasinputas conf.txt,andthedockwasrunusingthecommandprompt.Thedistancefromthebestmodeandtheaffinity(Kcal/mol)were determined[Sandeepetal.,2011][Morrisetal.,2008]

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3. RESULTS:

3.1) Target protein structure

ThemaintargetofthebreastcancercellwereHumanEstrogenReceptorAlpha(HERα)andHumanEpidermalGrowthFactor Receptor2(HER-2).Boththereceptors3DstructureswererecoveredfromProteinDataBank(PDB)and thecrystallography structurewerevisualizedinPyMolsoftwareasshowninfigure2andfigure3.

3.2)

Docking analysis

Figure2:-HumanEstrogenReceptor Alpha(HERα)withPDBID:2IOG

Figure3:-HumanEpidermalGrowthFactor Receptor2(HER-2)withPDBID:3PP0

Suganya et al (2014) demonstrated the docking of 19 different flavonoid original structures with Human Estrogen Receptor Alpha (HERα) and concluded that Chrysin and Equol have -11.01 kcal/mol and -10.83 kcal/mol of highest activation energy respectively.Hence,thesetwoflavonoids,ChrysinandEquol wereselected fordocking withHERα.Similarly,Chandrika etal (2016) demonstrated the docking of 150 flavonoids with HER2 receptors of breast cancer and concluded that the two citric fruit flavonoids such as Hesperetin and Naringenin were the best-docked results with HER2. Therefore, all the 10 modified structuresoffourflavonoidssuchasChrysin,Equol,Hesperetin,andNaringeninweresuccessfullydockedwithtworeceptors ofbreastcancer,HumanEstrogenReceptorAlpha(HERα)andHumanEpidermalGrowthFactorReceptor2(HER-2).

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Table -1: DockingresultsofHumanEstrogenReceptorAlpha(HERα)andHumanEpidermalGrowthFactorReceptor2(HER2)withmodifiedChrysinflavonoid.

Sr. no.

Name (IUPAC)

Structure modified Energy value (Kcal/mol) HERα HER2 1. -6.2 -6.8 2. -6.0 -6.3 3. -5.0 -6.3 4. -5.7 -6.5 5. -6.0 -5.6 6. -5.7 -5.7 7. -5.9 -6.6

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8. -5.7 -6.7 9. -5.9 -5.4 10. -5.4 -6.4 11. -5.3 -5.7

Table -2: DockingresultsofHumanEstrogenReceptorAlpha(HERα)andHumanEpidermalGrowthFactorReceptor2(HER2)withmodifiedEquolflavonoid.

Sr. no. Name (IUPAC) Structure modified Energy value (Kcal/mol)

HERα HER2 1. -6.3 -6.5 2. -5.5 -5.5 3. -5.3 -5.4

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4. -5.4 -5.3 5. -4.9 -6.5 6. -4.2 -5.0 7. -5.3 -6.0 8. -5.1 -4.8 9. -5.2 -6.0 10. -5.1 -5.4 11. -5.6 -6.0

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Table -3: DockingresultsofHumanEstrogenReceptorAlpha(HERα)andHumanEpidermalGrowthFactorReceptor2(HER2)withmodifiedHesperetinflavonoid.

Sr. no. Name (IUPAC)

Structure modified Energy value (Kcal/mol)

HERα HER2 1. -4.7 -5.9 2. -5.2 -5.7 3. -4.0 -5.1 4. -5.4 -6.0 5. -5.8 -5.4 6. -5.8 -5.9

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-6.1 8. -5.9 -4.7 9. -5.2 -6.1 10. -6.3 -7.0 11. -5.5 -6.4

Table -4: DockingresultsofHumanEstrogenReceptorAlpha(HERα)andHumanEpidermalGrowthFactorReceptor2(HER2)withmodifiedNaringeninflavonoid.

Sr. no. Name (IUPAC) Structure modified Energy value (Kcal/mol)

HERα HER2 1. -6.2 -6.3

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2. -5.7 -5.5 3. -5.2 -5.7 4. -5.3 -6.4 5. -6.3 -6.3 6. -5.6 -5.7 7. -5.3 -6.4 8. -5.5 -5.5

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-5.5 -6.2 10. -5.7 -5.8 11. -5.1 -6.2

TheactivationenergyfortheoriginalstructuresofChrysinandEquolretrievedas-6.2kcal/moland-6.3kcal/molrespectively with HERα and -6.8 kcal/mol and -6.5 kcal/mol with HER2 respectively. The activation energy of original structure of HesperetinandNaringeninretrievedas-4.7kcal/moleand-6.2kcal/molewithHERαrespectivelyandas-5.9kcal/moleand6.3kcal/molewithHER2respectively.The10modified structuresofeachflavonoid weredockedwith bothHERαandHER2 receptors,andtheresultsareshownintable1,2,3and4respectively.

The resultsofdocking were analysedand wereoutlinedthatamongall the 10modifiedstructuresof eachflavonoid, (2S)-8amino-2-(3-hydroxyphenyl)-6-iodo-3,4dihydro-2H-1benzopyran-4-oneisthebestmodifiedstructureofHesperetinflavonoid and2(S)-2-(2amino-4-hydroxyphenyl)-5-bromo-3,4-dihydro-2H-1-benzopyran-4-oneisbestmodifiedstructureofNaringenin flavonoid,asbothexhibitsthesamehighestactivationenergyof-6.3Kcal/molwithHERαreceptor,whichisgreaterthantheir original structure activation energy (-4.7 Kcal/mol and -6.2 Kcal/molrepectively), therefore these two modified structure of Hesperetin and Naringenin flavonoid is best suited to inhibit the activity of HERα receptor. Similarly,2-(2-amoniphenyl)5chloro-8-hydroxy-4H-chromen-4-onewhichisthebestmodifiedstructureofChrysinflavonoidexhibitstheactivationenergy of -6.0 Kcal/mol with HERα receptor, but is less than the original structure activation energy of Chrysin flavonoid (-6.2 Kcal/mol).Hence,therenosignificantdifferenceofactivationenergybetweenthemodifiedandoriginalstructureofChrysin flavonoid, to possess inhibitory activity of HERα receptor. None of the modified structures of Equol flavonoid exhibited the highest activation energy than the original structure, so the original structure with an activation energy of -6.3 Kcal/mol is bestsuitedtoinhibittheactivityofHERαreceptor.

Similarly, (2S)-8-amino-2-(3-hydroxyphenyl)-6-iodo-3,4dihydro-2H-1benzopyran-4-one which is best-modified structure of Hesperetin flavonoid exhibits the activation energy of -7.0 Kcal/mol, with HER2 receptor, that is greater than the activation energy of it’s original structure(-5.9 Kcal/mol). So, this best-modified structure shows good interaction with HER2 receptor and can be used to inhibit the activity of HER2 receptor. 2-(2-amino-4-chlorophenyl)-5-hydroxy-4H-chromen-4-one is the best-modified structure of Chrysin flavonoid and (3S)-5-chloro-3-(2-hydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-4-one is the best-modified structure of Equol flavonoid that exhibits the activation energy of -6.7 Kcal/mol and -6.5 Kcal/mol repectively, which is same as their original structure activation energy with HER2 receptor. Hence, these two modified structures of Chrysin and Equolflavonoid that can be used to show inhibitory activity with HER2 receptor but will not be

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significant as compared to the original structure of both. The best-modified structure Naringenin flavonoids are(2S)-2-(2hydroxyphrnyl)-7-sulfanyl-3,4-dihydro-2H-1-benzopyran-4-one and (2S)-2-(4-bromo-2-hydroxyphenyl)-7-chloro-3,4dihydro-2H-1-benzopyran-4-onethathaveasameactivationenergyof-6.4Kcal/mol,whichissameastheactivationenergyof originalstructureofNaringeninflavonoidwithHER2receptor.Hence,thesetwomodifiedstructureNaringeninflavonoidcan beusedtoshowinhibitoryactivitywithHER2receptorbutwillnotbesignificantascomparedtooriginalstructure.

4. CONCLUSION:

There is a need for new drugs that have biological activity. In this learning, the best modified flavonoid of Chrysin, Equol, Hesperetin, and Naringenin was found to be effective in binding to the HERα and HER2 receptors of breast cancer cells. The best modified structures of Naringenin, Chrysin and Equol flavonoid did not show a significant difference in their structure activation energy. The best modified form of hesperitin is (2S)-8-amino-2-(3-hydroxyphenyl)-6-iodo-3,4-dihydro-2H1benzopyran-4-one, which has an interaction energy of -6.3 kcal/mol and -7.0 kcal/mol, respectively, with the HERα and HER2receptorsofbreastcancer.Thisistheonlymodifiedflavonoidwithahigherinteractionenergybetweenitsoriginaland modified structures. Hence, it was concluded that this is the potent modified drug of Hesperetin flavonoid against breast cancer receptors called HERα and HER2.Further investigations are needed to determine the dosage of security and safety levelswithclinicaltrials.

ACKNOWLEDGEMENT

WeexpressourgratitudetoPrashantSinghParihar,forhisinterestinthispaper.Heexplainedthesolutionstoourproblems. Without his help, it was a matter of acute impossibility to propel in this endeavor His enthusiasm, knowledge, and exacting attention to detail have beenan inspirationandkept our work on track. It isa matterof profound privilege,and pleasureto payoursincereandheartfeltthankstohim.

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