research article investigation of flavonoids as potential...

Research ArticleIn Silico Investigation of Flavonoids as Potential TrypanosomalNucleoside Hydrolase Inhibitors

Christina Hung Hung Ha1 Ayesha Fatima2 and Anand Gaurav1

1Faculty of Pharmaceutical Sciences UCSI University 1 Jalan Menara Gading Taman Connaught Cheras56000 Kuala Lumpur Malaysia2Faculty of Medicine Quest International University Perak No 227 Plaza Teh Teng Seng Jalan Raja Permaisuri Bainun30250 Ipoh Perak Malaysia

Correspondence should be addressed to Christina Hung Hung Ha chrisabbyhagmailcom

Received 27 July 2015 Revised 17 October 2015 Accepted 20 October 2015

Academic Editor Huixiao Hong

Copyright copy 2015 Christina Hung Hung Ha et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Human African Trypanosomiasis is endemic to 37 countries of sub-Saharan Africa It is caused by two related species ofTrypanosoma brucei Current therapies suffer from resistance and public accessibility of expensive medicines Finding safer andeffective therapies of natural origin is being extensively explored worldwide Pentamidine is the only available therapy for inhibitingthe P2 adenosine transporter involved in the purine salvage pathway of the trypanosomatidsThe objective of the present study is touse computational studies for the investigation of the probable trypanocidal mechanism of flavonoids Docking experiments werecarried out on eight flavonoids of varying level of hydroxylation namely flavone 5-hydroxyflavone 7-hydroxyflavone chrysinapigenin kaempferol fisetin and quercetin Using AutoDock 42 these compounds were tested for their affinity towards inosine-adenosine-guanosine nucleoside hydrolase and the inosine-guanosine nucleoside hydrolase the major enzymes of the purinesalvage pathway Our results showed that all of the eight tested flavonoids showed high affinities for both hydrolases (lowest freebinding energy ranging from minus1023 to minus714 kcalmol)These compounds especially the hydroxylated derivatives could be furtherstudied as potential inhibitors of the nucleoside hydrolases

1 Introduction

Human African Trypanosomiasis (HAT) also known assleeping sickness is one of the 17 neglected tropical diseaseslisted by the WHO [1] It is endemic in sub-Saharan Africawhere the vector for its transmission the tsetse fly (Glossinaspecies) can be found HAT is caused by two subspeciesof Trypanosoma brucei (T b) namely Trypanosoma bruceigambiense and Trypanosoma brucei rhodesiense [2] Follow-ing several control measures theWorld Health Organizationreported a drop in the new cases [3] However many moreare believed to be unreported or undiagnosed especially thosewho live in remote areas

T b gambiense is responsible for the chronic form of thedisease that accounts for 98 of all reported cases while T brhodesiense causes the acute disease accounting for 2 of thereported cases [1 2] The infection is divided into two stages

for better therapeutic management In the first stage theparasite multiplies in the subcutaneous tissues after enteringthe host This stage which is also called the hemolytic phasecauses the first symptoms of headaches fever joint pains anditching In the second stage namely the neurological stagethe parasite crosses the blood-brain barrier and infects thecentral nervous system causing behavioral change confusionpoor sensory coordination and disturbances in the sleepcycle all of which are the hallmarks of the disease Theprolonged asymptomatic first stage of the disease sometimesmakes early diagnosis and treatment difficult

Suramin is the first-stage drug for rhodesiense The modeof action of suramin has been reported to be inhibition ofglycerol-3-phosphate oxidase and dehydrogenase [4] as wellas the inhibition of uptake of low-density lipoproteins and theconsequent hampering of cell division [5] Inhibitory effectsof suramin on dihydrofolate reductase L-a-glycerophosphate

Hindawi Publishing CorporationAdvances in BioinformaticsVolume 2015 Article ID 826047 10 pageshttpdxdoiorg1011552015826047

2 Advances in Bioinformatics

AMP IMP XMP GMPAMP

deaminaseIMP

dehydrogenaseGMP

synthetase

Adenosine

AK

Inosine

IAG-NH

Adenine

APRT

Hypoxanthine

IAG-NHIG-NH

HGPRT

Xanthine

XPRT

Guanosine

IAG-NHIG-NH

Adenine

HGPRT

Guanine deaminase

Methylthioadenosine phosphorylase

GMP reductaseS-AMP

S-AMP synthase

S-AMP lyase

P1

H2 H3P1

P1P2

P2 H2 H3

5998400 -Deoxy-5998400 -methylthioadenosine

Figure 1 PSP of T brucei showing key enzymes and transporters AK = adenosine kinase APRT = adenine phosphoribosyltransferaseHGPRT = hypoxanthine-guanine phosphoribosyltransferase XPRT = xanthine phosphoribosyltransferase and IG-NH = inosine-guanosinenucleoside hydrolase P1 P2 P1P2 H2 and H3 are purine base andor nucleoside transporters [16]

oxidase dihydrofolate dehydrogenase reverse transcriptasethymidine kinase trypsin and RNA polymerase were alsoreported [6 7] Pentamidine is effective for treating first-stagegambiense Its mode of action centers on the inhibition ofP2 adenosine transporter in the parasites hence inhibitingtheir replication Melarsoprol is the only drug recommendedfor the second stage of both types of diseases The primarytarget of melarsoprol was suggested to be the inhibitionof trypanothione reductase a key enzyme in detoxificationprocesses in the trypanosomes [8] However Wang pro-posed inhibition of phosphofructokinase a key enzyme inthe glycolytic pathway [7] Eflornithine (DFMO) has beensuccessful only in the second stage of gambiense infection[9] The drug is an inhibitor of ornithine decarboxylase(ODC) a key enzyme in polyamine biosynthesis [10] Alldrugs are available only in the intravenous form Nifurtimoxthe only orally administered drug acts by causing oxidativestress in the trypanosome Combination therapy may bemore effective than monotherapy for the treatment of late-stage T brucei gambiense trypanosomiasis The Nifurtimox-Eflornithine combination therapy was recently approvedby the WHO for use in late-stage T brucei gambiensetrypanosomiasis [11 12] There is none available for late-stage rhodesiense Most currently used drugs are toxic andresistance has been reported frequently [13]

Development of new safe and more efficacious drugs forHAT is an ongoing research because of the wide coveragearea and easy transmission between humans and animalSeveral studies on rational drug design report inhibition ofkey enzymes responsible for replication of the parasite Thepurine metabolism pathway of T brucei provides a valuabletarget in search of new selective antitrypanosomal drugs Incontrast to human hosts (mammals) all parasites are unableto synthesize purines de novo and solely rely on the purinesalvage pathway (PSP) to satisfy their purine needs which isessential for all stages of the parasite life cycle As shown inFigure 1 the key enzymes in this pathway are the nucleosidehydrolases (NH) These enzymes catalyze the cleavage of theN-glycosidic bond of nucleosides to yield purine bases Thereaction can be simplified as follows

nucleoside NH997888rarr purine base + pentose sugar (1)

where the nucleoside can be inosine guanosine andoradenosine depending on its specific preference of substratesTwo types of NH have been reported for T brucei the purinenucleoside specific inosine-adenosine-guanosine nucleosidehydrolase (IAG-NH) and the 6-oxopurine-specific inosine-guanosine nucleoside hydrolase (IG-NH) [14 15] Inhibitionof NH depletes the level of purine bases in T brucei retarding

Advances in Bioinformatics 3

Table 1 Test compounds and their hydroxylations

Test compounds Position of -OHFlavone mdash5-Hydroxyflavone 57-Hydroxyflavone 7Chrysin 57Apigenin 5741015840

Kaempferol 35741015840

Fisetin 373101584041015840

Quercetin 3573101584041015840

8

5

7

6

2

3

O1

4

A C

B

1998400

29984003998400

4998400

5998400

6998400

Figure 2 Basic flavonoid structure

their growth and multiplication In addition NH activity isabsent in mammal purine biosynthesis pathways [16]

Berg et al have shown significant trypanocidal effects ofnucleoside hydrolase inhibitors without exhibiting cytotox-icity to human cell lines This makes NH a good target fordeveloping new cure forHAT [16] Researchers also indicatedthat the NH inhibitors show isoenzyme selective inhibitiontowards IAG-NH and IG-NH due to the difference in activesite features and inhibition of either one enzyme alone is notsufficient to impair the PSP in the parasites [16]

Flavonoids are polyphenolic compounds having a com-mon benzo-120574-pyrone structure (Figure 2) They are presentin many plants including those commonly found in humandiet such as vegetables fruits grains wine and flowersFlavonoids are well known for their diverse biologicalactivities particularly having antioxidant anti-inflammatoryand antithrombogenic effects Studies have also shown thatflavonoids exert anticancer antibacterial and antivirus prop-erties and can be safely used for therapeutic purposes withoutcausing any visible toxic effects [17ndash22] Excellent antitry-panosomal and antileishmanial activities of flavonoids havealso been reported by various studies [23ndash25] Among allcompounds eight flavonoids of various levels of hydroxyla-tions namely flavone 5-hydroxyflavone 7-hydroxyflavonechrysin apigenin kaempferol fisetin and quercetin areselected for this study Flavone is the nonhydroxylatedcompound while the hydroxyflavones chrysin and apigeninrepresent the mono- di- and trihydroxylated derivatives offlavone On the other hand kaempferol fisetin and quercetinbelong to the flavan-3-ol subclass with kaempferol as the35741015840-tetrahydroxylated derivative quercetin having oneadditional hydroxylation at position 31015840 and fisetin having allsimilar hydroxylations as quercetin except for the missing5-OH Table 1 summarizes the test compounds and therespective positions of hydroxyl (-OH) groups

In silico screening offers the advantage of identifying leadcompounds from several potentially useful hits [26] Molec-ular docking offers a very efficient and fast method to do so[27] Researchers have employed the method successfully todetermine potentially useful binding sites and used the resultsto identify improve and perhaps develop drugs that fit betterinto the binding pocket Several simple pieces of softwaresuch as DOCK AutoDock and AutoDock Vina offer theadvantage of locating the plausible pockets efficiently [28 29]

Using docking technique the study looks at the potentialof flavonoids as inhibitors of nucleoside hydrolases whichcould be the probablemechanismof trypanocidal effect of thecompounds It is our interest to study the probable protein-ligand binding interaction and to draw structure-activityrelationships

2 Materials and Methods

The crystal structure of T b brucei IAG-NH (PDB ID 4I71)resolved at 128 A [15] and T b brucei IG-NH (PDB ID3FZ0) [30] were retrieved from the Protein Data Bank [31]Both structures had cocrystallized ligands Only chain A outof the four chains of 3FZ0 was used as a representative asall four chains are identical The protein was processed fordocking procedure using AutoDock Tools 156 All solventmolecules water molecules and the cocrystallized ligandand the allosteric inhibitor Ni2+ ion were removed from thestructure Kollman charges and polar hydrogens were addedThe calcium ion in the enzymes was also removed in orderto study the protein-ligand interactions without interferenceof the cation The files were generated as PDBQT formatPDBQT file of the ligands was generated with all the defaultvalues accepted Docking was performed by using AutoDock42 [29] with grid spacing set at 20 A and the grid points in119909- 119910- and 119911-axis set to 36 times 36 times 36 The grid is centeredat the cocrystallized ligands of the respective proteins Thesearch was done based on the Lamarckian genetic algorithmThe number of individuals in population is set to be 150with a maximum of 2500000 energy evaluations and 27000generations The rate of gene mutation is 002 while the rateof crossover is 08 For each ligand the docking runs were setto be 50 and the analysis is done based on binding energiesandRootMean SquareDeviation (RMSD) valuesThe ligandswere ranked in ascending order of free binding energies

The 3D structures of the ligands are downloaded fromPubChem The selected binding sites for both enzymes werethe ones reportedly occupied by the inhibitors AGV andBTB in the crystal structures [15 30] Controlled dockingwas performed with ligands AGV and BTB and two of thenatural substrates guanosine and inosine for validation ofthe experimental protocol All the test compoundswere listedin Table 2 with the structures generated using ChemSketch[32] featured in Figure 3

3 Results and Discussion

For all ligands docked the 50 runs were grouped into a rangeof 1-2 and 1ndash8multimember conformational clusters for IAG-NH and IG-NH respectively All docking conformations


AGV BTB

N

HO

HO

HO HO

OH

NNN

HOOH

OH

H2N

NH

(a)Guanosine Inosine

N

NN

O

O

HO

OH

OHNHN

NN

O

O

HO

OH

OH

NH2

NH

(b)

Flavone 5-Hydroxyflavone

7-Hydroxyflavone Chrysin

Apigenin Kaempferol

Fisetin Quercetin

O

O

O

O

HO

HO

OH

OH

OH

O

O

HO

HO

OH

OH

O

O

HO

HO

OH

OHO

O

HO

OH

OH

O

O

OH O

O

OH

OH

O

O OH

(c)

Figure 3 Structures of chemical compounds used in the study (a) Chemical structures of AGV and BTB the cocrystallized ligand of T bbrucei IAG-NH and IG-NH respectively (b) Chemical structures of inosine and guanosine the natural substrate of both T b brucei IAG-NH and IG-NH (c) Chemical structures of the compounds tested flavone 5-hydroxyflavone 7-hydroxyflavone apigenin fisetin kaempferolquercetin and chrysin

were visualized using AutoDock Tools 156 so as to ensurethe ligands were docked into the defined pocket Figure 4presents the lowest binding energy from the largest clusterchosen to represent the result of each docking regardlessof the cluster rank This is based on the rationale thatconformations in the largest cluster are more statistically

possible The estimated inhibition constant Ki calculated at29815∘K for the selected conformations was recorded as wellThe results are presented in Figure 5

Figure 6 illustrates the docking poses of the controlledmolecules PDB files of the docked position were generatedfor control and best compounds using AutoDock Tools [29]


Table 2 The test compounds chemical group classification andPubChem identification numbers

Compound Class PubChem IDAGVa Iminoribitol 25141279BTBb Tertiary amine 81462Guanosine Purine nucleoside 6802Inosine Purine nucleoside 6021Flavone Flavone 106805-Hydroxyflavone Flavone 681127-Hydroxyflavone Flavone 5281894Chrysin Flavone 5281607Apigenin Flavone 5280443Kaempferol Flavan-3-ol 5280863Fisetin Flavan-3-ol 5281614Quercetin Flavan-3-ol 5280343aAGV is (2R3R4S)-1-[(4-amino-5H-pyrrolo[32-d]pyrimidin-7-yl)methyl]-2-(hydroxymethyl)pyrrolidine-34-diolbBTB is 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-13-diol

minus12

minus10

minus8

minus6

minus4

minus2

0

Bind

ing

ener

gies

(kca

lmol

)

Compounds

Binding energy of the enzyme inhibitors

IAG-NH binding energyIG-NH binding energy

Flav

one

5-H

ydro

xyfla

vone

7-H

ydro

xyfla

vone

Chry

sin

Apig

enin

Kaem

pfer

ol

Fise

tin

Que

rcet

in

Gua

nosin

e

Inos

ine

AGV

BTB

Figure 4 Docking results showing lowest free binding energy ofcompounds with IAG-NH and IG-NH from T b brucei

which were then imported to LigPlot+ to generate two-dimensional ligand binding interaction plots and superim-posed to compare the bindings of different ligands on thesame protein and the same ligand on both NH [33] Ourresults indicated that the binding pocket of IAG-NH wasmade of Asp10 Asn12 Lys13 Asp14 Asp15 Asp40 Phe79Trp83 Thr137 Gly138 Met164 Gly165 Asn173 Glu184Trp185 Asn186 Leu210 Glu248 Arg252 Asp255 Ala256Tyr257 Trp260 and Asp261 As for IG-NH the binding siteresidues were Asp11 Asp15 Asp16 Asn40 Trp80 Phe83Leu131 Gly132 Met162 Asn171 Glu177 Phe178 Asn179Trp205 Phe247 Leu250 Thr254 Thr275 Cys276 Val277Val278 Pro279 and Asp280

Results also showed that AGV has the lowest free bindingenergy minus1119 kcalmol against IAG-NH among all the tested

0123456789

10

Inhi

bitio

n co

nsta

nt (120583

mol

)

Compounds

Inhibition constant of enzyme inhibitors

IAG-NH inhibition constantIG-NH inhibition constant

Flav

one

5-H

ydro

xyfla

vone

7-H

ydro

xyfla

vone

Chry

sin

Apig

enin

Kaem

pfer

ol

Fise

tin

Que

rcet

in

Gua

nosin

e

Inos

ine

AGV

BTB

Figure 5 The estimated inhibition constant (Ki) from dockingresults of the compounds with IAG-NH and IG-NH from T bbrucei

compounds The estimated Ki of AGV were 00063120583Magainst IAG-NH and 08154 120583Magainst IG-NHThese resultswere in agreement with the experimental data by Berg et althat showed the inhibition constant Ki of 0018120583Mand 32 120583Magainst IAG-NH and IG-NH respectively [16] The bindingenergy of AGV towards IG-NH (minus831 kcalmol) is lower thanIAG-NH (minus1119 kcalmol) but considerably higher whencompared to both natural substrates inosine and guanosine(binding energies minus531 kcalmol and minus706 kcalmol resp)This can be explained by the extensive hydrogen bondingof AGV in IAG-NH as compared to IG-NH as shown inFigure 7

The binding energy of BTB is comparatively lowertowards both enzymes minus49 and minus254 kcalmol with inhibi-tion constant of gt10 120583MThis is obvious from the structure ofBTB which lacks the pyrrolidine and pyrrole rings requiredby the hydrophobic pocket to bind effectively as presentin the natural substrates of the enzymes as well as AGVThe extensive OH groups bind to only four uncharged andnegatively charged amino acids in the binding pocket therebyproducing low binding energy and high binding constant(not shown)

All the eight test compounds showed considerably highselectivity (Figure 4) with estimated Ki at nanomolar concen-tration against IAG-NH enzyme Tasdemir et al [23] havedone in vitro study on trypanocidal activity of these com-pounds along with several other classes of flavonoids Theyreported that flavone 5-hydroxyflavone 7-hydroxyflavonechrysin apigenin fisetin kaempferol and quercetin indicateIC50of 64 106 66 53 51 33 92 and 83microgramsmL

respectively againstT b rhodesiensewheremelarsoprol usedas control had IC

50values of 00026microgramsmL in the

same experiment Tasdemir et al attributed the trypanocidal


(a) (b)

Figure 6 Docked poses of T b brucei NH with their cocrystallized ligands (stick figures) (a) IAG-NH with AGV (b) IG-NH with BTB

activity to the presence of hydroxyl groups on the flavonerings [23]

Our results also confirmed the observations of theauthors where strong hydrogen bonds were formed betweenthe hydroxyl groups on the flavonoids with polar amino acidresidues in the binding pocket of IAG-NH such as Asn173and Glu184 For example in case of flavone that had thelowest energy (minus828 kcalmol) compared to other hydrox-ylated derivatives since it has no hydroxyl groups to offerThe carbonyl oxygen remains the main functional groupto hydrogen bond with the residues in the binding pocketBinding energy increases with each increase in number ofhydroxyl groups from 0 in flavone (minus828 kcalmol) to 3 inapigenin (minus1023 kcalmol) This phenomenon could also beseen in interaction of AGV and guanosine with the sameprotein Quercetin with 5-hydroxyl groups had slightly lessbinding energy minus1012 kcalmol The reason for this changecould be attributed to the position of the OH group discussedfurther below

The position of -OH groups was also found to affectthe binding energy 5-OH position increases the bindingenergy of a flavone more than 7-OH For example flavoneand 5-hydroxyflavone have binding energies minus828 kcalmoland minus916 kcalmol as compared with 7-hydroxyflavone thatexhibits binding energy of minus873 kcalmol We observedfurther that hydroxylation at position 5 significantlyincreases the binding energy as seen in pairs of compoundsflavone and 5-hydroxyflavone minus828 kcalmol andminus916 kcalmol 7-hydroxyflavone and chrysin (5-OHand 7-OH) minus873 kcalmol and minus940 kcalmol and fisetin(no 5-OH) and quercetin (-OH at 3 5 7 31015840 and 41015840 positions)minus937 kcalmol and minus1012 kcalmol

Hydroxylation at positions 3 and 31015840 was also found to beunfavorable It can be seen that binding energy of apigeninis minus1023 kcalmol with no hydroxyl groups at both statedpositions while kaempferol has hydroxyl group at position3 and its binding energy decreases to minus967 kcalmol Thebinding energy of fisetin that has a hydroxyl group at 31015840 in

addition to position 3 is further decreased to minus937 kcalmolOur results also indicated that hydroxylation at 41015840 positioncould be responsible for improved binding energy such asin case of kaempferol and quercetin with binding energies ofminus967 kcalmol and minus1012 kcalmol Addition of hydroxyl topositions 3 and 31015840 decreased the binding energy such as thecase of quercetin but the presence of hydroxyl group at posi-tions 5 7 and 41015840 led to its comparatively better binding energySummarizing our results for IAG-NH flavonoids with threehydroxyl groups at positions 5 7 and 41015840 respectively as seenin apigenin seemed to be the most ideal combination

In case of IG-NH all compounds showed generallylower binding energies Analysing the effect of hydroxylationpattern of flavonoids on the binding energies for IG-NHflavone with no hydroxyl group had the lowest bindingenergy (minus714 kcalmol) among the flavonoids Increasingthe number of hydroxyl groups improved binding energiesHydroxylation at positions 5 and 41015840 remained importantwhile position 31015840 led to reduction of binding energy Thestrongest bound molecule was quercetin with minus861 kcalmolbinding energy

Besides the flavonoids also shared a similar pattern ofextensive hydrophobic interactions with the protein residuesIt can be proposed that the sum of both hydrogen bond andhydrophobic interactions contributed to the high affinity ofthese ligands towards the trypanosomal NH Tasdemir etal [23] had reported trypanocidal activity of the flavonoidsagainst the whole organism Combining our docking andtheir experimental results it can be said that inhibition ofnucleoside hydrolases could be the probable target of thetrypanocidal activity of flavonoids

Overall all flavonoids showed higher affinity towardsIAG-NH than IG-NH This could be due to the fact thatthe IAG-NH structure used for docking has a closed andnarrower binding pocket compared to that of IG-NH Thusligands that are more planar and sufficiently small are ableto fit into the IAG-NH pocket Upon visualizing the ligandswere closer to the amino acid residues in IAG-NH binding


(a) (b)

289

287269

304

279C9

C8

N7 C5

C4

C6

N6

N1

C2

N3C7

Agv401 Trp83

Trp260

Asp40

Asn186

Glu184

Asp261

Glu248

Asn173

Tyr257Asn12

Phe79

Asp14

Asp15

Thr137

Trp185

Arg252

4I71-AGV

C1998400

C2998400C3998400C4998400

C5998400

O2998400

O3998400

O5998400

N4998400

(c)

286

269

285

269

C9

C8

N7C5

C4

C6

N6

N1

C2

N3C7Agv401

Asn171

Asp15

Trp80

Phe178

Asn40

Asn179

Asp280

Asp11

Met162

Ser172

Pro279

Asp16

Leu131

Glu177

3FZ0 and AGV

C1998400

C2998400

C3998400

C4998400

C5998400

O2998400

O3998400

O5998400

N4998400

(d)

Figure 7 Docked poses of AGV with (a) IAG-NH and (b) IG-NH LigPlot figures of AGV docked with (c) IAG-NH and (d) IG-NH

pocket thus the calculated intermolecular forces appeared tobe higherHowever this selectivity towards IAG-NHcould befurther investigated by docking studies with flexible bindingpocket or molecular dynamics Figure 8 shows the dockedposition of the compounds of the highest binding energy forboth enzymes

It is difficult to directly correlate our results to IC50of the

flavonoids proposed by Tasdemir et al [23] This is becausethe control used in the experimental study melarsoprol isa known inhibitor of trypanothione reductase and all the

reported IC50

are compared with that target and our targetenzymes being different

Tasdemir et al had concluded that there was no specifictrend observed between the chemical structures and antitry-panosomal activities [23] Several other in vitro studies havealso reported excellent trypanocidal activities of methylatedand glycosylated flavonoid derivatives [23 24 33] A possibleexplanation to these observations is that the trypanocidalactivity of flavonoids may involve multitarget inhibitionthus structure-activity relationship of flavonoids differs for


(i) (ii)

(a)

287

305

304244

291282

323

C1

C2

C3

C4 C5

C6 C7C8

C9

O1

O2

O3O4

C10C11

C12

C13

C14

C15

O5

Apigenin

Trp83

Asn173

Asn186

Glu248

Tyr257

Glu184

Trp185

Asn12

Trp260

Asp261

Asp40

Lys13

Met164

(i) (ii)

4I71

277284

308

280

307260

C1

C2

C3

C4C5

C6C7

C8

C9

O1

O2

C10C11

C12

C13

C14

C15

O3

O4

O5

O6O7

Quercetin

Asn171

Asp280

Asp16

Asn179

Leu131

Asp15

Glu177

Phe178

Asn40

Met162

Asp11

Gly13

3FZ0 quercetin

(b)

Figure 8 (a) Docked position of (i) apigenin (stick figure) in the binding pocket of IAG-NH and (ii) quercetin (stick figure) in the bindingpocket of IG-NH (b) LigPlot figures of (i) apigenin docked in IAG-NH and (ii) quercetin docked in IG-NH Amino acid residues forminghydrophobic interactions were highlighted in red circles Amino acids contributing to hydrogen bonds were labelled green and hydrogenbonds were indicated as dotted lines with bond length labelled in green

each protein target This idea is supported by the fact thatflavonoids possess a wide range of bioactivities and are able toinhibit a large variety of enzymes which spans across almostall enzyme classes including hydrolases oxidoreductasesisomerases kinases and ligases [17 19 22]

From our results we showed that the purine salvagepathway enzymes could also be one of the targets Furtherdocking studies by screening a library of flavonoids foractivities against other target enzymes of trypanosomiasis arean ongoing research of our group Laboratory experimentsusing specific enzymes could be used to further confirmthe inhibition effect of flavonoids on each enzyme As the

laboratory data is still scarce a more cost-effective alternativeis to utilize molecular dynamics simulation for prediction ofthe protein-ligand interactions

4 Conclusion

Using computational docking studies we have proposed thatnucleoside hydrolases could be one of the trypanocidaltargets of the flavonoids proposed by Tasdemir et al intheir experimental work Using several flavonoids structurespossessing hydroxyl groups at various positions we havetried to provide insight into the effect of hydroxylation on


the free binding energy of the tested compounds Based onour results flavonoids have more affinity for IAG-NH ascompared with IG-NH The size of the binding pocket andthe lining residues favor planar hydrophobic residues withthree hydroxyl groups at 5 7 and 41015840 positions of the flavonestructure Further studies using molecular dynamics mayprovide better insight into the lead flavonoid structure forexperimental work

Abbreviations

HAT Human African TrypanosomiasisWHO World Health OrganizationPSP Purine salvage pathwayNH Nucleoside hydrolaseIAG-NH Inosine-adenosine-guanosine nucleoside

hydrolaseIG-NH Inosine-guanosine nucleoside hydrolaseAGV (2R3R4S)-1-[(4-Amino-5H-pyrrolo[32-

d]pyrimidin-7-yl)methyl]-2-(hydroxymethyl)pyrrolidine-34-diol

BTB 2-[Bis(2-hydroxyethyl) amino]-2-(hydroxymethyl)propane-13-diol

Disclosure

The presented work is solely the authorsrsquo original work and isnot copied from anywhere

Conflict of Interests

The authors declare that they have no conflict of interests inpreparation of this paper

Authorsrsquo Contribution

Christina Hung Hung Ha carried out the experimental workand drafted the paper Ayesha Fatima and Anand Gauravhelped to edit the paper All authors read and approved thefinal paper

References

[1] WHO ldquoThe 17 neglected tropical diseasesrdquo httpwwwwhointtrypanosomiasis africanen

[2] W N Setzer and I V Ogungbe ldquoIn-silico investigation ofantitrypanosomal phytochemicals from Nigerian medicinalplantsrdquo PLoS Neglected Tropical Diseases vol 6 no 7 Article IDe1727 2012

[3] P P Simarro A Diarra J A R Postigo J R Franco and JG Jannin ldquoThe human african trypanosomiasis control andsurveillance programmeof theworld health organization 2000ndash2009 the way forwardrdquo PLoS Neglected Tropical Diseases vol 5no 2 Article ID e1007 2011

[4] A H Fairlamb and I B R Bowman ldquoTrypanosoma bruceisuramin and other trypanocidal compoundsrsquo effects on sn-glycerol-3-phosphate oxidaserdquo Experimental Parasitology vol43 no 2 pp 353ndash361 1977

[5] E L M Vansterkenburg I Coppens J Wilting et al ldquoTheuptake of the trypanocidal drug suramin in combination withlow-density lipoproteins by Trypanosoma brucei and its possi-ble mode of actionrdquo Acta Tropica vol 54 no 3-4 pp 237ndash2501993

[6] J Pepin and F Milord ldquoThe treatment of human African try-panosomiasisrdquo Advances in Parasitology vol 33 pp 1ndash47 1994

[7] C C Wang ldquoMolecular mechanisms and therapeuticapproaches to the treatment of African trypanosomiasisrdquoAnnual Review of Pharmacology and Toxicology vol 35 pp93ndash127 1995

[8] C Walsh M Bradley and K Nadeau ldquoMolecular studies ontrypanothione reductase a target for antiparasitic drugsrdquoTrendsin Biochemical Sciences vol 16 pp 305ndash309 1991

[9] P P Simarro J Jannin and P Cattand ldquoEliminating humanAfrican trypanosomiasis where do we stand and what comesnextrdquo PLoS Medicine vol 5 no 2 article e55 2008

[10] C J Bacchi H C Nathan S H Hutner P P McCann andA Sjoerdsma ldquoPolyamine metabolism a potential therapeutictarget in trypanosomesrdquo Science vol 210 no 4467 pp 332ndash3341980

[11] G Priotto S Kasparian W Mutombo et al ldquoNifurtimox-eflornithine combination therapy for second-stage AfricanTrypanosoma brucei gambiense trypanosomiasis a multicentrerandomised phase III non-inferiority trialrdquo The Lancet vol374 no 9683 pp 56ndash64 2009

[12] G Priotto S Kasparian D Ngouama et al ldquoNifurtimox-eflornithine combination therapy for second-stage Try-panosoma brucei gambiense sleeping sickness a randomizedclinical trial in Congordquo Clinical Infectious Diseases vol 45 no11 pp 1435ndash1442 2007

[13] V Delespaux and H P de Koning ldquoDrugs and drug resistancein African trypanosomiasisrdquo Drug Resistance Updates vol 10no 1-2 pp 30ndash50 2007

[14] R Pelle V L Schramm and D W Parkin ldquoMolecular cloningand expression of a purine-specific N-ribohydrolase from Try-panosoma brucei brucei sequence expression and molecularanalysisrdquo Journal of Biological Chemistry vol 273 no 4 pp2118ndash2126 1998

[15] F Giannese M Berg P Van Der Veken et al ldquoStructuresof purine nucleosidase from Trypanosoma brucei bound toisozyme-specific trypanocidals and a novel metalorganicinhibitorrdquo Acta Crystallographica Section D Biological Crystal-lography vol 69 part 8 pp 1553ndash1566 2013

[16] M Berg L Kohl P Van der Veken et al ldquoEvaluation ofnucleoside hydrolase inhibitors for treatment of african try-panosomiasisrdquoAntimicrobial Agents and Chemotherapy vol 54no 5 pp 1900ndash1908 2010

[17] B H Havsteen ldquoThe biochemistry and medical significance ofthe flavonoidsrdquo Pharmacology andTherapeutics vol 96 no 2-3pp 67ndash202 2002

[18] S S Lim H-S Kim and D-U Lee ldquoIn vitro antimalarialactivity of flavonoids and chalconesrdquo Bulletin of the KoreanChemical Society vol 28 no 12 pp 2495ndash2497 2007

[19] P C H Hollman ldquoAbsorption bioavailability and metabolismof flavonoidsrdquoPharmaceutical Biology vol 42 supplement 1 pp74ndash83 2004

[20] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobiology Reviews vol 12 no 4 pp 564ndash582 1999

[21] N S Alrawaiq and A Abdullah ldquoA review of flavonoidquercetin metabolism bioactivity and antioxidant propertiesrdquo


International Journal of PharmTech Research vol 6 no 3 pp933ndash941 2014

[22] V P Litvinov P C H Hollman N S Alrawaiq et al ldquoA reviewof flavonoid quercetin metabolism bioactivity and antioxidantpropertiesrdquo International Journal of PharmTech Research vol 6no 3 pp 933ndash941 2014

[23] D Tasdemir M Kaiser R Brun et al ldquoAntitrypanosomal andantileishmanial activities of flavonoids and their analogues invitro in vivo structure-activity relationship and quantitativestructure-activity relationship studiesrdquo Antimicrobial Agentsand Chemotherapy vol 50 no 4 pp 1352ndash1364 2006

[24] M Bourjot C Apel M-T Martin et al ldquoAntiplasmodial antit-rypanosomal and cytotoxic activities of prenylated flavonoidsisolated from the stem bark of artocarpus styracifoliusrdquo PlantaMedica vol 76 no 14 pp 1600ndash1604 2010

[25] A M M Nour S A Khalid M Kaiser R Brun W E Abdallaand T J Schmidt ldquoThe antiprotozoal activity of methylatedflavonoids from Ageratum conyzoides Lrdquo Journal of Ethno-pharmacology vol 129 no 1 pp 127ndash130 2010

[26] I M Kapetanovic ldquoComputer-aided drug discovery and devel-opment (CADDD) in silico-chemico-biological approachrdquoChemico-Biological Interactions vol 171 no 2 pp 165ndash1762008

[27] H Alonso A A Bliznyuk and J E Gready ldquoCombiningdocking and molecular dynamic simulations in drug designrdquoMedicinal Research Reviews vol 26 no 5 pp 531ndash568 2006

[28] O Trott and A J Olson ldquoAutoDock Vina improving the speedand accuracy of docking with a new scoring function efficientoptimization and multithreadingrdquo Journal of ComputationalChemistry vol 31 no 2 pp 455ndash461 2010

[29] G M Morris H Ruth W Lindstrom et al ldquoAutodock4 andAutoDockTools4 automated docking with selective receptorflexibilityrdquo Journal of Computational Chemistry vol 30 no 16pp 2785ndash2791 2009

[30] A Vandemeulebroucke C Minici I Bruno et al ldquoStructureand mechanism of the 6-oxopurine nucleosidase from Try-panosoma brucei bruceirdquo Biochemistry vol 49 no 41 pp 8999ndash9010 2010

[31] H M Berman J Westbrook Z Feng et al ldquoThe protein databankrdquo Nucleic Acids Research vol 28 no 1 pp 235ndash242 2000

[32] Advanced Chemistry Development ChemSketch AdvancedChemistry Development Toronto Canada 2014

[33] R A Laskowski andM B Swindells ldquoLigPlot+ multiple ligand-protein interaction diagrams for drug discoveryrdquo Journal ofChemical Information and Modeling vol 51 no 10 pp 2778ndash2786 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


AMP IMP XMP GMPAMP

deaminaseIMP

dehydrogenaseGMP

synthetase

Adenosine

AK

Inosine

IAG-NH

Adenine

APRT

Hypoxanthine

IAG-NHIG-NH

HGPRT

Xanthine

XPRT

Guanosine

IAG-NHIG-NH

Adenine

HGPRT

Guanine deaminase

Methylthioadenosine phosphorylase

GMP reductaseS-AMP

S-AMP synthase

S-AMP lyase

P1

H2 H3P1

P1P2

P2 H2 H3

5998400 -Deoxy-5998400 -methylthioadenosine

Figure 1 PSP of T brucei showing key enzymes and transporters AK = adenosine kinase APRT = adenine phosphoribosyltransferaseHGPRT = hypoxanthine-guanine phosphoribosyltransferase XPRT = xanthine phosphoribosyltransferase and IG-NH = inosine-guanosinenucleoside hydrolase P1 P2 P1P2 H2 and H3 are purine base andor nucleoside transporters [16]

oxidase dihydrofolate dehydrogenase reverse transcriptasethymidine kinase trypsin and RNA polymerase were alsoreported [6 7] Pentamidine is effective for treating first-stagegambiense Its mode of action centers on the inhibition ofP2 adenosine transporter in the parasites hence inhibitingtheir replication Melarsoprol is the only drug recommendedfor the second stage of both types of diseases The primarytarget of melarsoprol was suggested to be the inhibitionof trypanothione reductase a key enzyme in detoxificationprocesses in the trypanosomes [8] However Wang pro-posed inhibition of phosphofructokinase a key enzyme inthe glycolytic pathway [7] Eflornithine (DFMO) has beensuccessful only in the second stage of gambiense infection[9] The drug is an inhibitor of ornithine decarboxylase(ODC) a key enzyme in polyamine biosynthesis [10] Alldrugs are available only in the intravenous form Nifurtimoxthe only orally administered drug acts by causing oxidativestress in the trypanosome Combination therapy may bemore effective than monotherapy for the treatment of late-stage T brucei gambiense trypanosomiasis The Nifurtimox-Eflornithine combination therapy was recently approvedby the WHO for use in late-stage T brucei gambiensetrypanosomiasis [11 12] There is none available for late-stage rhodesiense Most currently used drugs are toxic andresistance has been reported frequently [13]

Development of new safe and more efficacious drugs forHAT is an ongoing research because of the wide coveragearea and easy transmission between humans and animalSeveral studies on rational drug design report inhibition ofkey enzymes responsible for replication of the parasite Thepurine metabolism pathway of T brucei provides a valuabletarget in search of new selective antitrypanosomal drugs Incontrast to human hosts (mammals) all parasites are unableto synthesize purines de novo and solely rely on the purinesalvage pathway (PSP) to satisfy their purine needs which isessential for all stages of the parasite life cycle As shown inFigure 1 the key enzymes in this pathway are the nucleosidehydrolases (NH) These enzymes catalyze the cleavage of theN-glycosidic bond of nucleosides to yield purine bases Thereaction can be simplified as follows

nucleoside NH997888rarr purine base + pentose sugar (1)

where the nucleoside can be inosine guanosine andoradenosine depending on its specific preference of substratesTwo types of NH have been reported for T brucei the purinenucleoside specific inosine-adenosine-guanosine nucleosidehydrolase (IAG-NH) and the 6-oxopurine-specific inosine-guanosine nucleoside hydrolase (IG-NH) [14 15] Inhibitionof NH depletes the level of purine bases in T brucei retarding





Fisetin 373101584041015840

Quercetin 3573101584041015840

8

5

7

6

2

3

O1

4

A C

B

1998400

29984003998400

4998400

5998400

6998400













AGV BTB

N

HO

HO

HO HO

OH

NNN

HOOH

OH

H2N

NH


N

NN

O

O

HO

OH

OHNHN

NN

O

O

HO

OH

OH

NH2

NH

(b)



Apigenin Kaempferol

Fisetin Quercetin

O

O

O

O

HO

HO

OH

OH

OH

O

O

HO

HO

OH

OH

O

O

HO

HO

OH

OHO

O

HO

OH

OH

O

O

OH O

O

OH

OH

O

O OH

(c)








minus12

minus10

minus8

minus6

minus4

minus2

0

Bind

ing

ener

gies

(kca

lmol

)

Compounds



Flav

one

5-H

ydro

xyfla

vone

7-H

ydro

xyfla

vone

Chry

sin

Apig

enin

Kaem

pfer

ol

Fise

tin

Que

rcet

in

Gua

nosin

e

Inos

ine

AGV

BTB




0123456789

10

Inhi

bitio

n co

nsta

nt (120583

mol

)

Compounds



Flav

one

5-H

ydro

xyfla

vone

7-H

ydro

xyfla

vone

Chry

sin

Apig

enin

Kaem

pfer

ol

Fise

tin

Que

rcet

in

Gua

nosin

e

Inos

ine

AGV

BTB









(a) (b)











(a) (b)

289

287269

304

279C9

C8

N7 C5

C4

C6

N6

N1

C2

N3C7

Agv401 Trp83

Trp260

Asp40

Asn186

Glu184

Asp261

Glu248

Asn173

Tyr257Asn12

Phe79

Asp14

Asp15

Thr137

Trp185

Arg252

4I71-AGV

C1998400

C2998400C3998400C4998400

C5998400

O2998400

O3998400

O5998400

N4998400

(c)

286

269

285

269

C9

C8

N7C5

C4

C6

N6

N1

C2

N3C7Agv401

Asn171

Asp15

Trp80

Phe178

Asn40

Asn179

Asp280

Asp11

Met162

Ser172

Pro279

Asp16

Leu131

Glu177

3FZ0 and AGV

C1998400

C2998400

C3998400

C4998400

C5998400

O2998400

O3998400

O5998400

N4998400

(d)





reported IC50




(i) (ii)

(a)

287

305

304244

291282

323

C1

C2

C3

C4 C5

C6 C7C8

C9

O1

O2

O3O4

C10C11

C12

C13

C14

C15

O5

Apigenin

Trp83

Asn173

Asn186

Glu248

Tyr257

Glu184

Trp185

Asn12

Trp260

Asp261

Asp40

Lys13

Met164

(i) (ii)

4I71

277284

308

280

307260

C1

C2

C3

C4C5

C6C7

C8

C9

O1

O2

C10C11

C12

C13

C14

C15

O3

O4

O5

O6O7

Quercetin

Asn171

Asp280

Asp16

Asn179

Leu131

Asp15

Glu177

Phe178

Asn40

Met162

Asp11

Gly13

3FZ0 quercetin

(b)





4 Conclusion




Abbreviations





Disclosure






References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Fisetin 373101584041015840

Quercetin 3573101584041015840

8

5

7

6

2

3

O1

4

A C

B

1998400

29984003998400

4998400

5998400

6998400













AGV BTB

N

HO

HO

HO HO

OH

NNN

HOOH

OH

H2N

NH


N

NN

O

O

HO

OH

OHNHN

NN

O

O

HO

OH

OH

NH2

NH

(b)



Apigenin Kaempferol

Fisetin Quercetin

O

O

O

O

HO

HO

OH

OH

OH

O

O

HO

HO

OH

OH

O

O

HO

HO

OH

OHO

O

HO

OH

OH

O

O

OH O

O

OH

OH

O

O OH

(c)








minus12

minus10

minus8

minus6

minus4

minus2

0

Bind

ing

ener

gies

(kca

lmol

)

Compounds



Flav

one

5-H

ydro

xyfla

vone

7-H

ydro

xyfla

vone

Chry

sin

Apig

enin

Kaem

pfer

ol

Fise

tin

Que

rcet

in

Gua

nosin

e

Inos

ine

AGV

BTB




0123456789

10

Inhi

bitio

n co

nsta

nt (120583

mol

)

Compounds



Flav

one

5-H

ydro

xyfla

vone

7-H

ydro

xyfla

vone

Chry

sin

Apig

enin

Kaem

pfer

ol

Fise

tin

Que

rcet

in

Gua

nosin

e

Inos

ine

AGV

BTB









(a) (b)











(a) (b)

289

287269

304

279C9

C8

N7 C5

C4

C6

N6

N1

C2

N3C7

Agv401 Trp83

Trp260

Asp40

Asn186

Glu184

Asp261

Glu248

Asn173

Tyr257Asn12

Phe79

Asp14

Asp15

Thr137

Trp185

Arg252

4I71-AGV

C1998400

C2998400C3998400C4998400

C5998400

O2998400

O3998400

O5998400

N4998400

(c)

286

269

285

269

C9

C8

N7C5

C4

C6

N6

N1

C2

N3C7Agv401

Asn171

Asp15

Trp80

Phe178

Asn40

Asn179

Asp280

Asp11

Met162

Ser172

Pro279

Asp16

Leu131

Glu177

3FZ0 and AGV

C1998400

C2998400

C3998400

C4998400

C5998400

O2998400

O3998400

O5998400

N4998400

(d)





reported IC50




(i) (ii)

(a)

287

305

304244

291282

323

C1

C2

C3

C4 C5

C6 C7C8

C9

O1

O2

O3O4

C10C11

C12

C13

C14

C15

O5

Apigenin

Trp83

Asn173

Asn186

Glu248

Tyr257

Glu184

Trp185

Asn12

Trp260

Asp261

Asp40

Lys13

Met164

(i) (ii)

4I71

277284

308

280

307260

C1

C2

C3

C4C5

C6C7

C8

C9

O1

O2

C10C11

C12

C13

C14

C15

O3

O4

O5

O6O7

Quercetin

Asn171

Asp280

Asp16

Asn179

Leu131

Asp15

Glu177

Phe178

Asn40

Met162

Asp11

Gly13

3FZ0 quercetin

(b)





4 Conclusion




Abbreviations





Disclosure






References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


AGV BTB

N

HO

HO

HO HO

OH

NNN

HOOH

OH

H2N

NH


N

NN

O

O

HO

OH

OHNHN

NN

O

O

HO

OH

OH

NH2

NH

(b)



Apigenin Kaempferol

Fisetin Quercetin

O

O

O

O

HO

HO

OH

OH

OH

O

O

HO

HO

OH

OH

O

O

HO

HO

OH

OHO

O

HO

OH

OH

O

O

OH O

O

OH

OH

O

O OH

(c)








minus12

minus10

minus8

minus6

minus4

minus2

0

Bind

ing

ener

gies

(kca

lmol

)

Compounds



Flav

one

5-H

ydro

xyfla

vone

7-H

ydro

xyfla

vone

Chry

sin

Apig

enin

Kaem

pfer

ol

Fise

tin

Que

rcet

in

Gua

nosin

e

Inos

ine

AGV

BTB




0123456789

10

Inhi

bitio

n co

nsta

nt (120583

mol

)

Compounds



Flav

one

5-H

ydro

xyfla

vone

7-H

ydro

xyfla

vone

Chry

sin

Apig

enin

Kaem

pfer

ol

Fise

tin

Que

rcet

in

Gua

nosin

e

Inos

ine

AGV

BTB









(a) (b)











(a) (b)

289

287269

304

279C9

C8

N7 C5

C4

C6

N6

N1

C2

N3C7

Agv401 Trp83

Trp260

Asp40

Asn186

Glu184

Asp261

Glu248

Asn173

Tyr257Asn12

Phe79

Asp14

Asp15

Thr137

Trp185

Arg252

4I71-AGV

C1998400

C2998400C3998400C4998400

C5998400

O2998400

O3998400

O5998400

N4998400

(c)

286

269

285

269

C9

C8

N7C5

C4

C6

N6

N1

C2

N3C7Agv401

Asn171

Asp15

Trp80

Phe178

Asn40

Asn179

Asp280

Asp11

Met162

Ser172

Pro279

Asp16

Leu131

Glu177

3FZ0 and AGV

C1998400

C2998400

C3998400

C4998400

C5998400

O2998400

O3998400

O5998400

N4998400

(d)





reported IC50




(i) (ii)

(a)

287

305

304244

291282

323

C1

C2

C3

C4 C5

C6 C7C8

C9

O1

O2

O3O4

C10C11

C12

C13

C14

C15

O5

Apigenin

Trp83

Asn173

Asn186

Glu248

Tyr257

Glu184

Trp185

Asn12

Trp260

Asp261

Asp40

Lys13

Met164

(i) (ii)

4I71

277284

308

280

307260

C1

C2

C3

C4C5

C6C7

C8

C9

O1

O2

C10C11

C12

C13

C14

C15

O3

O4

O5

O6O7

Quercetin

Asn171

Asp280

Asp16

Asn179

Leu131

Asp15

Glu177

Phe178

Asn40

Met162

Asp11

Gly13

3FZ0 quercetin

(b)





4 Conclusion




Abbreviations





Disclosure






References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




minus12

minus10

minus8

minus6

minus4

minus2

0

Bind

ing

ener

gies

(kca

lmol

)

Compounds



Flav

one

5-H

ydro

xyfla

vone

7-H

ydro

xyfla

vone

Chry

sin

Apig

enin

Kaem

pfer

ol

Fise

tin

Que

rcet

in

Gua

nosin

e

Inos

ine

AGV

BTB




0123456789

10

Inhi

bitio

n co

nsta

nt (120583

mol

)

Compounds



Flav

one

5-H

ydro

xyfla

vone

7-H

ydro

xyfla

vone

Chry

sin

Apig

enin

Kaem

pfer

ol

Fise

tin

Que

rcet

in

Gua

nosin

e

Inos

ine

AGV

BTB









(a) (b)











(a) (b)

289

287269

304

279C9

C8

N7 C5

C4

C6

N6

N1

C2

N3C7

Agv401 Trp83

Trp260

Asp40

Asn186

Glu184

Asp261

Glu248

Asn173

Tyr257Asn12

Phe79

Asp14

Asp15

Thr137

Trp185

Arg252

4I71-AGV

C1998400

C2998400C3998400C4998400

C5998400

O2998400

O3998400

O5998400

N4998400

(c)

286

269

285

269

C9

C8

N7C5

C4

C6

N6

N1

C2

N3C7Agv401

Asn171

Asp15

Trp80

Phe178

Asn40

Asn179

Asp280

Asp11

Met162

Ser172

Pro279

Asp16

Leu131

Glu177

3FZ0 and AGV

C1998400

C2998400

C3998400

C4998400

C5998400

O2998400

O3998400

O5998400

N4998400

(d)





reported IC50




(i) (ii)

(a)

287

305

304244

291282

323

C1

C2

C3

C4 C5

C6 C7C8

C9

O1

O2

O3O4

C10C11

C12

C13

C14

C15

O5

Apigenin

Trp83

Asn173

Asn186

Glu248

Tyr257

Glu184

Trp185

Asn12

Trp260

Asp261

Asp40

Lys13

Met164

(i) (ii)

4I71

277284

308

280

307260

C1

C2

C3

C4C5

C6C7

C8

C9

O1

O2

C10C11

C12

C13

C14

C15

O3

O4

O5

O6O7

Quercetin

Asn171

Asp280

Asp16

Asn179

Leu131

Asp15

Glu177

Phe178

Asn40

Met162

Asp11

Gly13

3FZ0 quercetin

(b)





4 Conclusion




Abbreviations





Disclosure






References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)











(a) (b)

289

287269

304

279C9

C8

N7 C5

C4

C6

N6

N1

C2

N3C7

Agv401 Trp83

Trp260

Asp40

Asn186

Glu184

Asp261

Glu248

Asn173

Tyr257Asn12

Phe79

Asp14

Asp15

Thr137

Trp185

Arg252

4I71-AGV

C1998400

C2998400C3998400C4998400

C5998400

O2998400

O3998400

O5998400

N4998400

(c)

286

269

285

269

C9

C8

N7C5

C4

C6

N6

N1

C2

N3C7Agv401

Asn171

Asp15

Trp80

Phe178

Asn40

Asn179

Asp280

Asp11

Met162

Ser172

Pro279

Asp16

Leu131

Glu177

3FZ0 and AGV

C1998400

C2998400

C3998400

C4998400

C5998400

O2998400

O3998400

O5998400

N4998400

(d)





reported IC50




(i) (ii)

(a)

287

305

304244

291282

323

C1

C2

C3

C4 C5

C6 C7C8

C9

O1

O2

O3O4

C10C11

C12

C13

C14

C15

O5

Apigenin

Trp83

Asn173

Asn186

Glu248

Tyr257

Glu184

Trp185

Asn12

Trp260

Asp261

Asp40

Lys13

Met164

(i) (ii)

4I71

277284

308

280

307260

C1

C2

C3

C4C5

C6C7

C8

C9

O1

O2

C10C11

C12

C13

C14

C15

O3

O4

O5

O6O7

Quercetin

Asn171

Asp280

Asp16

Asn179

Leu131

Asp15

Glu177

Phe178

Asn40

Met162

Asp11

Gly13

3FZ0 quercetin

(b)





4 Conclusion




Abbreviations





Disclosure






References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

289

287269

304

279C9

C8

N7 C5

C4

C6

N6

N1

C2

N3C7

Agv401 Trp83

Trp260

Asp40

Asn186

Glu184

Asp261

Glu248

Asn173

Tyr257Asn12

Phe79

Asp14

Asp15

Thr137

Trp185

Arg252

4I71-AGV

C1998400

C2998400C3998400C4998400

C5998400

O2998400

O3998400

O5998400

N4998400

(c)

286

269

285

269

C9

C8

N7C5

C4

C6

N6

N1

C2

N3C7Agv401

Asn171

Asp15

Trp80

Phe178

Asn40

Asn179

Asp280

Asp11

Met162

Ser172

Pro279

Asp16

Leu131

Glu177

3FZ0 and AGV

C1998400

C2998400

C3998400

C4998400

C5998400

O2998400

O3998400

O5998400

N4998400

(d)





reported IC50




(i) (ii)

(a)

287

305

304244

291282

323

C1

C2

C3

C4 C5

C6 C7C8

C9

O1

O2

O3O4

C10C11

C12

C13

C14

C15

O5

Apigenin

Trp83

Asn173

Asn186

Glu248

Tyr257

Glu184

Trp185

Asn12

Trp260

Asp261

Asp40

Lys13

Met164

(i) (ii)

4I71

277284

308

280

307260

C1

C2

C3

C4C5

C6C7

C8

C9

O1

O2

C10C11

C12

C13

C14

C15

O3

O4

O5

O6O7

Quercetin

Asn171

Asp280

Asp16

Asn179

Leu131

Asp15

Glu177

Phe178

Asn40

Met162

Asp11

Gly13

3FZ0 quercetin

(b)





4 Conclusion




Abbreviations





Disclosure






References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(i) (ii)

(a)

287

305

304244

291282

323

C1

C2

C3

C4 C5

C6 C7C8

C9

O1

O2

O3O4

C10C11

C12

C13

C14

C15

O5

Apigenin

Trp83

Asn173

Asn186

Glu248

Tyr257

Glu184

Trp185

Asn12

Trp260

Asp261

Asp40

Lys13

Met164

(i) (ii)

4I71

277284

308

280

307260

C1

C2

C3

C4C5

C6C7

C8

C9

O1

O2

C10C11

C12

C13

C14

C15

O3

O4

O5

O6O7

Quercetin

Asn171

Asp280

Asp16

Asn179

Leu131

Asp15

Glu177

Phe178

Asn40

Met162

Asp11

Gly13

3FZ0 quercetin

(b)





4 Conclusion




Abbreviations





Disclosure






References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Abbreviations





Disclosure






References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article investigation of flavonoids as potential...

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