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Clinical Immunology (2010) 137, 347–356

The evaluation of catechins that contain a galloylmoiety as potential HIV-1 integrase inhibitorsFan Jiang a, Wei Chen a, Kejia Yi b, Zhiqiang Wu a, Yiling Si a,Weidong Han a, Yali Zhao a,⁎

a Department of Molecular Biology, Institute of Basic Medicine, Chinese PLA General Hospital, 28 Fu Xing Road,Hai Dian District, 100853 Beijing, Chinab Beijing Science Technology and Management College, Beijing

Received 22 April 2010; accepted with revision 11 August 2010Available online 15 September 2010

Abbreviations: CG, catechin gallagallate; GCG, gallocatechin gallate; Eepicatechin; GC, Gallocatechin; C, Caacid; IC50, their concentrations require⁎ Corresponding author. Fax: +86 10E-mail address: zhaoyl301@yahoo.c

1521-6616/$ – see front matter © 201doi:10.1016/j.clim.2010.08.007

KEYWORDSCatechin;HIV integrase;Inhibitor

Abstract Four catechins with the galloyl moiety, including catechin gallate (CG), epigalloca-techin gallate (EGCG), gallocatechin gallate (GCG), and epicatechin gallate (ECG), were found toinhibit HIV-1 integrase effectively as determined by our ELISA method. In our docking study, it isproposed that when the HIV-1 integrase does not combine with virus DNA, the four catechins maybind to Tyr143 and Gln148, thus altering the flexibility of the loop (Gly140-Gly149), which could

lead to an inhibition of HIV-1 integrase activity. In addition, after combining HIV-1 integrase withvirus DNA, the four catechins may bind between the integrase and virus DNA, consequently,disrupt this interaction. Thus, the four catechins may reduce the activity of HIV-1 integrase bydisrupting its interaction with virus DNA. The four catechins have a highly cooperative inhibitoryeffect (IC50=0.1 μmol/L). Our study suggests that catechins with the galloyl moiety could be anovel and effective class of HIV-1 integrase inhibitors.© 2010 Elsevier Inc. All rights reserved.

te; EGCG, epigallocatechinCG, epicatechin gallate; EC,techin hydrate; DKA, Diketod for 50% inhibition.66937516.om.cn (Y. Zhao).

0 Elsevier Inc. All rights reserv

1. Introduction

Highly active antiretroviral therapy (HAART) is now thestandard for the treatment of acquired immunodeficiencysyndrome (AIDS). It is based on a combination of drugs thattarget human immunodeficiency virus-1 (HIV-1) reversetranscriptase and HIV-1 protease [1]. However, HAART

ed

often leads to multidrug resistance and is not well-toleratedby patients [2]. HIV-1 integrase is the third important viralenzyme and critical for productive HIV-1 infection. There isno mammalian homologue of HIV-1 integrase, potentiallymaking anti-integrase therapeutics of low toxicity [3,4]. In2007 the first HIV-1 integrase target drug, Raltegravir, wasapproved by the Food and Drug Administration (FDA) [5].Based on a 48-week clinical trial, raltegravir, a HIV-1integrase inhibitor showed good efficacy and safety fortreatment-naive [6] and experienced patients [7]. Thus, HIV-1 integrase has become an attractive drug target recently.However, raltegravir resistance mutations have been foundin clinical trials. These mutations are associated with failureof raltegravir in clinical trials [8].

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348 F. Jiang et al.

Worldwide, money spent on HIV/AIDS research raisedfrom $300 million in 1996 to $10 billion in 2007. Bringing asingle drug to market costs an average of $2 billion. Afterraltegravir was approved for market, much research focusedon the development of raltegravir analog HIV-1 integraseinhibitors. However, these raltegravir analogs have had a lowsuccess rate against raltegravir-resistant HIV strains [9].

Catechins are the main compounds in green tea andconsist mainly of epicatechin (EC), epigallocatechin (EGC),epicatechin gallate (ECG), and epigallocatechin gallate(EGCG). Half of the major catechins are epimerised tocatechin (C), gallocatechin (GC), catechin gallate (CG), andgallocatechin gallate (GCG) during heat treatment forsterilization [10]. It has been reported that catechins havevarious health benefits, such as antioxidant, cancer-pre-venting properties, antiviral activities, etc. [11–13].

Recently, EGCG, the primary catechin, has been shown toinhibit a broad spectrum of HIV-1 subtypes at physiologicallyrelevant concentrations (0.1–10 μM) without harming humancells. Thus, EGCG is a candidate alternative therapy tocontrol HIV-1 infection [14].

Previous studies have reported that EGCG inhibits HIV-1by targeting HIV-1 reverse transcriptase and protease.However, the concentration of EGCG necessary to inducethese effects is higher than its physiological concentration[15–17]. Williamson et al. [18] showed that EGCG inhibitsHIV-1 viral attachment by blocking gp120-CD4 at lowconcentration. However, Yamaguchi et al. [15] onlydetected weak inhibition of viral attachment (EGCGIC20=100 μmol). Kawai et al. [19] also reported weakEGCG-dependent inhibition of viral attachment to gp120-CD4 (IC28=100 μmol).

Thus, although catechins effectively inhibit HIV-1 infec-tion in preclinical experiments, the actual mechanism ofinhibition remains unclear.

To further investigate catechin-dependent inhibition ofHIV-1 infection, we used an ELISA method to study theinhibition of HIV-1 integrase by four catechins that contain agallate moiety. To investigate the cooperative inhibitoryeffect of these catechins, we combined the four catechins atthe same concentration. Moreover, we chose EC, a majorcatechin that does not contain the gallate moiety, tocompare the inhibitory effect. In addition, we used amolecular docking study to investigate the inhibitorymechanism of catechins with and without the gallate moietyon HIV-1 integase.

2. Materials and methods

2.1. Reagents

(−)-Epigallocatechin gallate (EGCG) (Sigma, E4143), (−)-epicatechin gallate (ECG) (Sigma, E3893), (−)-gallocatechingallate (GCG) (Sigma, G6782), (−)-catechin gallate (CG)(Sigma, C0692), and (−)-epicatechin (EC) (Sigma, E4018) wasdiluted with sterile distilled water, respectively. Moreover,we made a complex solution by combining EGCG, ECG, GCG,and CG to equal concentrations. Sodium azide is included inthe Xpress HIV-1 Integrase Assay Kits (Express Biotech

International, USA), as a positive control compound thatinhibits HIV-1 integrase activity.

2.2. HIV-1 integrase assay

The Xpress HIV-1 Integrase Assay Kits (Express BiotechInternational, USA) were used to measure the inhibitoryeffects of catechins on HIV-1 integrase activity. Streptavidin-coated 96-well plates were coated with a double-strandedHIV-1 LTR U5 donor substrate (DS) oligonucleotide containingan end-labeled biotin. Full-length recombinant HIV-1 inte-grase protein (200 nM, purified frombacteria)was then loadedonto the oligo substrate. EGCG, ECG, GCG, CG, EC, thecomplex solution, or sodium azide was added to the reactionplates and then a double-stranded target substrate (TS) oligocontaining 3′-end modifications was added to the plate. TheHRP-labeled antibody was directed against the TS 3′-endmodification and the absorbance due to the HRP antibody–TMB peroxidase substrate reaction was measured at 450 nm.

2.3. Model building

Two X-ray structures of the core domain of HIV-1 integrase(PDB ID: 1BL3 [20] and PDB ID: 1QS4 [21]) and a theoreticalmodel (PDB ID: 1WKN) of full-length HIV-1 integrase incombination with viral DNA published by De Luca et al. [22]were obtained from Protein Data Bank with all crystallo-graphic waters removed [23].

The 3D sdf files of EGCG (CID_65064), GCG(CID_199472),CG(CID_6419835), ECG(CID_107905), EC(CID_72276), (−)-gallocatechin (GC) (CID_9882981) and 2D sdf file of (+)-catechin hydrate (C) (CID_107957) were obtained fromPubchem compound database. The three dimensional struc-tures of catechins with polar hydrogen and chiralities werebuilt using the Dundee PRODRG2 Server. Energy minimizationof each ligand was performed using the steepest descent fora minimum of 50,000 steps. A ffgmx GROMACS force field wasdeveloped using the Dundee PRODRG2 Server [24].

The protonation sites and all hydrogens of 1WKN wereprepared using CHARMM v22 [22]. The nonpolar hydrogens of1WKN were merged using AutoDockTOOlS (version 1.5.1).1QS4 and 1BL3 structures with polar hydrogen were preparedfor docking using AutoDockTOOlS (version 1.5.1). Gasteigercharges and grid boxes were prepared for protein structuresusing AutoDockTOOlS (version 1.5.1). Flexible torsions in theligands were assigned with the AUTOTORS module automat-ically [25,26].

2.4. Two-step blind docking

The receptors were divided into two parts. Two grid boxes(x: 50 Å, y: 52 Å, z: 50 Å) covered the top and lower half of1BL3 protein. One grid box (x: 50 Å, y: 52 Å, z: 50 Å) coveredthe top half of 1QS4 protein. Another grid box (x: 52 Å, y:52 Å, z: 50 Å) covered the lower half of 1QS4 protein. Onegrid box(x: 50 Å, y: 60 Å, z: 50 Å) covered the top half of1WKN protein. Another grid box (x: 66 Å, y: 60 Å, z: 50 Å)covered the lower half of 1WKN protein. Autodock vinaprogram (version 1.0 BETA) was selected for the dockingprocess with default parameters [25]. In the first step, we

Figure 2 Concentration-dependent inhibition of HIV-1 inte-grase by a mixture of the four catechins with the galloyl moietyto equal concentrations.

349The evaluation of catechins that contain a galloyl moiety as potential HIV-1 integrase inhibitors

collected the five top-scoring binding sites for eachcompound.

In the second step, the grid boxes were 16×16×16 Å cubeswith grid points, centered on the best binding sites obtainedfrom the first step. Autodock vina program (version 1.0 BETA)was again used for the docking process with defaultparameters [25,26].

2.5. Specific Mg ions for binding sites docking study

We chose three single Mg sites in the 1BL3 model and twodouble Mg sites in the 1WKN model. The five Mg ions wereselected to be the center of the grid box, and each grid boxwas 16×16×16 Å. Autodock vina program (version 1.0 BETA)was also used for the docking experiment using defaultparameters [25,26].

The docking results were analyzed and visualized by usingPyMOL program (version 0.97).

3. Results

3.1. ElISA-based method

A report by the XpressBio company used the Xpress HIV-1Integrase Assay Kit (Express Biotech International, USA) tomeasure the efficacy of raltegravir inhibition. The IC50 ofraltegravir for HIV-1 integrase was 0.26 μmol/L [27]. Usingthe same ELISA method, we found that the IC50 of EGCG andCG was 0.96 μmol/L and 0.56 μmol/L, respectively (Fig. 1);these were slightly higher than raltegravir. GCG and ECG alsoappeared to exhibit good inhibitory effects, with IC50 valuesof 2.4 μmol/L and 3.02 μmol/L (Fig. 1). Compared tocatechins with the galloyl moiety, EC, a catechin withoutthe galloyl moiety, showed a weak inhibitory effect. The IC50

for EC was found to be above 344.5 μmol/L. Interestingly,the IC50 value of the combination of four catechins contain-ing a galloyl moiety was 0.1 μmol/L (Fig. 2), which is betterthan raltegravir. The 0.1 μmol/L mixture included an equalvolume of 0.1 μmol/L EGCG, 0.1 μmol/L ECG, 0.1 μmol/LGCG, and 0.1 μmol/L CG. The IC50 of sodium azide, a positivecontrol compound, is 0.75%.

In our study, it was shown that catechins that contain agalloyl moiety may have an inhibitory effect similar to

Figure 1 Concentration-dependent inhibition of HIV-1 inte-grase by catechins with the galloyl moiety.

raltegravir. Moreover, the mixture of different catechinscontaining a galloyl moiety may more effectively inhibit HIV-1 integrase than raltegravir.

3.2. Blind docking study

We initially used blind docking to investigate the bindingsites for raltegravir and catechin. We list the identity anddocking energy of residues of HIV-1 integrase and bases ofvirus DNA that have polar contact with raltegravir orcatechins in Table 1.

As shown in Table 1,we have observed that HIV-1 integrasecombines with virus DNA (1WKN) and raltegravir andcatechins containing a galloy moiety bind this site betweenHIV-1 integrase and virus DNA with high efficacy, so as todisturb the interaction of HIV-1 integrase with virus DNA.

When HIV-1 integrase does not combine with virus DNA(1QS4 and 1BL3), raltegravir makes polar contact withresidue Gln168.

When HIV-1 integrase does not contact virus DNA (1QS4 and1BL3), each catechin makes polar contacts with the loop(Gly140-Gly149) within the HIV-1 integrase core domain. Theresidues in the loop (Gly140-Gly149) are indicated by bold textin Table 1. Furthermore, catechins that contain a galloymoietymost frequently dockwith residues Tyr143 and Gln148.

3.3. Specific Mg ions binding sites docking study

The function of Mg ions is important, and the biding sitesaround the Mg ions are a focus of many investigators [28,29].To investigate the effect of raltegravir and catechins on theMg ion site in HIV-1 integrase, we chose three single Mg ionsites in the 1BL3 model and two doulbe Mg ion sites in the1WKN model. The residues that make contact with ralte-gravir and catechins are listed in Table 2.

As shown in Table 2, we observed that raltegravir andcatechins make polar contact with the five Mg ions, but thedocking efficacy was lower than other sites from Table 1.This implies that a different inhibitory mechanism may bemore important than disturbing the Mg ions during ralte-gravir and catechin-induced HIV-1 integrase inhibition.

As shown in Table 2, catechins not onlymade polar contactwith the Mg ions and residues that directly contact the Mgions, such as Asp116, Asp64, and Glu152, but also made polar

Table 1 Energy values from blind docking.

A B C D E F G H

Catechins with the galloyl moietyCG −8.4 Chain B: His114, −8.8 Chain B: Gln168; 4 5 −9.4 C5, A6, G7, C44, C46, Gly247

Tyr143, Gln148, Chain C: Tyr143,Asn144 Gln148

EGCG −8.5 Chain B: Asp116, −8.0 Chain A: Lys136; 3 5 −10 A6, C46, T47, Gly247Tyr143, Gln148, Chain C: Gly140,Asn144 Tyr143

GCG −8.4 Chain B: Val79, −8.9 Chain C: Tyr143, 2 3 −10 C5, A6, A45Lys186, Arg199, Gln148Ala196, His183;Chain C: Gly140

ECG −8.3 Chain B: Asp116, −8.3 Chain C: Ala86, 2 3 −9.2 T8, A43, A45Tyr143, Asn144, Gln177, Arg107Gln148

Catechins without the galloyl moietyC −7.9 Chain B: Gln62, −7.9 Chain A: Gln137; 0 3 −9.2 Unshown data

Leu63, His114, Chain C: Phe139Ser147,Gly149, Asn144

GC −7.9 Chain B: Tyr143, −7.8 Chain A: Gln95; 1 3 −8.8 Unshown dataAsn144, Gln63, Chain B: His171His114, Gly140

EC −8.3 Chain B: Leu63, −7.8 Chain A: Gln95; 2 3 −9.0 G11, G12Gln62, Gln148, Chain B: Thr174;Asn144 Chain C: Tyr143

Raltegravir −8.5 Chain B: Gln62; −8.5 Chain B: Gln168; 0 0 −10.1 A6, T47, C5His114 Chain A: Gln95

(A) Final docked energy of best conformation for each catechin from 1QS4 (kcal/mol). (B) Residues with polar contact for each catechinthat are involved in the best binding site from 1QS4. (C) Final docked energy of the best conformation for each catechin from 1BL3 (kcal/mol). (D) Residues with polar contact for each catechin that are involved in the best binding site from 1BL3. (E) The amount of Tyr143 orGln148 has polar contact with catechins from 1QS4 and 1BL3. (F) The amount of residues in the loop (140–149) has polar contact withcatechins from 1QS4 and 1BL3. (G) Final docked energy of the best conformation for each catechin from 1WKN (kcal/mol). (H) Residueswith polar contact for each catechin that are involved in the best binding site from 1WKN.

350 F. Jiang et al.

contact with residues in the loop domain, which are indicatedby bold text in Table 2. However, raltegravir only made polarcontact with Mg ions and residues that directly contact theMgions, such as Asp116, Asp64, and Glu152, but did not makepolar contact with residues in the loop domain.

3.4. Structural features of catechins and raltegravirinteract with integrase/DNA

As shown in Table 1, EGCG had the best docking efficacy inthe 1QS4 structure and GCG had best docking efficacy in the1BL3 structure. We analyzed the relationship of EGCG and

Notes to Table 2:(A) Final docked energy of the best conformation for each catechin incontact for each catechin involved in the best binding site in Mg ioconformation for each catechin in Mg ion site in chain B of 1BL3 (kcal/mthe best binding site in Mg ion site in chain B of 1BL3. (E) Final dockedchain C of 1BL3 (kcal/mol). (F) Residues with polar contact for each ca1BL3. (G) Final docked energy of the best conformation for each catecpolar contact for each catechin involved in the best binding site in Mconformation for each catechin in Mg ion site in chain B of 1WKN (kcal/the best binding site in Mg ion site in chain B of 1WKN.

GCG structure with binding sites in the HIV-1 integrase andcompared EGCG and GCG with raltegravir. The hydroxylgroup of EGCG and GCG played an important role in thebinding to loop domain in the 1QS4 and 1BL3 models(Schematic 1 and Schematic 5). In the 1QS4 and 1BL3models, the binding site area of raltegravir was similar toEGCG and GCG (Schematic 3 and Schematic 6). However,raltegravir made polar contact with different residues incomparison to EGCG and GCG (Schematic 2 and Schematic 4).

In the 1WKN model, EGCG was the best catechin thatbinds with 1WKN with the lowest energy and made the mostnumber of polar contacts with residues. Comparison of EGCGwith raltegravir found that EGCG and raltegravir had very

Mg ion site in chain A of 1BL3 (kcal/mol). (B) Residues with polarn site in chain A of 1BL3. (C) Final docked energy of the bestol). (D) Residues with polar contact for each catechin involved in

energy of the best conformation for each catechin in Mg ion site intechin involved in the best binding site in Mg ion site in chain C ofhin in Mg ion site in chain A of 1WKN (kcal/mol). (H) Residues withg ion site in chain A of 1WKN. (I) Final docked energy of the bestmol). (J) Residues with polar contact for each catechin involved in

351The evaluation of catechins that contain a galloyl moiety as potential HIV-1 integrase inhibitors

similar binding sites between the HIV-1 integrase and virusDNA, as shown in Schematic 9. We also noted that the galloylmoiety of EGCG had the same binding function as the hydroxy

Table 2 Energy values from specific Mg ions binding sites dockin

A B C D E

Catechins with the galloyl moietyCG −6.2 Chain A: −6.3 Chain B: −7

Asp116, Asn117,Asp64, Asp116,Thr115, Asp64Glu152,Gln62

EGCG −7.3 Chain A: −6.5 Chain B: −7Gly140,Asp64, Val150,Cys65, Asp116,His114 Leu63

GCG −6.6 Chain A: −6.2 Chain B: −7Glu152, Asn117,Asn117, Thr115,Asp116, Asp116Mg,Asp64

ECG −6.8 Chain A: −6.6 Chain B: −7Ile151, His114,Glu152, Ieu63,Mg, Val150Thr115

Catechins without the galloyl moietyC −6.4 Chain A: −6.1 Chain B: −6

Mg, Glu138,Asp116, Phe139,His114 His114,

Asp116,Mg,Leu63

GC −6.5 Chain A: −6.1 Chain B: −6Asp64, Asp116,Asp116, Mg,Thr115, His114,His114, Gln62,Gln62, Asp64,Mg

EC −6.1 Chain A: −6.1 Chain B: −6Asp116, Asp116,His114 Val150,

Ile151,Gln62,Glu152

Raltegravir −7.2 Chain A: −6.4 Chain B: −7Asp116, His114,Mg, Gln62,His67, Val150,Thr66, Glu152,Asp64, Asp64,Ser119, Asp116Asn120

ketone and 1,3,4-oxadiazole moieties of raltegravir. Bothgalloyl of EGCG and hydroxy ketone and 1,3,4-oxadiazole ofraltegravir made polar contacts with A6, T47, and bases in

g study.

F G H I J

.1 Chain A: −5.9 Chain A: −4.9 Chain B:Thr122, Phe139, Gln148,Lys127 Gln148, Asp116,Chain C: Glu92, Asn117Asp64, Glu152Asn117,Mg

.4 Chain C: −5.6 Chain A: −4.8 Chain B:Mg, Gln148, Cys65,Cys65, Thr115, Asn155Asn155 Phe139,

His67.7 Chain C: −5.3 Chain A: −5.4 Chain B:

Mg, Gly140, Gln148,Asp64, Asn117, His67,Asn155 Cys65, Asn117

Asp64,Glu152

.4 Chain A: −5.6 Chain A: −4.9 Chain B:Thr122, Gln148, Cys65,Chain C: His67 Asn117Asp64,Asn155,Glu92

.6 Chain A: −4.9 Chain A: −5.7 Chain B:Thr122, Glu92 Lys159,Chain C: Lys156,Gln148 Glu152

.7 Chain C: −4.9 Chain A: −5.4 A25,Asp64 Gly140, Chain B:

Gln148, Lys156,Asn117 Glu152,

Lys159,His67

.9 Chain A: −5.1 Chain A: −5.4 Chain B:Thr122, His67, Cys65,Ser119 Cys65 Glu92

.2 Chain A: −6.2 Chain A: −5.9 Chain B:Thr122, ASP64, GLN152,Thr124, ASN117, PHE139Chain C: ASP116Thr66

Schematic 3 Docking of EGCG and raltegravir in the X-raycrystal structure of HIV-1 integrase. Raltegravir is shown in red;EGCG is shown in green. 1QS4 is shown in gray. Residues thatmake polar contact with EGCG are shown in blue. Residues thatmake polar contact with raltegravir are shown in yellow. Mg ionis shown in magenta.

Schematic 1 2D representation of EGCG making polarcontacts with the 1QS4 structure.

352 F. Jiang et al.

virus DNA (Schematic 7 and Schematic 8). Furthermore,EGCG had two more polar binding sites than raltegravir(Schematic 7 and Schematic 8).

Therefore, in the 1QS4 and 1BL3models, the hydroxyl groupof catechin, EGCG and GCG made polar contacts with the loopdomain, which is different from raltegravir. In the 1WKNmodel, EGCG had a very similar polar contact in comparison toraltegravir, but EGCG had more binding sites than raltegravir.

4. Discussion

HIV-1 integrase protein is responsible for the insertion of HIVcDNA into the genome of infected cells. There are two mainsteps for the integration reaction: 3′-processing and strandtransfer. During 3′-processing, endonucleolytic cleavageexposes the 3′-ends of the viral cDNA, allowing stand transfer

chematic 2 2D representation of raltegravir making polarontacts with the 1QS4 structure.

Schematic 4 2D representation of raltegravir making polarcontacts with the 1BL3 structure.

Sc

to be carried out. In this step, HIV-1 integrase joins the 3′-OHcDNA ends to the 5′ phosphate of the acceptor DNA [2,3].Some studies have indicated that the loop (Gly140-Gly149) isessential for strand transfer. Moreover, the flexibility of theloop (Gly140-Gly149) is a critical determinant of HIV-1integrase activity [30–32].

Some residues of the loop (Gly140-Gly149) are lost in themajority of published X-ray structure of core domain of HIV-1integrase [21,23,30]. Thus, we chose to use two differentHIV-1 integrase structures in our study. The first structurefrom 1QS4 pdb file lack loop residues 141–144 in chain A,141–142 in chain B, and 141–143 in chain C [21]. In contrast,the second structure from 1BL3 pdb file contains the full loop

Schematic 5 2D representation of GCG making polar contactswith the 1BL3 structure.

Schematic 7 2D representation of raltegravir making polarcontacts with the 1WKN structure.

353The evaluation of catechins that contain a galloyl moiety as potential HIV-1 integrase inhibitors

in chain C, but lacks loop conformation in chain B and chain A[20]. 1WKN is a theoretical model was published by De Lucaet al. [22]. 1WKN contains the C-terminal, N-terminal, andcore domain of HIV-1 integrase. In addition, 1WKN modelsthe interaction of HIV-1 integrase with viral DNA. In thistheoretical model, two Mg2+ ions were placed in the coredomain of each chain of HIV-1 integrase. Hare et al [33]published the X-ray structure of full-length integrase incombination with its cognate DNA in 2010. However, that X-ray structure was of a prototpye foamy virus and not HIV.Thus, we used 1WKN, the HIV-1 theoretical model.

Schematic 6 Docking of GCG and raltegravir in the X-raycrystal structure of HIV-1 integrase. Raltegravir is shown in red.GCG is shown in green. 1BL3 is shown in gray. Residues that makepolar contact with GCG are shown in blue. Residues that makepolar contact with raltegravir are shown in yellow. Mg ion isshown in magenta.

We conclude with the results in Tables 1 and 2. In general,it was shown that the final docked energy of catechins thatcontain the galloyl moiety was better than catechins that donot contain the galloly moiety, which is consistent with theirinhibitory potency, as measured by our ELISA method. Thereappears to be a relationship between the binding potencyand the inhibitory potency, such that the catechins with thegalloyl moiety are more potent at inhibitors of HIV-1integrase. In the study of Bessong et al. [34], it was alsoreported that catechins without the galloyl moiety exhibitedonly weak activity against HIV-1 integrase (IC65=100 μmol),consistent with our study.

In different models, catechins could bind the loopdomain. In particular, catechins that contain the galloylmoiety may efficiently dock with the loop domain of HIV-1integrase that does not combine with virus DNA. Theinfluence of loop domain may be the most different aspectof catechin and raltegravir mode of inhibition. It is suggestedthat when the HIV-1 integrase does not combine with virusDNA, catechins may bind to Tyr143 and Gln148, thus altering

Schematic 8 2D representation of EGCG making polarcontacts with the 1WKN structure.

Schematic 9 Schematic representing the docking of EGCG and raltegravir in the X-ray crystal structure of HIV-1 integrase.Raltegravir is shown in red. EGCG is shown in green. 1WKN is shown in gray. Viral DNA bases only have polar contact with EGCG areshown in blue. Residues have polar contact with EGCG are shown in pink. Viral DNA bases have polar contact with raltegravir and EGCGare shown in yellow. Viral DNA bases only have polar contact with raltegravir are shown in cyan. Mg ion is shown in magenta.

354 F. Jiang et al.

the flexibility of the loop (Gly140-Gly149), which could leadto an inhibition of HIV-1 integrase activity.

Raltegravir and catechins also have a similar inhibitorymechanism. The strongly disturb HIV-1 integrase interactionwith virus DNA and they also have influence on the Mg ionbinding sites.

Our study initially used blind docking to investigate theraltegravir and catechins, and then limited the docking sitearound the five different Mg ions sites to investigate themechanism of raltegravir and catechin HIV-1 integrase inhibi-tion. Our method may be more thorough than previous studiesthat investigated the influence of drugs on HIV-1 integrase.

As shown in Table 1, CG and GCG had a better bindingefficacy to the loop in chain C of HIV-1 integrase, and ECGand EGCG had better binding efficacy to the loop in chain B ofHIV-1 integrase. Thus, if we combine the four catechins thatcontain a galloyl moiety, the loop domain in both chain B andchain C will be inhibited and would consequently influenceHIV-1 integrase function more thoroughly.

As shown in Table 2, catechins that contain a galloylmoiety influenced Mg ions and the residues around the Mgions. Thus, if catechins that contain a galloyl moiety have acompetitive effect on loop domain, some may alsoinfluence Mg ions or residues around the Mg ions. Thus, acombination of different catechins that contain a galloylmoiety may decrease the function of HIV-1 integrase moreeffectively than a single catechin. This hypothesis isconfirmed by our ELISA experiment. In our ELISA-basedmethod, the complex solution, which included four cate-chins that contain a galloyl moiety, had a strongerinhibitory effect than a single catechin. The presence ofmultiple binding sites may help catechins with the galloylmoiety continue to inhibit HIV-1 integrase even whenTyr143 and Gln148 have been mutated.

In our study, we determined the ability of catechinsthat contain a galloyl moiety to inhibit HIV-1 integrase.One clinical study has determined that the safety of an800-mg daily of administration of EGCG and polyphenon E(a defined, decaffeinated green tea polyphenol mixture)[35]. After an 800-mg daily oral administration of EGCG,the peak of concentration in human plasma (Cmax) is 5–6times greater than the IC50 of EGCG against HIV-1integrase. Thus, in clinical trials, EGCG may effectivelyinhibit HIV-1 integrase. After 10 days of accumulation ofEGCG in human body, the Cmax of EGCG was even higherthan raltegravir [36]. Furthermore, when EGCG wascombined with other catechins, the Cmax was increased[37], and the efficacy HIV-1 integrase inhibition alsoincreased. Indeed, the efficacy of HIV-1 integrase inhibi-tion of complex of catechins containing a galloyl moietywas better than raltegravir as determined by our ELISAmethod. In our docking study, we determined that theinhibitory mechanism of catechins that contain a galloymoiety and raltegravir is different. Furthermore, ingeneral, catechins appeared to be weak and nonspecificinhibitors of the oxidation, hydroxylation, and dealkylationreactions mediated by CYP1A1, CYP1A2, CYP2A6, CYP2C19,CYP2B1, CYP2B2, CYP2E1, CYP3A1, CYP3A2, and CYP3A4isozymes as determined in vitro [38].This implies thatcatechins can be used with other drugs.

Thus, our study suggests that catechins containing agalloy moiety may be a highly potent complement HIV-1integrase inhibitor.

The recently approved clinical integrase inhibitors arealmost all derived from diketo acid (DKA),a syntheticcompound that selectively inhibits the strand transfer stepof integration [2,39]. Conversely, catechins are naturalcompounds found in tea that are well-known for their

355The evaluation of catechins that contain a galloyl moiety as potential HIV-1 integrase inhibitors

healthy effects and safety. Thus, catechins may provide asafe and well-tolerated drug.

Furthermore, catechins are cheap natural compounds.EGCG (purity≥98%), the most popular catechin containing agalloyl moiety, is less than $5 each gram on Chinese market.After EGCG is mixed into polyphenon E (EGCG purity≥60%),the price is just $0.06, which is nearly 700 times cheaperthan raltegravir. Thus, catechins that contain a galloylmoiety may reduce the prescription cost for HIV-1 patientsand may be popular among low-income populations.

Acknowledgment

The authors thank Professor Hong Qu from Peking Universityfor her computer assistance in part of this study.

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