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Anti-HIV-1 activity of phlorotannin derivative 8,4‴dieckol from Korean brown alga Ecklonia cava a

a

b

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Fatih Karadeniz , Kyong-Hwa Kang , Jae W. Park , Sun-Joo Park & Se-Kwon Kim

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Marine Bioprocess Research Center, Pukyong National University, Busan, Republic of Korea b

OSU Seafood Research and Education Center, Astoria, OR, USA

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Department of Chemistry, Pukyong National University, Busan, Republic of Korea

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Department of Marine Bio Convergence Science, Pukyong National University, Busan, Republic of Korea Published online: 12 Jun 2014.

To cite this article: Fatih Karadeniz, Kyong-Hwa Kang, Jae W. Park, Sun-Joo Park & Se-Kwon Kim (2014) Anti-HIV-1 activity of phlorotannin derivative 8,4‴-dieckol from Korean brown alga Ecklonia cava, Bioscience, Biotechnology, and Biochemistry, 78:7, 1151-1158, DOI: 10.1080/09168451.2014.923282 To link to this article: http://dx.doi.org/10.1080/09168451.2014.923282

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Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 7, 1151–1158

Anti-HIV-1 activity of phlorotannin derivative 8,4‴-dieckol from Korean brown alga Ecklonia cava Fatih Karadeniz1, Kyong-Hwa Kang1, Jae W. Park2, Sun-Joo Park3 and Se-Kwon Kim4,* 1

Marine Bioprocess Research Center, Pukyong National University, Busan, Republic of Korea; 2OSU Seafood Research and Education Center, Astoria, OR, USA; 3Department of Chemistry, Pukyong National University, Busan, Republic of Korea; 4Department of Marine Bio Convergence Science, Pukyong National University, Busan, Republic of Korea

Received November 20, 2013; accepted February 27, 2014

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http://dx.doi.org/10.1080/09168451.2014.923282

8,4‴-dieckol is a natural product which has been isolated from brown alga, Ecklonia cava. This polyphenolic compound is a phlorotannin derivative with a broad range of bioactivities. Its inhibitory activity on human immunodeficiency virus type-1 (HIV-1) was tested and the results indicated that 8,4‴-dieckol inhibited HIV-1 induced syncytia formation, lytic effects, and viral p24 antigen production at noncytotoxic concentrations. Furthermore, it was found that 8,4‴-dieckol selectively inhibited the activity of HIV-1 reverse trancriptase (RT) enzyme with 91% inhibition ratio at the concentration of 50 μM. HIV-1 entry was also inhibited by 8,4‴-dieckol. According to data from this study, 8,4‴-dieckol is an effective compound against HIV-1 with high potential for further studies. These results suggest that it might be used as a drug candidate for the development of new generation therapeutic agents, although further studies on the mechanism of inhibition should be addressed. Key words:

Ecklonia cava; phlorotannin; 8,4‴-dieckol syncytia; HIV-1 RT inhibition

High amounts of secondary metabolites produced by microorganisms, plants, and marine organisms have been of much attention as bioactive substances for disease treatment.1,2) Among these diseases, acquired immunodeficiency syndrome (AIDS) stands as one of the most important diseases worldwide with about 35.3 million people infected by human immunodeficiency virus type-1 (HIV-1).3) HIV-1 is identified as the causative agent of AIDS and up to now there are significant advances in rational drug design and highly active compounds can be synthesized.4) However, HIV drug resistance, side effects, and the need for long-term antiviral treatment urge the inevitable development of new anti-HIV agents, targets, and therapies.5–7) In this regard, natural occurring products are still known as the richest source of bioactive compounds. Natural *Corresponding author. Email: [email protected] Fatih Karadeniz and Kyong-Hwa Kang contributed equally to this work. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

products promote excessive availability for discovering anti-HIV treatment as they contain massive amount of potential for being a consistent source for successful drug discovery.8) There are quite big amounts of marine-based natural products which are reported to have bioactivities like antifungal and antimicrobial effects.9,10) In addition, several natural products from marine organisms have been reported to express bioactivity against prevalent diseases such as cancer, diabetes, and obesity.11–13) So far, numerous compounds isolated from natural resources have been reported to exhibit significant antiHIV activity and inhibit HIV-1 activity in almost every stage of the viral life cycle.14) Polysaccharides and phlorotannins isolated from the marine algae show distinct inhibitory activity against HIV-1 reproduction as well as viral infection.15,16) In our present research, we reported the anti-HIV-1 activity of phloroglucinol derivative 8,4‴-dieckol from marine brown alga, Ecklonia cava. The results obtained from the experiments indicated that 8,4‴-dieckol inhibited the cytopathic effects of HIV-1 including HIV-1 induced syncytia formation, lytic effects, and viral p24 antigen production, as well as exhibited RT enzyme inhibitory and HIV-1 entry activities.

Materials and methods General materials. Nevirapine, dextran sulfate, and polyethylene glycol (MW 8000 Da) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Primary and secondary antibodies for immunoblot analysis were purchased from SantaCruz Biotechnology (CA, USA). HIV-1 p24 antigen capture ELISA kit was obtained from Perkin-Elmer Life Sciences (Boston, MA, USA). Reverse transcriptase activity assay kit was purchased from InvitroGen (CA, USA). Luciferase gene reporter assay kit was obtained from Promega Corp. (Madison, WI). Cell culture medium (RPMI 1640), penicillin/streptomycin, fetal bovine serum

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(FBS), and other cell culture materials were obtained from Gibco BRL, Life Technology (NY, USA) and Sigma Chemical Co. (St. Louis, MO, USA). 8,4‴-dieckol was isolated and elucidated as a part of previous research along several other phlorotannin derivatives (Fig. 1). Hence, structural characteristics of 8,4‴-dieckol have been reported earlier.17) Cell and viruses. Peripheral blood mononuclear cells (PBMCs) from healthy donors were obtained using Ficoll-Hypaque (Sigma Chemical Co., St. Louis, MO, USA) density gradient centrifugation. PBMCs were resuspended in RPMI 1640; supplemented with 10% FBS, penicillin (100 U/mL), streptomycin (100 μg/mL), 2 mM glutamine and 10 mM HEPES; and stimulated with 10 μg/mL phtohemagglutinin (PHA, Sigma Chemical Co., St. Louis, MO, USA) during 3 days and further maintained in culture medium containing 50 U/mL of recombinant IL-2 (Sigma Chemical Co., St. Louis, MO, USA). The viral isolate HIV-1Ba-L stocks were prepared in PHA-activated PBMCs from healthy donors. C8166 cell line from Dr G. Farrar was provided by the EU Programme EVA Centre for AIDS Reagents, NIBSC, UK. TZM-bl, a HeLa derivative, was obtained from Dr John C. Kappes, Dr. Xiaoyun Wu, and Tranzyme Inc. H9 and H9/HIV-1IIIB cell lines were obtained through the American Type of Culture Collection (Manassas, VA, USA). HIV-1LAV, Triple Drug Resistant HIV-1 (RTMDR1), HIV-1RF, and HIV-1Ba-L were obtained through Reference Reagent Program and the Centre for AIDS Reagents supported by EU Programme EVA/MRC.

Cell viability assay. The cytotoxicity of 8,4‴-dieckol was determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) formazan assay, a method based on reduction as described previously.18) H9 cells were seeded to the wells of a 96-well flat-bottomed microtiter plate at a density of 105 cells/mL. After incubation for overnight, the cells were treated with various concentrations of 8,4‴-dieckol and kept at 37 °C. After a 72 h incubation period, MTT was added to each well and the plate was incubated for another 4 h at 37 °C. Optical density was measured at 540 nm with a microplate reader. The optical density of formazan formed by untreated cells was taken as 100% of viability. Inhibition of syncytia formation. HIV-1 induced syncytia formation analysis was carried out as described previously.19) Briefly, C8166 cells were infected with different X4 tropic HIV-1 strains at a multiplicity of infection (M.O.I.) of 0.1 and incubated for 2 h. After viral adsorption, cells were washed twice with PBS to remove unabsorbed particles, resuspended in complete medium and seeded (105 cells/mL) to a 48well plate in aliquots of 400 μL in triplicate. One hundred microliters of serial dilutions of compound in complete medium were transferred to the microtiter plate and the plate was incubated at 37 °C for 4 days. The number of syncytia was counted using an inverted microscope. Inhibition of cytopathic effect of HIV-1. In order to detect the anti-HIV-1 activity of 8,4‴-dieckol on acutely infected C8166 cells, a tetrazolium-based (MTT) colorimetric method was applied as described before.20,21) C8166 cells were suspended in complete RPMI 1640 medium and infected with HIV-1 at a M.O.I. of 0.1. After 2 h of incubation, the cells were washed twice with PBS, resuspended in culture medium and 400 μL of aliquots were added (105 cells/mL) to each well of a 48-well flat-bottomed microtiter plate. One hundred microliters of medium containing test compound at various concentrations or nevirapine was added to the microtiter plate and the cell viability was determined by the MTT method after a 6-day incubation period at 37 °C. Western blot analysis. H9 cells were cultured in cell culture plates (106 cells/mL) and treated with various concentrations of the test compound for 2 h. The cells were infected with HIV-1IIIB at a M.O.I. of 0.1 and incubated at 37 °C for 4 days. After the incubation period cells were centrifuged at 1000 rpm for 10 min. Following the harvest of supernatant, the cells were washed with cold PBS and lysed with 500 μL of lysis buffer (Sigma Chemical Co., St. Louis, MO, USA). The supernatant containing virus was centrifuged as described by Lee et al.22) Briefly, the virus was pelleted from supernatant by mixing with 30% polyethylene glycol (PEG) (50% v/v) in 0.4 M NaCl. The mixture was centrifuged for 45 min at 15,000 rpm. The viral pellet was lysed and used for immunoblot analysis.

Fig. 1.

Chemical structure of 8,4‴-dieckol from E. cava.

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Protein concentrations were determined using a protein assay kit (BioRad Laboratories, Hercules, CA). Cell or viral lysates were separated by denaturating SDS-PAGE in 25 mM Tris, 192 mM glycine, 0.1% SDS with a 4% stacking, and 10% separating gel. Separated proteins were blotted onto a nitrocellulose membrane and blocked in T-BST containing 5% skim milk powder. The membrane was subjected to mouse antip24 monoclonal antibody (1:500, SantaCruz) and horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (1:5000, SantaCruz), respectively. The antibodies were visualized by chemiluminescence (Fujifilm Life Science, Tokyo, Japan).

six-day incubation period at 37 °C. Nevirapine was used as a drug control.

p24 antigen capture ELISA. PBMCs or H9 cells were seeded in 24-well flat-bottomed microtiter culture plates (3 × 106 cells/mL). H9 cells were infected with HIV-1IIIB and HIV-1RTMDR1 whereas PBMCs were infected with HIV-1Ba-L at a M.O.I. of 0.1 for 1 h at 37 °C. After the infection, the cells were treated with/ without various concentration of the test compound or nevirapine and incubated for 7 days. Intracellular HIV1 p24 amount was quantitated with p24 antigen capture ELISA using a commercial kit (Perkin-Elmer Life Sciences, Boston, MA) according to the instructions of the manufacturer. The standard curve was prepared using p24 protein obtained from Perkin-Elmer Life Sciences (Boston, MA).

Luciferase gene reporter assay. The ability of 8,4‴-dieckol to inhibit HIV-1 replication was further investigated by luciferase gene reporter assay using TZM-bl cells. TZM-bl cells are HeLa cell clones that express CD4, CCR5 and CXCR4 constitutively. The cells contain integrated genes for luciferase and β-galactosidase under control of an HIV-1 LTR, and their susceptibilities to infection by both R5 and X4 tropic viruses are comparable to those of PBMCs. Anti-HIV-1 activity in TZM-bl cells were performed as described previously.23) Briefly, one day before infection, 5 × 104 cells were seeded in a 24-well flat bottomed microtiter plate to adhere. Cells were treated with various concentrations of 8,4‴-dieckol, infected with HIV-1 at a M.O.I. of 0.1 and maintained for 48 h at 37 °C and 5% CO2. After incubation, cells were washed with cold PBS and lysed in 300 μL of lysis buffer (Promega Corp., Madison, WI). Twenty microliters of cell lysate was mixed with 100 μL of luciferase substrate (Promega) and light emission was measured for 10 s in a luminometer (Tecan Austria GmbH, Austria).

Reverse transcriptase activity assay. In order to evaluate the inhibitory effect of 8,4‴-dieckol on HIV-1 reverse transcriptase enzyme, a commercial fluorescence RT assay kit (InvitroGen) was applied according to the manufacturer’s instructions. Briefly, viral particles from the infected culture supernatants were mixed with 30% PEG (50% v/v) in 0.4 M NaCl and pelleted by centrifugation at 15,000 rpm for 45 min. Five microliters of viral lysate was transferred to each well of a 96-well microtiter plate containing indicated concentrations of test compound or nevirapine as a positive control. Twenty microliters of reaction mixture containing a template/primer hybrid, poly(A)/d(T)16, and dTTP as a triphosphate substrate was added to the wells and the plate was incubated for 1 h at 37 °C. Two microliters of 200 mM EDTA was added to each well to terminate the polymerization reaction. Fluorescence intensity was measured at 480 nm (excitation) and 520 nm (emission) with a GENios® microplate reader (Tecan Austria GmbH, Austria) after the addition of 173 μL of fluorescent PicoGreen® reagent, which selectively binds to dsDNA or DNA–RNA heteroduplexes over single stranded nucleic acids or free nucleotides. Delayed addition of 8,4‴-dieckol to HIV-1IIIB infected cells. C8166 cells were cultured into individual wells of a 48-well plate (105 cells/mL). Diluted HIV-1IIIB stock supernatants were added to appropriate wells to yield a final M.O.I. of 0.1. At defined time periods following the infection, cells were treated with 50 μM of 8,4‴-dieckol. Cell viability was determined by the MTT method after a

Effect of 8,4‴-dieckol on cell–cell fusion. Uninfected C8166 cells (105 cells/mL) and H9 cells, chronically infected with HIV-1IIIB were incubated together in a flat-bottomed 48-well microtiter plate (ratio 10:1) in the presence of various concentrations of 8,4‴-dieckol or complete medium alone in a humidified atmosphere of 5% CO2 at 37 °C. The plate was incubated for 72 h and the number of syncytia was determined microscopically.

Statistical analysis. All experiments were carried out at least in triplicate and the results are reported as means ± standard deviation.

Results Effect of 8,4‴-dieckol on T-cell viability and syncytia formation 8,4‴-dieckol exhibited no cytotoxicity up to 500 μM and the CC50 value of 8,4‴-dieckol was higher than 1000 μM (Fig. 2(A)). Primarily, 8,4‴-dieckol was evaluated for its anti-HIV properties according to its potential to inhibit syncytia formation on C8166 cells infected with syncytia, inducing X4 tropic HIV-1 strains (HIV-1IIIB, HIV-1RF and HIV-1LAI). Microscopic image of HIV-1RF-induced syncytia inhibitory activity of 8,4‴-dieckol was shown in Fig. 2(B). Syncytia formation on C8166 cells was observed and recorded at the end of day 2 and day 4 after the primary infection with viral particles (Fig. 2(C) and (D)). The presence of 8,4‴-dieckol significantly inhibited syncytia formation in C8166 cells in a dose-dependent manner.

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Fig. 2. Cytotoxicity and syncytia inhibitory effect of 8,4‴-dieckol. Notes: (A) Cell viability assay on H9 cells. Cytotoxic effect of 8,4‴-dieckol on the H9 cells was determined by MTT formazan assay. The optical density of formazan formed by untreated cells was taken as 100% of viability. (B) Microscopic image of C8166 cells acutely infected with HIV1RF. C8166 cells were infected with HIV-1RF at a M.O.I. of 0.1 for 2 h. Infected cells were washed twice with PBS, followed by test compound treatment and incubated for 3 days. (I) HIV-1RF infected control, (II) 8,4‴ dieckol-50 μM, (III) Blank, (IV) Nevirapine-2 μM. The number of syncytia was counted at day 2 (C) and day 4 (D) using an inverted microscope (100X). C8166 cells were infected with HIV-1IIIB, HIV-1RF or HIV-1LAI at a M.O.I. of 0.1 for 2 h and unabsorbed viral particles were removed by washing with PBS twice. After treatment with/without 8,4‴-dieckol, cells were incubated for 4 days.

Protective effect of 8,4‴-dieckol on HIV-1-induced lytic effect Cytoprotective activity of 8,4‴-dieckol on C8166 cells from the lytic effect of HIV-1 was evaluated with a formazan-based cell viability assay (Fig. 3). According to the data obtained from cell viability assay, 8,4‴dieckol could successfully maintain cell viability and inhibit HIV-1 induced lytic effects significantly. A dose-dependent inhibition of HIV-1 infection of multiple isolates was exhibited by 8,4‴-dieckol and at the highest concentration of test compound treated, the cell viability was more than 90% at the end of six days. Effect of 8,4‴-dieckol on p24 antigen production Data obtained from syncytia formation and cytoprotective effect of 8,4‴-dieckol was consistent with the data obtained from p24 antigen capture ELISA and immunoblot analysis of p24 antigen. 8,4‴-dieckol effectively inhibited p24 antigen production on cells which were infected with X4 or R5 tropic viruses. Moreover, RTMDR1, a triple HIV-1 RT drug resistant virus which displays co-resistance to AZT, didanosine and nevirapine, p24 antigen production was inhibited at significant levels whereas nevirapine could not inhibit the

Fig. 3. Protective effect of 8,4‴-dieckol on C8166 cells acutely infected with HIV-1. Notes: C8166 cells were infected with HIV-1IIIB, HIV-1RF or HIV1LAI for 2 h and unadsorbed viral particles were removed by washing. Cells were treated with indicated concentrations of 8,4‴-dieckol and incubated for 6 days. Cell viability was determined by MTT assay 6 days after initial infection. Nevirapine (2 μM) was used as a positive control.

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p24 on H9 cells infected with RTMDR1 (Fig. 4(A)). In addition to p24 antigen capture ELISA data, inhibition of p24 protein production was further supported with data from immunoblot analysis. Immunoblot data indicated a dose-dependent inhibition of p24 protein produced by H9 cells acutely infected by HIV-1IIIB (Fig. 4(B)). Effect of 8,4‴-dieckol on viral infection Inhibitory effect of 8,4‴-dieckol on viral infection was also determined by measuring the virus-induced luciferase activity in infected TZM-bl cells (Fig. 5(A)). The replication of multiple HIV-1 strains was repressed when 8,4‴-dieckol was introduced to the cell culture. At the highest concentration of 8,4‴-dieckol treated, the luciferase production in TZM-bl cells was inhibited more than 80% for all viral strains except for RTMDR1 which was occurred at the ratio of 76.33%. Anti-HIV-1 mechanism of 8,4‴-dieckol Further experiments were carried out to better understand the anti-HIV-1 mechanism of 8,4‴-dieckol. As shown in cell–cell fusion experiments (Fig. 5(B)), a significant inhibition of syncytia formation could not be observed. However, a similar inhibition profile to nevirapine, which is an inhibitor of HIV-1 reverse transcriptase enzyme, was obtained when the addition of 8,4‴-dieckol to the cell culture was delayed (Fig. 6). Subsequently, the inhibitory effect of 8,4‴-dieckol on reverse transcriptase enzymes of several HIV-1 isolates was investigated (Fig. 7). 8,4‴-dieckol significantly inhibited the activity of reverse transcriptase enzyme dose-dependently. 8,4‴-dieckol inhibited 91% activity of HIV-1IIIB RT enzyme and approximately 80% for rest of the HIV-1 strains tested. Furthermore, the RT enzyme activity of HIV-1RTMDR1 strain was inhibited at a ratio of 76.1% while nevirapine did not exhibit any

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significant inhibitory activity on HIV-1RTMDR1 reverse transcriptase.

Discussion In the present study, anti-HIV-1 properties of 8,4‴dieckol, a phlorotannin derivative obtained from marine brown alga E. cava, was investigated. 8,4‴-dieckol was shown to be an effective compound against multiple HIV-1 strains. 8,4‴-dieckol inhibited HIV-1 induced syncytia formation and exhibited significant HIV-1 RT enzyme inhibitory activity. Moreover, it showed significant cytoprotective effect on C8166 cells at noncytotoxic concentrations. Numerous polyphenolic compounds have gained attention due to their structural diversity as well as their significant anti-HIV properties and a considerable number of them have been identified as HIV inhibitors. However, cell toxicity and exhibiting poor inhibitory activity against replicating virus were reported as their main drawbacks.24) Yu et al.25) screened polyphenolic compounds such as gallotannins, ellagitannins, condensed tannins, complex tannins, and other related compounds for anti-HIV RT activity, in which some of the tannins exhibited unique RT inhibitory activities. The tannins are believed to exhibit their mode of anti-HIV action by inhibiting polymerase and ribonuclease activity of HIV reverse transcriptase enzyme.25,26) Phlorotannins are tannin derivatives which contain several phloroglucinol units linked to each other in different ways. So far, phlorotannins are mostly isolated from red and brown alga.27) Numerous bioactivities of phlorotannins have been reported up to date such as antioxidant, anti-inflammatory,28) antibacterial,29) and anti-MMP activities.30) Furthermore, Ahn et al.31) claimed that phloroglucinol derivative 8,8’-bieckol inhibited the activity of recombinant RT and protease

Fig. 4. Effect of 8,4‴-dieckol on HIV-1 p24 production. Notes: (A) Determination of p24 protein in culture supernatant. H9 cells infected with HIV-1IIIB or HIV-1RTMDR1 and PBMCs were infected with HIV-1Ba-L at a M.O.I. of 0.1. After sample or Nevirapine (2 μM) treatment, the cells were incubated for 7 days. Amount of p24 antigen production was determined with p24 antigen capture ELISA. (B) Immunoblot analysis of HIV-1 p24 antigen production. H9 cells infected with HIV-1IIIB for 7 days. Mouse anti-p24 antibody was used to detect p24 protein in the cells and cell culture supernatant. Nevirapine (2 μM) was used as a positive control.

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Fig. 7. HIV-1 reverse transcriptase enzyme inhibitory activity of 8,4‴-dieckol. Notes: The effect of 8,4‴-dieckol on HIV-1RT activity inhibition was determined by fluorescent RT activity assay. Nevirapine (2 μM) was used as a drug control.

Fig. 5. Inhibition of gene-based HIV-1 replication and cell–cell fusion by 8,4‴-dieckol. Notes: (A) Inhibitory effect of 8,4‴-dieckol on viral infection. TZM-bl cells infected with HIV-1IIIB, HIV-1RF, HIV-1LAI, HIV-1RTMDR1 or HIV-1Ba-L at a M.O.I. of 0.1. After sample treatment, the cells were incubated for 2 days. The inhibitory effect of 8,4‴-dieckol on HIV-1 was detected with the inhibition of luciferase amount produced by infected cells. (B) Effect of 8,4‴-dieckol on cell–cell fusion. Uninfected C8166 cells were co-cultured with H9 cells chronically infected with HIV-1IIIB (Ratio-10:1) in the presence of various concentrations of 8,4‴-dieckol and incubated for 72 h. The number of syncytia was determined microscopically (100X). Dextran Sulfate (DS-100 μg/mL) was used as a positive control.

Fig. 6. Delayed addition of 8,4‴-dieckol to the cell culture. Notes: C8166 cells were infected with HIV-1IIIB at a M.O.I. of 0.1, treated with 50 μM of 8,4‴-dieckol and incubated for 6 days. Cell viability was determined by MTT assay 6 days after initial infection. Nevirapine (2 μM) was used as a positive control.

of HIV-1 in vitro. Artan et al.32) reported the enhanced HIV-1 inhibitory effect of a phloroglucinol derivative, 6,6′-bieckol, from E. cava. As indicated in Figs. 2 and 3, 8,4‴-dieckol significantly inhibited HIV-1 induced syncytia formation and exhibited cytoprotective effect on C8166 cells. Three X4 tropic isolates of HIV were tested and all of them were successfully inhibited by 8,4‴-dieckol at similar levels. However, the number of syncytia in infected C8166 cells was recorded to be increasing by time which demonstrates that the inhibitory activity of 8,4‴dieckol is lost by time, and it should be reintroduced to the cell culture. The inhibition of replication of HIV was further investigated by virus-induced luciferase activity assay. According to data obtained from this experiment, the replication of multiple strains of HIV including a triple-drug-resistant virus, HIV-1RTMDR1, was inhibited by 8,4‴-dieckol. Production of p24 antigen is an important indicator of viral replication on infected cells. The inhibitory effect of 8,4‴-dieckol on viral p24 protein production was determined by p24 antigen capture ELISA and immunoblot analysis (Fig. 4). The amount of p24 protein produced by HIV-1Ba-L, HIV-1IIIB, and HIV1RTMDR1 was effectively suppressed by 8,4‴-dieckol. The immunoblot data of HIV-1IIIB p24 core protein was consistent with the data obtained from ELISA (Fig. 4(A)). Production of p24 protein from both cell and culture supernatant was inhibited by 8,4‴-dieckol. In order to illustrate the mode of action of 8,4‴-dieckol, further experiments were conducted. To investigate the entry step of HIV-1, cell–cell fusion experiment was carried and the number of syncytia was recorded. Uninfected C8166 cells were incubated with H9 cells chronically infected with HIV-1IIIB, and 8,4‴-dieckol did not exhibit significant syncytia formation (Fig. 5(A)). As shown in delayed addition of 8,4‴-dieckol experiments, the compound effectively inhibited HIV-1 replication when it was treated up to six hours postinfection (Fig. 5(B)). An RT inhibitor, nevirapine exhibited a similar HIV-1 inhibitory effect in delayed-addition

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experiments. Nevirapine and 8,4‴-dieckol showed significant anti-HIV-1 activity when treated as late as 6 h post-infection. This data suggested that 8,4‴-dieckol may inhibit HIV-1 replication by targeting reverse transcriptase enzyme. The anti-HIV-1 RT activity of 8,4‴dieckol was investigated by using isolated reverse transcriptase enzymes from several strains. 8,4‴-dieckol was shown to be an effective inhibitor of HIV-1 RT according to the data obtained from reverse transcriptase activity assay. Reverse transcriptase enzymes of both R5 and X4 tropic viruses were significantly inhibited by 8,4‴-dieckol. More importantly, 76.1% activity of HIV-1RTMDR1 reverse transciptase, which is resistant to AZT, didanosine and nevirapine, was also inhibited by 8,4‴-dieckol at a concentration of 50 μM. These results clearly demonstrate that 8,4‴-dieckol possesses the ability to inhibit the replication of HIV-1 isolates with common drug-resistance mutations to RT inhibitors. The results using drug-resistant isolate suggest that 8,4‴-dieckol shows anti-HIV RT effect possibly through binding the RT sites in different locations or conformations than that of Nevirapine. In conclusion, 8,4‴-dieckol is a tannin derivative exhibiting relatively lower cytotoxicity in comparison to other polyphenols and significant HIV-1 reverse transcriptase inhibition activity. In addition, the ability to act against drug-resistant HIV-1 strains makes 8,4‴-dieckol a promising lead compound for further development of novel anti-HIV-1 agents with enhanced efficiency compared to on-the-market drugs.

Acknowledgments This research was supported by a grant from the Marine Bioprocess Research Center of the Marine Biotechnology Program funded by the Ministry of Oceans and Fisheries, Republic of Korea.

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Anti-HIV-1 activity of phlorotannin derivative 8,4‴-dieckol from Korean brown alga Ecklonia cava.

8,4‴-dieckol is a natural product which has been isolated from brown alga, Ecklonia cava. This polyphenolic compound is a phlorotannin derivative with...
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