Mem Inst Oswaldo Cruz, Rio de Janeiro, 103(5) August 2008
Anti-HSV-1 and anti-HIV-1 activity of gallic acid and pentyl gallate
ILaboratório de Virologia Aplicada, Departamento de Ciências Farmacêuticas
IILaboratório de Síntese e Estrutura-Atividade, Departamento de Química
IIILaboratório de Virologia Aplicada, Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Catarina, Campus Universitário Trindade, 88040-900 Florianópolis, SC, Brasil
IVDepartamento de Virologia Clínica, Universidade de Göteborg, Suécia
VLaboratório de Imunologia Clínica, Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro, RJ, Brasil
VILaboratório de Virologia Molecular, Departamento de Biologia Celular e Molecular, Universidade Federal Fluminense, Niterói, RJ, Brasil
The synthetic n-alkyl esters of gallic acid (GA), also known as gallates, especially propyl, octyl and dodecyl gallates, are widely employed as antioxidants by food and pharmaceutical industries. The inhibitory effects of GA and 15 gallates on Herpes Simplex Virus type 1 (HSV-1) and Human Immunodeficiency Virus (HIV-1) replication were investigated here. After a preliminary screening of these compounds, GA and pentyl gallate (PG) seemed to be the most active compounds against HSV-1 replication and their mode of action was characterized through a set of assays, which attempted to localize the step of the viral multiplication cycle where impairment occurred. The detected anti-HSV-1 activity was mediated by the inhibition of virus attachment to and penetration into cells, and by virucidal properties. Furthermore, an anti-HIV-1 activity was also found, to different degrees. In summary, our results suggest that both compounds could be regarded as promising candidates for the development of topical anti-HSV-1 agents, and further studies concerning the anti-HIV-1 activity of this group of molecules are merited.
Herpes Simplex Virus type 1 (HSV-1) is an enveloped DNA virus that causes one of the most common viral infections in humans, leading to a variety of diseases ranging from mild to severe and sometimes life-threatening (White & Fenner 1994, Whitley & Rozman 2001). Although several nucleoside analogues have been approved for clinical use, such as acyclovir, immunocompromised patients are at increased risk of severity and recurrent infections, since resistant strains have recently been observed (Brady & Bernstein 2004). Therefore, it is desirable to develop new antiviral agents in order to substitute or complement currently available drugs.
The synthetic n-alkyl esters of gallic acid (GA), also known as gallates, especially propyl, octyl and dodecyl gallates, are widely employed as antioxidants by the food and pharmaceutical industries (van der Heijden et al. 1986, Kubo et al. 2002a). Besides the antioxidant activity, other biological activities have been described for this group of molecules, mainly anticancer mechanisms (Fiuza et al. 2004, Kitagawa et al. 2005, Frey et al. 2006, Veluri et al. 2006) as well as antibacterial and antifungal properties (Fujita & Kubo 2002, Kubo et al. 2002b, c, 2003, 2004, Stapleton et al. 2004). However, there are few reports about the antiviral activity of these compounds. In 1988, a study described the inhibition of HSV-1 and HSV-2 replication by methyl gallate (Kane et al. 1988). In 2002, as part of the screening of phenolic compounds against HIV-1 integrase, GA was found to be active (Ahn et al. 2002). More recently, several biological activities of a group of gallates were described by our research group, and various structure-activity relationships regarding their anti-HSV-1, antioxidant and genotoxic effects were proposed (Savi et al. 2005). Furthermore, the pronounced anti-HSV-1 activity of octyl gallate, and its inhibitory effect against RNA viruses were also recently described (Uozaki et al. 2006, Yamasaki et al. 2007).
In the present study, GA and 15 gallates were re-evaluated for anti-HSV-1 and anti-HIV-1 activities, followed by selection of the most active anti-HSV-1 compounds and the determination of the viral multiplication step(s) upon which these compounds act.
MATERIALS AND METHODS
Compounds - GA and 15 gallates (with increasing number of carbons in the alkyl chain; Fig. 1) were synthesized as previously described (Savi et al. 2005). The compounds (50 mM) were dissolved in dimethyl sulphoxide, stored at -20°C protected from light, and further diluted in culture medium prior to use. Acyclovir was purchased from Sigma (St. Louis, USA).
Cells and viruses - For the anti HSV-1 activity screening, Vero cells were grown in minimum essential medium (MEM; Cultilab, São Paulo, Brazil) supplemented with 10% fetal bovine serum (FBS; Cultilab, São Paulo, Brazil), penicillin (100 U/ml), streptomycin (100 µg/ml) and amphotericin B (25 µg/ml; Cultilab, São Paulo, Brazil). Cell cultures were maintained at 37°C in a humidified 5% CO2 atmosphere. The HSV-1 strain KOS (Faculty of Pharmacy, University of Rennes, France) was propagated in Vero cells, while stock viruses were prepared as previously described (Simões et al. 1999). After three cycles of freezing/thawing, the fluids were titrated on the basis of PFU count as previously described (Burleson et al. 1992) and stored at -80°C until use. Purified extracellular HSV-1 particles were obtained as previously described (Karger & Mettenleiter 1993), with minor modifications. The specific activity was 1.29 x 10-2 (counts per minute [cpm]/PFU).
For the anti-HIV-1 activity screening, peripheral blood mononuclear cells (PBMCs) from healthy human donors were obtained by density gradient centrifugation (Histopaque; Sigma, St. Louis, USA) from buffy coat preparations. PBMCs were resuspended in RPMI 1640 (LGC Bio, São Paulo, Brazil) supplemented with 10% FBS (Hyclone, Logan, USA), penicillin and streptomycin (Cultilab, São Paulo, Brazil), 2 mM glutamine and 10 mM HEPES (Sigma, St. Louis, USA), stimulated with 5 mg/ml of phytohemagglutinin (PHA; Sigma, St. Louis, USA) for two to three days, and further maintained in culture medium containing 5 U/ml of recombinant human interleukin-2 (Sigma, St. Louis, USA). HIV-1 isolate Ba-L (R5-tropic, subtype B; Cirne-Santos et al. 2008) was used to infect cells. Virus isolates were prepared in PHA-activated PBMCs from human healthy donors.
Cytotoxicity evaluation - Confluent cells were exposed to different concentrations of compounds for 72 h. After incubation, cell viability was assessed by a MTT [3-(4,5-dimethylthiazol-2,5-diphenyl tetrazolium bromide] assay (Mosmann 1983). The 50% cytotoxic concentration (CC50) was defined as the concentration (µM) that reduced cell viability by 50% when compared to untreated controls.
Screening of in vitro anti-HSV-1 activity - Confluent Vero cells were infected with HSV-1 at a MOI of 0.5 and treated with non-cytotoxic concentrations of each compound for 72 h. Acyclovir (10 µM) was used as a positive control. The same method used to evaluate cell viability with MTT was followed with appropriate modifications (Takeuchi et al. 1991), and the 50% inhibitory concentration (IC50) was defined as the concentration (µM) that reduced the absorbance of infected cells to 50% when compared to untreated controls.
Viral plaque number reduction assay - This assay followed the procedures previously described (Kuo et al. 2001) with minor modifications. Acyclovir (10 µM) was used as a positive control. Approximately 100 PFU of HSV-1 were adsorbed for 1 h at 37°C on confluent Vero cells. Cultures were then washed twice with PBS and overlaid with MEM containing 1.5% carboxymethylcellulose and different concentrations of compounds. After 72 h, cells were fixed and stained with naphtol blue-black and plaques were counted. The IC50 was defined as the concentration (µM) that inhibited 50% of viral plaque formation when compared to untreated controls.
Virucidal assay - This assay followed procedures that have been previously described (Ekblad et al. 2006). Mixtures of GA or pentyl gallate (PG) and 4.0 x 104 PFU of HSV-1 in serum-free MEM were co-incubated for 20 min at 37°C in a water bath prior to the dilution of the mixture to non-inhibitory concentrations of compound (1:100), and the residual infectivity was determined by a viral plaque number reduction assay, as described above.
Attachment and penetration assays - The attachment assay followed procedures that have been described previously (Ekblad et al. 2006) with minor modifications. Different concentrations of GA or PG were mixed with purified radiolabelled HSV-1 and incubated for 15 min at 37°C. Then, the virus-compound mixtures were adsorbed for 2 h at 4°C on confluent cells. Subsequently, cultures were washed twice with PBS supplemented with 1 mM CaCl2 and 0.5 mM MgCl2 (PBS-A) and lysed with PBS-A containing 5% sodium dodecyl sulphate (SDS), after which the lysates were transferred to scintillation vials for radioactivity quantification. The penetration assay followed the procedures previously described (Cheng et al. 2004) with minor modifications. Approximately 100 PFU of HSV-1 were adsorbed for 2 h at 4°C on confluent cells, washed twice with PBS and incubated in the presence or absence of different concentrations of GA or PG at 37°C to maximize the penetration of viruses. After 10 min, cultures were treated for 1 min with warm citrate-buffered saline (pH 3) to inactivate the unpenetrated viruses and the inhibitory activity of the compounds was determined by a viral plaque number reduction assay.
Western blotting analysis - This assay followed the procedures described previously (Kuo et al. 2001) with minor modifications. Confluent cells were infected with HSV-1 at a MOI of 0.1 and after 1 h of adsorption at 37°C, cultures were incubated in the presence or absence of 125 µM of GA or PG. At 18 h post-incubation, cellular proteins were extracted, dissolved (~50 µg; determined by the Bradford procedure) in the dissociation buffer (2% SDS, 5% b-mercaptoethanol, 0.125 M Tris-HCl, 30% glycerol, 0.8% bromophenol blue, and 100 µg/ml phenylmethylsulfonyl fluoride; Sigma, St. Louis, USA) and boiled for 5 min. The proteins were then resolved by 10% SDS-PAGE and transferred to Immobilon PVDF® membranes (Millipore, Billerica, USA). Membranes were blocked overnight with 10% nonfat dry milk in Tris-Base saline buffer, and then incubated with the following antibodies: mouse monoclonal antibodies raised against HSV-1 g proteins gD (Santa Cruz Biotechnology, Santa Cruz, USA) and gC (Sjogren-Jansson & Jeansson 1985, 1990); rabbit polyclonal antibody raised against HSV-1 g protein VP5 (kindly provided by G Cohen and R Eisenberg, University of Pennsylvania, Philadelphia); and goat polyclonal antibody raised against HSV-1 a protein ICP27 (Santa Cruz Biotechnology, Santa Cruz, USA). Specific reactive proteins were detected by a diaminobenzidine (DAB) colorimetric reaction, after incubation with rabbit anti-mouse, swine anti-rabbit or donkey anti-goat immunoglobulin secondary antibodies, respectively, linked to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, USA).
Drug combination assay - This assay followed procedures that have been previously described (Gong et al. 2004) with minor modifications. A viral plaque number reduction assay was performed as described above, and the treatment consisted of GA, PG or acyclovir alone (0.125 x IC50, 0.25 x IC50, 0.5 x IC50, 1 x IC50, 2 x IC50), and a mixture of varying concentrations of each compound in a fixed ratio (i.e., 0.5 x IC50 of GA + 0.5 x IC50 of acyclovir; 0.25 x IC50 of GA + 0.25 x IC50 of PG). Based on inhibitory activities, the synergistic or antagonistic antiviral effect among compounds was determined using the software MacSynergy II (kindly provided by Mark Prichard, University of Michigan, USA), through a three-dimensional analytical method (Prichard et al. 1993).
Screening of in vitro anti-HIV-1 activity - This assay followed the procedures described previously (Cirne-Santos et al. 2008) with minor modifications. Peripheral blood mononuclear cells (PBMCs) were initially exposed to viral suspensions containing 5-10 ng/ml of HIV-1 p24 Ag during 2-3 h. Cells were washed, resuspended in complete medium, plated in 96-well culture plates (2.0 x 105 cells/well) in triplicate, and treated with 50 µM of GA and gallates. After seven days at 37°C in 5% CO2, viral replication was assessed by measuring the HIV-1 p24 Ag presence in culture supernatants by an ELISA capture assay (ZeptoMetrix Co., Buffalo, USA).
Statistical analysis - The mean values ± standard deviations are representative of three independent experiments. For the determination of CC50 and IC50 values, non-linear regressions of concentration-response curves were used.
Cytotoxicity evaluation and anti-HSV-1 activity - GA and 15 gallates were evaluated for their cytotoxicity and anti-HSV-1 activity in Vero cells by the MTT assay. Considering the selectivity indices (SI) obtained in this preliminary screening, the most active compounds were: GA (39.1), methyl (3.5), ethyl (6.3), propyl (5.1), butyl (5.4) and pentyl (9.2) gallates. These compounds were selected to have their antiviral activity confirmed by a viral plaque number reduction assay. For these purposes, acyclovir was used as a positive control. The results of the antiviral activity confirmation are summarized in Fig. 1. As shown, GA and PG presented lower IC50 values among the compounds evaluated and at the highest concentrations tested (125 µM), only these two compounds thoroughly inhibited HSV-1 replication. Thus, the following study was focused on these compounds.
Effect of GA and PG on HSV-1 attachment and penetration - To elucidate whether the anti-HSV-1 activity of GA and PG was related to the blockage of the early events of viral infection, their effects on viral attachment to and penetration into cells were investigated. Fig. 2 shows the observed results, for which both compounds were active: GA presented a greater effect in the attachment assay (Fig. 2A), with an IC50 value of 23.9 ± 9.4 µM, and PG was more active in the penetration assay (Fig. 2B), with an IC50 value of 9.1 ± 3.2 µM. Furthermore, in order to identify if the cells or the virus particles were affected, cells were pre-treated (3 h) with compounds. The compounds were then washed out, and thereafter cells were infected with 100 PFU of HSV-1. The results showed that the pre-treatment of cells with both compounds did not reduce HSV-1 infectivity (data not shown), suggesting that the virus particles were targeted.
Virucidal activity of GA and PG - The virus-inactivating activity of these compounds in the absence of cells was evaluated (Fig. 3). The co-incubation of 4.0 x 104 PFU of HSV-1 with different concentrations of compounds followed by the dilution of the mixture to non-inhibitory concentrations revealed that both GA and PG caused complete inactivation of HSV-1 infectivity at relatively low IC50 values.
Effect of GA and PG on HSV-1 protein synthesis - We analyzed whether GA and PG inhibition of HSV-1 replication was related to the blockage of viral protein synthesis. Accordingly, the expression of ICP27, gC, gD and VP5 viral proteins in infected Vero cells was also evaluated. As shown in Fig. 4, while uninfected cells (Lane 1) did not express these proteins, all were detectable in HSV-1 infected cells (Lane 2). Treatment with GA (Lane 3) and PG (Lane 4) suppressed the expression of these proteins. These results showed that both GA and PG inhibited HSV-1 replication when cells were already infected.
Effect of GA and PG in combination with acyclovir - The treatment of infected cells with a mixture of GA or PG and acyclovir revealed that neither a synergistic nor an antagonistic antiviral effect among compounds was observed (data not shown).
Effect of GA and other gallates on HIV-1 multiplication - In addition, GA and 15 gallates were screened for anti-HIV-1 activity. As shown in Table, the higher percentages of inhibition were found to be among the compounds with no more than five carbons in the alkyl moiety. A similar association between the size of the alkyl moiety and the antiviral activity was found for HSV-1.
In a previous study, our research group described some biological effects of GA and its alkyl esters (gallates). Although structure-activity relationships regarding the anti-HSV-1, antioxidant, and genotoxic effects were proposed, the steps of viral replication affected by these compounds were not determined (Savi et al. 2005). In this study, GA and 15 gallates were evaluated for their in vitro anti-HSV-1 activity by a more robust methodology (viral plaque assay), and the best candidates had their mode of action studied. The screening process started with the evaluation of cytotoxic effects of these compounds in Vero cells followed by the determination of their antiviral activity by MTT assay. GA and gallates with as much as five carbons in the alkyl chain presented the optimum anti-HSV-1 activity (higher SI values) among the evaluated compounds, and therefore, their anti-HSV-1 activity was confirmed by a viral plaque number reduction assay (Fig. 1), in which GA and PG were chosen due to their higher percentages of HSV-1 inhibition.
Therefore, the anti-HSV-1 activity of GA and PG was scrutinized. The combination of GA and PG with acyclovir resulted in no interaction (data not shown). The pretreatment of cells with GA and PG did not affect viral infectivity (data not shown). Thus, neither GA nor PG affects the viral infection process through their binding to cell membrane molecules. However, the addition of compounds concomitantly with the virus, at specific conditions, revealed that both compounds affect virus attachment to and penetration into cells (Fig. 2). These observations suggest that GA and PG affect HSV-1 infectivity possibly by detaching viruses that have already bound to cells, perhaps through the disturbance of viral glycoproteins.
In order to verify these findings, the virucidal activity of compounds was evaluated. After 20 min of co-incubation at 37°C, both compounds caused complete inactivation of HSV-1 in the absence of cells at low IC50 values (Fig. 3). The direct virucidal activity was also found for other gallate derivatives, such as epigallocatechin-3-gallate and octyl gallate (Song et al. 2005, Uozaki et al. 2006).
Subsequently, we examined the effects of both compounds when the viruses have already penetrated into cells and started their replication. The results showed that the expression of ICP27, gC, gD, and VP5 proteins was inhibited in infected cells treated with GA and PG (Fig. 4). To clarify these results and eliminate any possibility of cytotoxicity of compounds on infected cells, the viability of infected Vero cells treated with GA and PG was evaluated by a trypan blue dye exclusion method. The results showed that viability was not substantially affected when compared to untreated infected controls (data not shown).
These results suggested that the inhibition of the expression of viral proteins by GA and PG may be attributed to their virucidal effect on the new progenies of virus, avoiding the cell-to-cell spread of infection.
During the course of our studies, Uozaki et al. (2006) described anti-HSV-1 activity of octyl gallate with a moderate cytotoxicity. In our study, the cytotoxicity presented a similar profile, although the antiviral activity was found only for the gallates with as much as seven carbons in the alkyl moiety. The discrepancies between the results of these studies may come from the different cell lines employed and methodologies used, as already supposed by the authors (Uozaki et al. 2006).
The anti-HIV-1 activity of GA and gallates was also screened (Table I). The cytotoxicity of GA and gallates on PBMCs did not present a clear concentration-response pattern, with CC50 values higher then 1,000 µM (except GA), while the cytotoxic effects on Vero cells were superior and increased along with the number of carbons in the alkyl moiety, reaching a maximum effect at the compound with 11 carbons (undecyl gallate; data not shown). In relation to anti-HIV-1 activity, the results showed that GA and gallates with no more than five carbons in the alkyl moiety presented higher percentages of viral replication inhibition (³ 78%). A similar association between the size of the alkyl moiety and the antiviral activity was found for HSV-1, suggesting that the lipophilicity of these molecules may be involved in their biological properties, as already suggested (Rosso et al. 2006).
In summary, the anti-HSV-1 activity of GA and PG was attributed to the inhibition of virus attachment to and penetration into cells, as well as to their virucidal effects. These results suggest that both compounds could be regarded as promising candidates for the development of topical anti-HSV-1 agents, and further studies concerning the anti-HIV-1 activity of this group of molecules are merited.
To the Hemotherapy Service of the Hospital Clementino Fraga Filho (UFRJ), for providing buffy coats.
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