Inhibition of Herpes Simplex Viruses by Cationic Dextran Derivatives

Article Cite This: J. Med. Chem. 2017, 60, 8620-8630

pubs.acs.org/jmc

Inhibition of Herpes Simplex Viruses by Cationic Dextran Derivatives Magdalena Pachota,†,‡ Katarzyna Klysik,§ Aleksandra Synowiec,† Justyna Ciejka,‡,§ Krzysztof Szczubiałka,§ Krzysztof Pyrć,†,‡ and Maria Nowakowska*,§ †

Microbiology Department, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland ‡ Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7A, 30-387 Krakow, Poland § Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland S Supporting Information *

ABSTRACT: Human herpesviruses are among the most prevalent pathogens and currently there are no drugs available that could cure diseases induced by them. The most widely utilized antiherpes drugs, acyclovir and its derivatives, have serious limitations, such as low bioavailability and severe side effects. The current paper reports on the synthesis and characterization of cationic dextran derivatives (DEXxDSy) of various molecular weights and various degrees of substitution with ammonium groups, which were tested as antiherpes agents. DEXxDSy showed high effectiveness against HSV-1 and HSV-2 viruses, as found using a variety of techniques. Importantly, no toxicity was observed for these compounds in the range of active concentrations, demonstrating their potential as antivirals. The mechanism of action of DEXxDSy was assessed. We hypothesize that they may limit virus transmission, as extensive examination showed that they hamper the interaction between the virus and the cellular attachment receptor.

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INTRODUCTION

Primary HSV infection begins with epithelial cells, usually oral or ocular for HSV-1 and genital for HSV-2. The virus is easily spread through direct contact, that is, kissing or sexual intercourse. After a productive infection in the mucosal tissue, HSV proceeds to enter nerve endings and, employing retrograde transport, makes its way to the dorsal root neuron nuclei, where it establishes latency, a state of lifelong infection. Latency, a hallmark of the Herpesviridae family, is facilitated by a failure of immediate early gene transcription caused by ineffective axonal transport of viral regulation factors, such as VP16 and IFN-α activity in sensory neurons.9,10 Once the virus reaches the latent state, the infection seems to be incurable and may be reactivated, either spontaneously or by stress stimuli.11 The entry of virus into host cells is the first and critical step in the viral pathogenesis.12,13 It is mediated by an interplay of cellular and viral proteins, which differ in the number and characteristics between viral species. HSV-1 encodes several glycoproteins responsible for cell surface binding and virus entry. Glycoproteins gB and gC are responsible for initial binding of the virus at the cell surface, involving heparan sulfate proteoglycans (HSPGs). Virions devoid of these glycoproteins demonstrate significantly impaired infectivity.12 gB was also found to bind PILR-α, a particle considered to be a HSV-1 coreceptor.14 Glycoprotein D (gD) is considered to be the main entry mediator and several receptors for gD have been identified. The most important are nectin-1 and nectin-2,

Herpes simplex viruses type 1 and 2 (HSV-1 and HSV-2, respectively) belong to the Herpesviridae family encompassing more than 100 viruses, eight of which affect humans.1 They are also widespread in the human population, as it is estimated that 50−90% of the population worldwide may be infected.2 HSV-1 is mainly associated with watery blisters in the mouth area (lips, tongue, oral mucosa), while HSV-2 mostly affects the genital region.3,4 Both of them are, however, also associated with potentially fatal viral encephalitis and stromal keratitis, an ocular disease, which is a leading cause of cornea-derived blindness in developed countries.5−7 The HSV virion is a spherical, complex structure, about 200 nm in diameter. The inner core, containing the viral DNA stabilized by polyamines, is encapsulated by an icosahedral capsid, which is further coated with an unstructured protein layer, called the tegument. The whole structure is surrounded by a lipid bilayer decorated with viral surface glycoproteins. HSV is a linear dsDNA virus, with genome of 152 and 155 kbp for HSV-1 for HSV-2, respectively. Two major genomic regions, L (long) and S (short), both flanked with inverted repeats, may be distinguished. The HSV genome encodes for at least 90 transcriptional units, which are sorted into 3 classes: α, immediate early; β, early; and γ, late genes. The α genes are located in the inverted repeats of both L and S components and encode for proteins regulating expression of early and late genes. The β and γ genes are distributed within the L and S region.8 © 2017 American Chemical Society

Received: August 19, 2017 Published: September 28, 2017 8620

DOI: 10.1021/acs.jmedchem.7b01189 J. Med. Chem. 2017, 60, 8620−8630

Journal of Medicinal Chemistry

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Scheme 1. Synthesis of the Cationic Derivatives of DEX

highly cationic polyethylenimine, SP-012, that provided a protective effect against genital herpes caused by HSV-1 and HSV-2 in mice after topical application with magnitude of the responses comparable to that of ACV.34 The inhibitory effects of polyanionic substances on the replication of HSV were reported already five decades ago;35,36 however, the antiviral action of the compounds was considered to be largely nonspecific.37 The examples of polyanions that inhibit HSV were dendrimers containing sulfonic groups,38 poly(sodium styrenesulfonate) (PSSS)39 and its copolymer with maleic acid,40 poly(methacrylic acid) functionalized with ribavirin,41 and telomerized ω-acryloyl anionic surfactants.42 Natural polymers having anti-HSV activity include polyphenols from almond peel,43 sulfated lignins,44 and a variety of sulfated polysaccharides such as λ-carrageenans, sulfated cellulose nanocrystals (CNC), xylofuranan sulfate, ribofuranan sulfate, dextran sulfate, galactan sulfate, xylomannan sulfate, heparin, pentosan polysulfate, and fucoidan.45−47 Polymer-based hybrid coatings containing silver, zinc, and copper48 or obtained from thermally reduced graphene (TRGO) functionalized with dendrimeric polyglycerol sulfate (dPGS)49 were also found to bind and neutralize HSV. In the current paper, we present the results of our studies on antiviral activity of cationic dextran derivatives. A large library of cationic dextran derivatives differing in molecular weight and degree of substitution was synthesized and shown to actively block virus infection in vitro. Detailed study on the mechanism of action revealed that these compounds blocked very early stage of virus infection, that is, anchoring of virions on the cell surface. Careful analysis allowed us to select the effective polymeric inhibitor that was only sparingly internalized by the cells.

herpesvirus entry mediator (HVEM), and 3-O sulfated HSPG.15−18 The in vivo experiments carried out using single and double knockout mice have demonstrated the dependence of HSV-2 on nectin-119 and HSV-1 on HSPG receptor12 for their entry to the cells and spread. Interestingly, the in vitro experiments carried out using human corneal fibroblasts have shown that HSV- 2, unlike HSV-1, does not use the HSPG receptor for cell penetration.20 Two other glycoproteins taking part in HSV-1 entry, gH and gL, form a heterodimer, which dissociates upon binding to its receptor, αvβ6- or αvβ8-integrin. This interaction induces a change in gH conformation, which allows virus entry into the cell and directs the virus to acidic endosomes.21 No effective HSV vaccine is available, and therefore current research is oriented toward the development of antiviral compounds limiting the primary infection and supporting further treatment. Currently, the approved anti-HSV therapies involve mainly nucleoside analogues, such as acyclovir (ACV), which interfere with viral DNA synthesis reducing virus replication and shedding. Low bioavailability of this drug led to the development of derivatives with better pharmacokinetic parameters, for example, selenoacyclovir, valacyclovir, famciclovir, and ganciclovir.22−24 Most of these drugs undergo bioconversion within the cell to ACV. An alternative for treatment of some ACV-resistant HSV infections is foscarnet, which hampers viral polymerase activity by binding to the pyrophosphate binding site.25 All mentioned compounds are oriented on the same molecular target, and therefore some cross resistance is observed.26,27 For these reasons, there is a need for the development of novel antiherpes drugs with different mechanism of action. The site of entry of herpesviruses seems to be a promising target, as inhibition of early steps of the virus infection may limit not only infection but also virus transmission. In the current study, we made an effort to develop a nontoxic cationic polymer that could prevent virions from entering the cell via formation of multivalent interactions either with the virion (direct inactivation) or with the cell. Application of polymers for inhibition of HSV infection was proposed before and involved synthetic polycations and polyanions, as well as peptides/proteins. The examples of polycations showing anti-HSV activity include cationic polysaccharides conjugated with oligoamines via reductive amination,28 poly(L-lysine) and poly(L-arginine) derivatives, which inhibited early stages of HSV replication,29 poly(amidoamines) substituted with agmatine,30,31 viologen dendrimers,32 and a terpolymer of methyl methacrylate, N,Ndimethylaminoethyl methacrylate, and butyl methacrylate (Eudragit, E100), which destabilized virus membrane,33 and

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RESULTS Selection of Polymers. The primary tests involved cationic polysaccharides such as chitosan, dextran, pullulan, and hydroxypropylcellulose modified with GTMAC, which was based on the assumption that positively charged macromolecules interact with negatively charged HSV-1 virus envelope.12,50,51 Based on our previous experience with cationically modified polysaccharide interaction with glycosaminoglycans (GAGs)52 and available literature, we assumed that these polycations would interact with HS molecules present at the host cell serving as entry receptors for HSV-1 and attachment factor for both herpes simplex viruses. A number of positively charged polymers were tested, and it was revealed that the strongest inhibition of HSV-1 and HSV-2 infection was seen for dextrans (DEXxDSy) modified with 8621



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GTMAC (data not shown). These polymers were selected for further study. Synthesis of DEXxDSy Derivatives. In order to systematically study the subject, an extended library of polymers was synthesized (see Tables S1−S2 in Supporting Information for synthesis conditions), varying in the chain length and the content and density of positively charged GTMAC residues. Cationic DEX derivatives (DEXxDSy) were synthesized by the GTMAC epoxide ring opening reaction in basic conditions followed by the substitution of the hydrogen atom of the hydroxyl groups with GTMAC. The synthesis of cationic derivatives of DEX is presented in Scheme 1. The structure of the DEXxDSy polymers was confirmed using elemental analysis (EA, Table S3 in Supporting Information) and FTIR spectroscopy. EA revealed the presence of nitrogen in the obtained polymers, which unambiguously confirms the successful substitution of DEX with GTMAC. DS values were calculated based on the elemental composition of the polymers (Table S3 in Supporting Information). In the FTIR spectra of all GTMAC-substituted polymers, a weak band at 1477 cm−1 appeared that could be attributed to the presence of asymmetric angular binding of the methyl groups in GTMAC (Figures S1−S3 in Supporting Information), thereby confirming substitution of DEX with GTMAC. In the NMR spectra of the dextrans substituted with GTMAC, signals were found at 3.1 ppm, ascribed to the protons of the trimethylammonium group of GTMAC, and a signal at 4.2 ppm, ascribed to the proton of the CH group closest to OH group in GTMAC (Figures S4−S6 in the Supporting Information). These signals were absent in the parent dextrans thereby confirming successful substitution of these polymers with GTMAC. The zeta potential was determined for the synthesized polymers in 1 mg/mL solutions in 0.015 M NaCl at 25 °C. All measurements were carried out in triplicate, and results are presented in Table S4 in Supporting Information. As expected, the DEX derivatives have a positive zeta potential resulting from the presence of the cationic quaternary amino groups of GTMAC attached to the main dextran chain. The values of zeta potential ranged from +7 to +35 mV and generally increased with increasing DS of the polymers studied. Cytotoxicity. In order to evaluate the usability of developed compounds, cytotoxicity of DEXxDSy was determined using XTT assay, which relies on the ability of the mitochondrial enzymes to convert the substrate (2,3-bis(2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carboxanilide) into colored formazan salts. The assay was performed on Vero E6 cell line, which is an accepted model for HSV-1 and HSV-2 infection. Briefly, cells were incubated for 48 h in the presence of different DEXxDSy polymers. Results presented in Figure 1 clearly show that the polymers of lower MW or DS are characterized by lower toxicity. Antiviral Activity of DEXxDSy. In order to assess inhibitory activity of the tested polymers, virus replication assay (assay 0) was performed for all polymers at 500 μg/mL (polymers that were toxic at that concentration were excluded from the study). Figure 2 demonstrates the decreased virus replication in the presence of both cationically modified 40 kDa DEX (DEX 40 DS y ) and cationically modified 100 kDa DEX (DEX100DSy), for polymers with DS = ∼20−40%. On the other hand, cationically modified 6 kDa DEX showed no significant antiviral activity in the range of DS values studied. Further, correlation between polymer concentration and efficacy was tested using titration and qPCR for DEX100DS40, a

Figure 1. Cytotoxicity of the tested polymers. Cell viability was assessed using XTT assay 2 days p.i. (postinoculation). Background fluorescence was subtracted for all the samples. Cell viability was calculated in reference to untreated cells (control). Derivatives of dextran with MW of 6 kDa (A), 40 kDa (B), and 100 kDa (C) with various DS were tested in the range of concentrations 50−2000 μg/ mL. Results are presented as average ± SD. Similar results were obtained in at least two replicates in three independent experiments.

polymer showing high anti-HSV-1 activity. The results are presented in Figure 3. Calculated IC50 values were 10.91 μg/ mL for HSV-1 and 45.61 μg/mL for HSV-2, as determined by virus titration and 19.30 μg/mL for HSV-1 and 16.29 μg/mL for HSV-2, as determined by qPCR. Mechanism of Action. An effort was made to elucidate the antiviral mechanism of action for modified dextrans. For this purpose, a set of functional assays, described in the Experimental Section, was performed. For mechanistic studies, DEX100DS40 polymer was selected, as it combines high efficiency with low cytotoxicity. First, we investigated the influence of DEX100DS40 on HSV-1 virus particles (Assay I). No significant reduction of viral titer was detected after pretreatment of virions with the polymer. Next, the possibility that the polymer renders the cell resistant to the infection was tested (Assay II). Decrease in virus yield (∼50%) was noted using both read-out methods. Subsequently, 8622



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functional assays suggested inhibition at early steps of replication. The ability of the studied polymers to bind HS was assessed with a colorimetric method using Azure A, a cationic dye. Azure A in the solution of HS forms aggregates absorbing at 513 nm, while the monomeric form of the dye (prevalent in the absence of HS) absorbs at 630 nm. Addition of DEX100DS40 caused the disruption of dye aggregates resulting in the increase in intensity of the absorption at 630 nm (monomeric band) and in the decrease of absorption at 513 nm (aggregate absorption band). The plot showing changes in the absorption spectra of Azure A upon addition of HS is shown in Figure 5. In addition, complexation process was quantified and dependence of the concentration of free (unbound) HS on the mass ratio of DEX100DS40 to HS present in solution is presented in Figure 6. The amount of DEX100DS40 required to bind 1 mg of HS was calculated to be equal to 1.30 mg. Such calculations were performed for all cationic DEX derivatives, and the results are given in Table S5 (see Supporting Information). Obtained data demonstrated clearly that HS is complexed with all DEXxDSy polymers. The general trend was that cationic DEXxDSy of a given MW bound HS stronger when its DS was higher. Among DEXxDSy of comparable DS, the derivatives of DEX with MW of 40 kDa showed the strongest HS binding properties. To confirm whether the interactions between cationic dextrans and HS present on the cell surface play a role in the inhibition of virus entry, flow cytometry analysis was carried out, as described in the Experimental Section. Data was normalized against a control of cells infected without preincubation with a cationic dextran. The signal recorded from the infected sample treated with DEX100DS40 was vastly

Figure 2. Influence of DS and MW of DEXxDSy on their anti-HSV-1 activity. Cationic dextran derivatives with MW of 6, 40, and 100 kDa with different DS were present throughout the infection at 500 μg/mL. Two days p.i., the amount of viral DNA in cell culture supernatant was assessed by qPCR. Difference in viral yield is presented on y-axis as log removal value (LRV), showing the relative decrease in the amount of virus in cell culture media compared to the control. Results are presented as average ± SD. Similar results were obtained in at least two replicates in three independent experiments.

the ability of DEX100DS40 to block viral adhesion to the cell was examined (Assay III). A decrease in viral titer reaching ∼90% for virus titration and 100% for plaque assay was observed. Finally, we tested whether DEX100DS40 may interfere with virus replication during the later phases of the infection (Assay IV), and ∼95% decrease in virus titer was observed for titration and ∼99% for plaque assay. Obtained results are summarized in Figure 4. Interaction between the Cationic DEX and HSV Attachment Receptor. The interaction between DEXxDSy polymers and heparan sulfate (HS) was studied, as results from

Figure 3. Concentration-dependent inhibition of HSV-1 (A, C) and HSV-2 (B, D) replication by DEX100DS40. Viral yield in samples was quantified by titration (A, B) or qPCR (C, D). For virus titration, the virus quantity is presented as TCID50 log decease and for qPCR as log decrease of viral DNA copy number per milliliter. Results are presented as average ± SD. Similar results were obtained in at least two replicates in three independent experiments. 8623



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Figure 4. Inhibition of HSV-1 by DEX100DS40 at different stages of the replication cycle. Functional assays were carried out as described in the Experimental Section, and the outcome was monitored by titration (A) and by plaque assay (B). Obtained results were normalized to the control sample of polymer-untreated cells. Results are presented as average ± SD. Similar results were obtained in at least two replicates in three independent experiments.

Figure 5. UV−vis absorption spectra of Azure A (7.2 × 10−5 M) in the presence of 0.2 mg/mL of HS and different concentrations of DEX100DS40 [mg/mL].

Figure 7. HSV-1 attachment in the presence of DEX100DS40 as determined with flow cytometry. Cells were preincubated with the polymer for 1 h, then infected with HSV-1 or mock sample for 2 h in the absence of DEX100DS40. vir ctrl, cells not treated with the polymer and infected with HSV-1; mock, cells not infected with HSV-1; vir +DEX100DS40, cells treated with the polymer and infected with HSV-1. Value on y axis represents the percentage of cells that internalized the virus, relative to the control sample (not treated with the polymer). Results are presented as average ± SD. Similar results were obtained in at least two replicates in three independent experiments.

°C, then cells were rinsed with 1 × PBS, fixed, and stained with phalloidin and DAPI as described in the Experimental Section. Even though all tested polymers (DEX6DS41, DEX40DS37, and DEX100DS40) were present inside the cell, the intracellular presence of DEX100DS40 seemed to be limited, compared to the polymers of lower MW. However, detailed study should be carried out to quantify the polymer internalization. The results are presented in Figure 9. Comparison of Antiviral Activity of DEX100DS40 and Acyclovir (ACV). To compare antiviral activity of DEX100DS40 with that of ACV, the most widely used anti-HSV drug, virus replication assay (Assay 0) was performed. The results are presented in Figure 10. In the tested range of concentrations DEX100DS40 was found to hamper HSV-1 infection as efficiently as ACV. However, despite the different mode of action of these compounds, no synergistic effect was observed (data not shown).

Figure 6. Dependence of the relative concentration of free (unbound) HS on the weight ratio of DEX100DS40 and HS.

limited, reaching the intensity observed for mock-treated sample. The results are presented in Figure 7 and suggest that interaction between modified dextrans and HS present at the cell surface hinders the viral adhesion to the host cell. To further verify the ability of cationic dextrans to inhibit HSV attachment to cell surface, confocal images were obtained for cells prepared as described for Assay II. After washing off the unbound virus particles, the cells were fixed and stained for visualization of HSV and F-actin. Figure 8 demonstrates maximal projections of XY stacks. Subcellular Localization of Dextran Derivatives. To test whether polymers may enter the cell, fluorescent derivatives of DEXxDSy were used. Briefly, cells were incubated with 250 μg/mL of fluorescently labeled DEXxDSy for 1 h at 37

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DISCUSSION AND CONCLUSIONS In the manuscript, we describe development, evaluation and optimization of cationic polymers as novel inhibitors of herpes simplex viruses. We assumed that the interaction between a 8624



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Figure 8. Modified dextrans block HSV-1 attachment to susceptible cells. Cells preincubated in the absence of DEX100DS40 (A) or in the presence of 500 μg/mL DEX100DS40 (B) were incubated with HSV-1 virions. Blue, DNA; red, f-actin; green, HSV-1. Maximal projections of collected stacks are presented. Scale bar: 10 μm. Similar results were obtained in at least two replicates in three independent experiments.

Figure 9. Subcellular localization of dextran derivatives. Fluorescent derivatives of DEX6DS41 (A), DEX40DS37 (B), and DEX100DS40 (C). Cells were incubated with the compounds for 1 h at 37 °C. Blue, DNA; red, f-actin; green, dextran. Images present single slices of XY stacks. Scale bar: 10 μm. Similar results were obtained in at least two replicates in three independent experiment.

pullulan, and hydroxypropylcellulose) substituted with GTMAC by systematically testing the inhibition of HSV-1 related cytopathic effect (CPE) by these polymers and selected cationic dextrans (DEXxDSy) as the most active against HSV. As properties of modified polymers may vary significantly depending on the backbone length and degree of substitution (DS) with the cationic groups, we synthesized a library of polymers varying in the chain length and charge density. We have shown that the anti-HSV activity of DEXxDSy increased with growing length of the polymer backbone, expressed here as the molecular weight (MW) and with the degree of substitution (DS). Increase in the polymer effectiveness with increasing MW and/or DS may be explained by the fact that longer polymer chain adopts the globular conformation with high surface density resulting from large number of charged residues present. Most likely for similar reasons, polymer length correlated with higher cytotoxicity. Optimization of MW and DS of DEXxDSy showed that only polymers with MW of at least 40 kDa and with DS > ∼40% showed marked inhibitory properties. However, molecules with higher DS showed higher cytotoxicity. To balance the effectivity and toxicity, we selected the largest polymer of 100 kDa with DS of 40% (DEX100DS40). This decision was also based on the fact that the cellular uptake of this polymer was markedly lower compared to that of the 40 kDa polymer. Even though the polymers were designed to hamper virus attachment/entry to the susceptible cell, we decided to first employ broad functional assays that would allow us to reliably determine the mechanism of DEX100DS40 antiviral action. The study showed that DEX100DS40 indeed inhibits virus replication

Figure 10. Comparison of DEX100DS40 antiviral activity with that of ACV. Viral yield in samples was quantified by qPCR. The virus quantity is presented as log decrease of viral DNA copy number per milliliter. Results are presented as average ± SD. Similar results were obtained in at least two replicates in three independent experiments.

cationically substituted polymer and negatively charged heparan sulfate (HS), a glycosaminoglycan (GAG) that serves as an attachment factor for HSV viruses and is important for the initiation of the infection,53,54 would inhibit HSV infection. Previous studies targeting or mimicking this interaction involved synthetic30,31 and natural55 polymers and peptides,56,57 which have several disadvantages, starting from price, through immunogenicity to low stability and limited effectiveness. Based on this assumption, we performed a preliminary screening of a series of polysaccharides (chitosan, dextran, 8625



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and y is the degree of substitution defined as the number of GTMAC groups attached to DEX per 100 glucose repeating units. In the second synthetic procedure, both the reaction time and GTMAC volume were varied (Table S2). The first polymer sample in each series was obtained by reacting 2 g of DEX dissolved in 100 mL of distilled water with initial volume of GTMAC (20 or 40 mL) for 1 or 2 h. Then, a defined volume of the reaction mixture was withdrawn to isolate a polymer sample while to the rest of the reaction mixture the defined volume of GTMAC was added, and the resulting mixture was heated while mixing for the defined time period. The withdrawal/GTMAC addition/heating cycles were repeated two or three times. To isolate the polymer, the reaction mixtures were neutralized to pH ∼ 7 by the addition of 1 M HCl, cooled, and transferred to dialysis tubes (MW cutoff values of 3.5, 12.8, and 14 kDa, depending of the MW of DEX). The dialysis was carried out against distilled water until the conductivity of the liquid surrounding the tube decreased below 10 μS. The polymers obtained were isolated from the solution using the freeze-drying technique. The substitution of DEX with GTMAC was confirmed using EA, IR spectroscopy, and zeta potential measurements. The DS values of the polymers obtained covered almost the whole possible range, that is, from 2% to 98%. As polymers obtained in different syntheses were used throughout the study and obtaining exactly the same DS value in syntheses performed in identical conditions could not be achieved, DEX100DS40 stands for polymers with DS ± 2%. Synthesis of Fluorescently Labeled DEXxDSy. DEX6DS41, DEX40DS37, and DEX100DS40 were fluorescently labeled as described previously.58 Briefly, 50 mg of the respective polymer was dissolved in 2 mL of warm DMSO and 1 drop of pyridine and 3 mg of FITC in 2 mL of DMSO were added. The mixture was heated to 95 °C for 2 h under continuous stirring. Then, DMSO was removed by dialysis against water, and the product was lyophilized. Determination of the Degree of Substitution (DS) of DEX Derivatives with GTMAC. The DS of the DEXxDSy was determined with elemental analysis (EA). The ratio of N/C in each polymer was determined, and the DS was calculated using the following equation:

cycle at early stages, most likely interacting with HS molecules, rendering the cells resistant to virus attachment and/or entry. Even though our results clearly show that developed polymers should interfere with early steps of the virus replication, inhibition of virus replication was also observed if the polymer was added at later stages. One may assume this may result from the inhibition of multicycle replication due to hindrance of subsequent transmission events. However, an additional mechanism involving interference with virus replication, assembly, or release may not be ruled out. In order to confirm our observations, we decided to perform analysis of virus adhesion to the cell surface by flow cytometry and confocal microscopy. Both methods showed that virus particle attachment to the cell surface was vastly limited, consistent with our concept that cationically modified polymers bind to the attachment receptors limiting virus’s ability to bind and penetrate the cell. Further, we observed that DEX derivatives form complexes with HS in 1 × PBS buffer (pH = 7.4), which is consistent with our previous observation that cationic dextran derivatives reverse the anticoagulant activity of heparin in vitro and in vivo due to its complexation.58−60 DEX100DS40 showed effectiveness in inhibiting HSV-1 infection similar to that of ACV. Although the mode of action of these two compounds is completely different, they did not exhibit any synergistic activity. Summarizing, the present study allowed for the development of novel inhibitors of HSV entry, which hinder interaction between the virus and the cell. Further, one may assume that polymers may hamper entry of HSV-1 via HS receptor during ocular infections. We believe that the developed polymers are able to interfere with HSV-1 and HSV-2 transmission and limit virus spread between cells and as such may be used for prophylaxis or in combination with replication inhibitors.

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DS[%] = 100 × (457.34 × (N/C)3 + 3.007 × (N/C)2

EXPERIMENTAL SECTION

+ 5.5951 × (N/C))

Materials. Dextran (DEX, MW of 6, 40, and 100 kDa, from Leuconostoc spp., Sigma), glycidyltrimethylammonium chloride (GTMAC, Sigma), heparan sulfate (HS), Azure A chloride (Fluka, Fluka standard), PBS (tablet, Sigma), fluorescein 5(6)-isothiocyanate (FITC, Sigma), sodium chloride (analytical grade, POCh), NaOH (POCh), and HCl (POCh) were used as received. All compounds were at least 95% pure, as determined using NMR and HPLC. Water was distilled twice and deionized using the Millipore Simplicity system. Apparatus. FTIR spectra were obtained on a Bruker IFS 48 spectrometer. Elemental analysis (EA) was performed on a EuroEA 3000 elemental analyzer. The UV/vis absorption spectra were obtained at room temperature in 1 cm quartz cuvettes using the single beam diode array Hewlett-Packard 8452A spectrophotometer with a resolution of 2 nm in the range of 190−820 nm. Zeta potential measurements were performed using Zetasizer Nano ZS instrument (Malvern Instruments). The sample was illuminated with a 633 nm laser, and the intensity of light scattered at an angle of 173° was measured with an avalanche photodiode. Synthesis of Cationically Modified Dextran (DEX). The synthesis of cationic derivatives of dextran is presented in Scheme 1. Cationic derivatives of DEX were obtained by the substitution of hydroxyl groups with GTMAC as described previously60 with some modifications. We have applied two synthetic protocols. In the first synthetic procedure, 0.5 g of DEX was dissolved in 25 mL of distilled water, then NaOH (2.5 mmol) was added. The solution was stirred with a magnetic stirrer and heated to 80 °C. In the next step different volumes of GTMAC were added, and the mixtures were kept at 80 °C for 4 h while being stirred. The MW values of DEX, volume of GTMAC, and reaction temperature used to obtain each DEX derivative are given in Table S1 (see Supporting Information). The products are denoted as DEXxDSy where x is the MW of DEX in kDa

where N/C is the ratio of the mass fraction of nitrogen to carbon. Studies on the Interactions between the DEXxDSy and HS using a Colorimetric Method. The mixtures containing a constant concentration of HS (0.2 mg/mL) and 1 × PBS solutions of various DEXxDSy at different concentrations (0.06−0.27 mg/mL) were prepared. The mixtures were shaken for 10 min and centrifuged at 3000 rpm for 10 min to separate the insoluble complex of HS with DEXxDSy. Then, 0.1 mL of the supernatant was added to the mixture of 0.9 mL of 1 × PBS and 1 mL of 8.0 × 10−5 M Azure A solution, and the UV−vis spectra were measured. Cell Culture. Vero E6 cells (Cercopithecus aethiops kidney epithelial, ATCC CRL-1586) were routinely maintained under Dulbecco-modified Eagle’s medium (DMEM, high glucose, Life Technologies) supplemented with 3% heat-inactivated fetal bovine serum (FBS, Life Technologies), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C in atmosphere containing 5% CO2. Virus Preparation and Titration. HSV-1 strain 17+ and HSV-2 strain HG52 were obtained from Public Health England (0104151v and 0104152v, respectively). Virus stocks were generated by infecting Vero E6 cells monolayers for 48 h, then lysing the cells with two freeze−thaw cycles. Collected lysates were aliquoted and stored at −80 °C. Mock samples were prepared in the same manner, using mockinfected cells. Virus stocks were quantified by titration on Vero E6 cells (48 h infection, 37 °C), according to Reed and Muench method.61 Following a 48 h incubation, the number of cytopathic effect (CPE)positive wells was counted, and the TCID50 was calculated. Cell Viability Assay. Cell viability was assessed on Vero E6 cell line using XTT Cell Viability Assay Kit (Biological Industries) according to vendor’s protocol. Briefly, cells were seeded in 96-well 8626



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times with 1 × PBS to remove unbound virus particles, and fresh medium supplemented with the polymer was added. Cells were cultured for 48 h at 37 °C; then medium samples were collected, and viral yield was assessed. Since the polymer is absent during the early stages of infection (virus adsorption/attachment and entry), a decline in the viral yield compared to the control in this test indicates that the antiviral activity of the polymer is due to inhibition of later stages of virus replication cycle (e.g., virus replication, assembly, or egress). qPCR. Viral DNA was isolated from supernatants using Viral DNA/ RNA Isolation Kit (A&A Biotechnology, Poland). Virus yield was then determined by quantitative real-time PCR (qPCR). The reaction was carried out in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad), in 10 μL of reaction mixture consisting of 1 × Kapa Probe Fast qPCR Master Mix, specific probe labeled with 6-carboxyfluorescein (FAM) and Black Hole Quencher 1 (BHQ1) (sequence 5′FAM CCG CCG AAC TGA GCA GAC ACC CGC GC BHQ1-3′, 100 nM), and primers (450 nM each, sequences, sense primer 5′-CAT CAC CGA CCC GGA GAG GGA C-3′, antisense primer 5′-GGG CCA GGC GCT TGT TGG TGT A-3′) and 2.5 μL of viral DNA in 10 μL. The temperature profile included 3 min at 95 °C, followed by 37 cycles of 2 s at 95 °C and 20 s at 60 °C. For quantification, DNA standards were prepared. Briefly, a fragment of the DNA polymerase gene, conserved among HSV-1 and HSV-2 strains, was amplified using the primers mentioned above and cloned into pTZ57R/T (Thermo Scientific, Poland) plasmid using InsTAclone PCR cloning kit (Thermo Scientific, Poland). The plasmid was propagated in E. coli TOP10 (Life Technologies, Poland), purified with GeneJET Plasmid Miniprep Kit (Thermo Scientific, Poland) and linearized by digestion with KpnI restriction enzyme. Concentration of the linearized DNA was determined spectrophotometrically, and the number of copies per milliliter was calculated. Six subsequent serial dilutions were then used as qPCR reaction template. In this article, the data from quantitative PCR are presented as log removal values (LRVs) in order to enable comparison of results obtained from different assays. LRV was calculated according to the following formula: LRV = −log(ci/c0) where ci is the number of viral RNA copies per milliliter in the sample in the culture treated with a given polymer and c0 is the number of viral RNA copies per milliliter in control sample (untreated cells). Flow Cytometry. Vero E6 cells were seeded in 6-well plates and cultured for 2 days at 37 °C in atmosphere containing 5% CO2. For analysis, cells were trypsinized and fixed with 4% PFA in 1 × PBS for 20 min. Subsequently, the cells were permeabilized by 20 min incubation in 0.1% Triton X100 in 1 × PBS, and nonspecific binding was blocked by overnight incubation with 5% BSA in 1 × PBS at 4 °C. Protein labeling was carried out by 2 h incubation with primary rabbit anti-HSV VP5 antibody (20-HR50, Fitzgerald Industries, USA) diluted 1:500, followed by 1 h incubation with secondary goat anti-rabbit antibody, conjugated with AlexaFluor 488 (A11001, Invitrogen, Poland). After washing with 0.1% Tween-20 in 1 × PBS, the cells were resuspended in 1 × PBS and analyzed with FACS Calibur (Becton Dickinson) using Cell Quest software. Confocal Microscopy. Vero E6 cells were seeded on coverslips in 6-well plates and cultured for 2 days at 37 °C with 5% CO2. For analysis of virus adhesion and entry, assays II and III were carried out, respectively. Immediately after washing away unbound virions, cells were fixed with 4% PFA in 1 × PBS for 20 min. Subsequently, the cells were permeabilized by 20 min incubation in 0.1% Triton X100 in 1 × PBS and nonspecific binding sites were blocked by overnight incubation in 5% BSA in 1 × PBS at 4 °C. To visualize virus particles, cells were incubated for 2 h with primary rabbit anti-HSV VP5 antibody (20-HR50, Fitzgerald Industries, USA) diluted 1:500 in 1 × PBS with 0.5% Tween 20, followed by 1 h incubation with secondary goat anti-rabbit antibody, conjugated with AlexaFluor 488 (A11001, Invitrogen, Poland). F-actin was costained with AlexaFluor 633-conjugated phalloidin (Invitrogen, Poland). Nuclear DNA was labeled with 0.1 μg/mL 4′,6′-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, Poland). Immunostained cultures were mounted on glass slides with ProLong Diamond Antifade Mountant (Life technologies, Poland). Fluorescent images were acquired under a

plates and cultured for 48 h. Subsequently medium was discarded, and cells were overlaid with fresh medium supplemented with DEXxDSy polymers or control samples. Culture was carried out for 48 h at 37 °C, and after that, incubation medium was refreshed again, and 25 μL of activated XTT reagent was added to each well. Following a 2 h incubation at 37 °C, media were transferred to a new 96-well plate and absorbance was measured at λ = 480 nm using a spectrophotometer (Flexstation 3, Molecular Devices). The results were normalized against a control of untreated cells (100% viability). Virus Replication Assay (Assay 0). Vero E6 cells were seeded in 6-well plates and cultured for 2 days at 37 °C in atmosphere supplemented with 5% of CO2. Subsequently, media was discarded, and cells were overlaid with fresh media supplemented with DEXxDSy polymers or control samples. Following a 30 min incubation, cells were infected with HSV-1 or HSV-2 virus at TCID50 = 400/mL or mock-infected. Infection was carried out for 2 h at 37 °C. Subsequently, unbound virus particles were removed by triple washing with sterile 1 × PBS, and culture media supplemented with polymers was applied. Samples were analyzed 2 days p.i. Plaque Assay. Twenty-four hours prior to the infection, cells were seeded in 6-well plates. On day 0, subconfluent cells were infected with 10-fold serial dilutions of the virus and incubated for 1 h at 37 °C. Subsequently, cells were washed once with 1 × PBS and overlaid with 2 mL of media supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 0.33% methylcellulose. After 72 h, media were discarded, and 1 mL of 0.1% crystal violet solution in 50:50 water/ ethanol mixture was added to each well. Following a 10 min incubation at room temperature, cells were washed twice with water and plaques were counted. Mechanism of Anti-HSV Action of DEXxDSy. A series of assays was employed to determine the mechanism of antiviral activity of DEXxDSy. The tests differ mainly in the sequence in which the cells, the virus, and the polymer may interact. Assay I: Does the Compound Inactivate the Virus? Virus stock samples were incubated with DEXxDSy polymers (500 μg/mL) for 1 h at 22 °C under constant mixing. Subsequently, the samples were diluted to decrease the polymer concentration below its active range (25 μg/mL). In negative control samples, cell culture medium was added instead of the polymer solution. The samples were then titrated on fully confluent Vero E6 cells to assess viral yield. This test indicates whether the antiviral effect results from a direct interaction between the polymer and the virus. Assay II: Does the Compound Protect the Cell? Fully confluent Vero E6 cells were overlaid with polymer solution and incubated for 1 h at 37 °C. Subsequently, the polymer was removed by triple wash with 1 × PBS and virus at TCID50 = 400/mL or mock at equal volume was added. After 2 h infection at 37 °C, the virus was washed away, fresh culture medium was applied, and the cells were cultured for 48 h. Samples of cell culture supernatant were collected, and viral yield was assessed. As no unbound polymer is present during the exposure to virus, inhibition of infection in this test indicates the influence of the polymer on the cell (e.g., cell surface coating, induction of innate immune responses). Assay III: Does the Compound Block Virus−Receptor Interaction? Fully confluent Vero E6 cells were chilled on ice, overlaid with an icecold solution of the DEXxDSy polymer (500 μg/mL) containing the virus at TCID50 = 400/mL or mock at equal volume in cell culture medium. Under these conditions, virions can attach to their receptor on the cell surface but cannot enter the cell, as the intracellular transport is inhibited at lower temperatures. Following 1 h incubation at 4 °C, the polymer solution was removed, cells were washed three times with ice-cold 1 × PBS to remove any unbound polymers and virions, overlaid with fresh medium, and cultured for 48 h at 37 °C. Then, samples of cell culture supernatant were collected, and viral yield was assessed. If the polymer inhibits the virus−receptor interaction, a decline in the viral yield is observed compared to the control. Assay IV: Does the Compound Hamper Infection at Later Stages? Fully confluent Vero E6 cells were infected with virus at TCID50 = 400/mL or mock for 2 h at 4 °C. Subsequently, cells were rinsed three 8627



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ZEISS LSM 710 (release version 8.1) confocal microscope and acquired with ZEN 2012 SP1 (black edition, version 8.1.0.484) software. Stacks acquisition parameters were as follows: frame size 1024 × 1024, step size 0.35 μm, pixel size 0.10 μm. For image processing, ImageJ FIJI version was used. Statistical Analysis. All the experiments were performed in triplicate, and the results are expressed as mean ± SD. To determine the significance of the obtained results, a comparison between groups was made using the Student’s t test. P values

Inhibition of Herpes Simplex Viruses by Cationic Dextran Derivatives

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