Highly Effective and Safe Polymeric Inhibitors of Herpes Simplex Virus

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Biological and Medical Applications of Materials and Interfaces

Highly effective and safe polymeric inhibitors of herpes simplex virus in vitro and in vivo Magdalena Pachota, Katarzyna K#ysik-Trzcia#ska, Aleksandra Synowiec, Shotaro Yukioka, Shin-ichi Yusa, Mateusz Zaj#c, Barbara Zawilinska, Tomasz Dzieci#tkowski, Krzysztof Szczubia#ka, Krzysztof Pyrc, and Maria Nowakowska ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10302 • Publication Date (Web): 09 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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ACS Applied Materials & Interfaces

Highly effective and safe polymeric inhibitors of herpes simplex virus in vitro and in vivo Magdalena Pachotaa,b, Katarzyna Kłysik-Trzciańskac, Aleksandra Synowiecb, Shotaro Yukiokad, Shin-Ichi Yusad, Mateusz Zającc, Barbara Zawilinskae, Tomasz Dzieciątkowskif, Krzysztof Szczubialkac, Krzysztof Pyrca,*, Maria Nowakowskac,*

a

Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian

University, 30-387 Krakow, Poland. b

Microbiology Department, Faculty of Biochemistry, Biophysics and Biotechnology,

Jagiellonian University, 30-387 Krakow, Poland. c

Department of Physical Chemistry, Faculty of Chemistry, Jagiellonian University, 30-

387 Krakow , Poland d

Department of Applied Chemistry, Graduate School of Engineering, University of

Hyogo, Himeji, Hyogo, 671-2280 Japan e Department of Virology, Chair of Microbiology, Faculty of Medicine, Jagiellonian University Medical College, 31-121 Krakow, Poland f Chair and Department of Medical Microbiology, Warsaw Medical University, 02-004 Warsaw, Poland

KEYWORDS: herpes simplex virus, polymeric inhibitors, PEGx-b-PMAPTACy, antiviral activity, polymer cell membrane interaction, in vitro and in vivo experiments

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ABSTRACT:

Series

of

poly(ethylene

glycol)-block-poly(3-(methacryloylamino)propyl

trimethylammonium chloride) (PEG-b-PMAPTAC) water-soluble block copolymers consisting of poly(ethylene glycol) (PEG) and poly(3-(methacryloylamino)propyl trimethylammonium chloride) (PMPTAC) were obtained by reversible addition−fragmentation chain-transfer (RAFT) polymerization and demonstrated to function as highly effective herpes simplex virus type 1 (HSV1) inhibitors as shown by in vitro tests (Vero E6 cells) and in vivo experiments (mouse model). Half maximal inhibitory concentration (IC50) values were determined by qPCR to be 0.36 ± 0.08 µg/ml for the most effective polymer PEG45-b-PMAPTAC52 and 0.84 ± 1.24 µg/ml for the less effective one, PEG45-b-PMAPTAC74. The study performed on the mouse model showed that the polymers protect mice from lethal infection. The polymers are not toxic to the primary human skin fibroblast (HSF) cells up to the concentration of 100 μg/m and to the Vero E6 cells up to the 500 μg/ml. No systemic or topical toxicity was observed in vivo, even with mice treated with concentrated formulation (100 mg/ml). The mechanistic studies indicated that polymers interacted with the cell and blocked the formation of the entry/fusion complex. Physicochemical and biological properties of PEGx-b-PMAPTACy make them promising drug candidates.

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Introduction Herpes simplex virus is one of the most prevalent human pathogens. The infection is usually transmitted by direct, intimate contact between the susceptible individual and the infectious patient1. Herpes simplex virus type 1 (HSV-1) is associated mainly with oropharyngeal infection, as it is transmitted mostly by the oral route, while herpes simplex virus type 2 (HSV-2) infection is usually the consequence of the exposition of genital, perigenital or anal skin sites2. The primary infection of the mucosa leads to ulceration and blistering, but at the same time the virus enters sensory neurons; traveling along neural cells it reaches dorsal root ganglia, where it establishes latent, lifelong infection3. Subsequently, an interplay between the immune system and the viral machinery begins. At reactivation, the virus is anterogradely transported to the mucosal tissues at the primary infection site. The clinical manifestation is, however, largely dependent on the immune competence of the individual4–6. The HSV infection is commonly considered to be mild, but in some cases it spreads and may localize to different sites, causing e.g., keratitis, which in some cases leads to blindness, oftentimes fatal encephalitis, or even systemic disease7–9. Page 3 of 36



While no protective vaccine has been developed7, drugs are available. Acyclovir (ACV) was the first, approved in the early 1980s; later a number of derivatives were developed. ACV and its derivatives are nucleoside analogs that inhibit herpesviral polymerase. Intriguingly, administered drugs are inactive until they reach the infected cell. Once this happens, the compounds become phosphorylated by virally encoded thymidine kinase and cellular kinases, leading to the formation of biologically active triphosphates10,11. Nucleoside analogs have been used in clinic already for a long time and therefore emergence of resistance mutants has become a problem12–14. For this reason, efforts are taken to develop novel compounds with different molecular targets, so their activity should not be affected by cross-resistance15–19. Polyoxymetalates (Cs2K=Na[SiW9Nb3O40]) have shown broad antiviral activity against several viruses, including HSV-1. That was explained considering the protection of cell surface at which polyoxymetalates were localized20. Graphene oxide (GO) derivatives (partially reduced sulfonated GO) were observed to inhibit HSV-1 infections by mimicking the cell surface receptor heparan sulfate, and competing with the latter in binding HSV-1. However, the inhibition did not affect cell-to-cell spreading21. Carbon nanodots obtained by Page 4 of 36


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hydrothermal carbonization of 4-aminophenylboronic acid hydrochloride were shown to act as entry inhibitor of HSV-1 in in vitro experiments on African green monkey kidney cancer cells (Vero) and human lung cancer cell (A549) 22. It is worth to mention, that none of the abovementioned drugs is able to cleanse the patient’s body from the infection. Considering the topical nature of the HSV infection, polymers constitute a promising alternative to small-molecule inhibitors. Natural polymers were previously proposed as antiherpesviral drugs. These included sulfated lignins, polyphenols from almond peel, and a number of sulfated polysaccharides23–25. Further studies led also to the development of synthetic polymers with antiviral properties. Early stages of infection are blocked by cationic polysaccharides conjugated with oligoamines, poly(L-lysine) and poly(L-arginine) derivatives26. Terpolymer of methyl methacrylate, N,Ndimethylaminoethyl methacrylate, and butyl methacrylate (Eudragit, E100), poly(amidoamines) substituted with agmatine, and viologen dendrimers were reported to act directly on the viral particle, destabilizing its membrane27–29. Also, polyanionic polymers were proven effective, and compounds like dendrimers containing sulfonic groups, poly(sodium styrenesulfonate) (PSSNa) and its copolymer with maleic acid, Page 5 of 36



poly(methacrylic acid) functionalized with ribavirin, and telomerized ω-acryloyl anionic surfactants were shown to inhibit herpesviral infection in vitro and in vivo30–34. However, the above polymers have several drawbacks, which include high toxicity (e.g., cationic polymers), disqualifying side effects (e.g., anticoagulative properties of polyanions), or simply low effectivity (generally only 2-3 logs inhibition in vitro). Previously, we reported cationic dextran derivatives as effective inhibitors of herpesviral infection35. These polymers block virus adherence to the cell surface by the glycosaminoglycans (GAGs) and block subsequent steps of the infection. Here, we developed a novel and superior to the previously reported, class of block copolymers, composed of poly(ethylene glycol) (PEG) and poly(3-(methacryloylamino) propyltrimethylammonium (PMPTAC) blocks. We show that they are non-toxic in vitro and in vivo, possibly due to the presence of PEG block in the polymer structure36, which sterically shields the excess positive surface charge of the PMAPTAC block37. The new polymers, denoted as PEGx-b-PMAPTACy, where x and y are the degrees of polymerization of the respective blocks, effectively inhibit HSV-1 infection by interfering with virus-cell interaction, what translates to 6 logs of viral yield decrease. Last but not least, the Page 6 of 36


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polymers are effective in vivo and protect the mice from lethal HSV-1 infection. We believe that the PEGx-b-PMAPTACy polymers are promising drug candidates to be used in complementary or substitutive therapy in HSV-1 affected individuals.

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Materials and Methods Synthesis of cationic block polymers A typical synthesis of PEGx-b-PMAPTACy (Scheme 1), where x and y are the degrees of polymerization (DP) of PEG and PMAPTAC blocks, respectively, was given in Supporting Information). The number-average molecular weight (Mn) estimated from gel-permeation chromatography (GPC) and 1H NMR, DP (shown as x and y values of respective polymers) estimated from 1H NMR, and molecular weight distribution (Mw/Mn) estimated from GPC, are given in Table S1 in Supporting Information. All polymers were well soluble in aqueous media. There was no aggregation observed up to the concentration as high as 1 g/l, as indicated by the monotonic (almost linear) increase in the specific conductivity of aqueous polymers solution on their concentration (Figure S2 in Supporting Information). DLS experiments allowed to observe the self-organization of the positively charged macromolecules which adopted spherical conformation of large dispersity. There is no clear dependence of these parameters on the polymer molecular weight (Table S2 and Table S3 in Supporting Information).

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Scheme 1. Synthesis of PEGx-b-PMAPTACy block copolymers

Cells and virus Vero E6 cells (African green monkey kidney epithelial, ATCC CRL-1586) were cultured in 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), streptomycin (100 μg/ml) and ciprofloxacin (5 µg/ml) at 37°C in an Page 9 of 36



atmosphere containing 5% CO2. Primary human skin fibroblasts (HSF) were maintained in Dulbecco-modified Eagle’s medium (DMEM, high glucose, Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Life Technologies), 1% non-essential amino acids (NEAA, Life Technologies), penicillin (100 U/mlL), streptomycin (100 μg/ml) and ciprofloxacin (5 µg/ml) at 37°C in an atmosphere containing 5% CO2. HSV-1 strain 17+ (0104151v) and HSV-2 strain HG52 (0104152v) were acquired from Public Health England. Clinical HSV-1 strain 207 was isolated from a patient at the Department of Microbiology, Collegium Medicum, Jagiellonian University. All described above clinical isolates of aciclovir-resistant HSV-1 strains were isolated from immunosuppressed patients hospitalized at the Department of Haematology, Oncology and Internal Medicine, Medical University of Warsaw38. Virus stocks were produced by infecting fully confluent Vero E6 cells with the virus at TCID50 (50% tissue culture infectious dose) = 400 for two days. The cells were then lysed by two freeze-thaw cycles, the lysates were aliquoted and stored at -80°C. Mock samples were generated by the same protocol, using uninfected cells. The TCID50 of virus stocks was determined by titration on Vero E6 cells according to Reed and Muench Page 10 of 36


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method39. The number of CPE (cytopathic effect) positive wells was calculated two days post-infection (p.i.).

Virus replication assay Vero E6 cells were seeded in 96-well plates 48 h before the experiment. At the day of the assay, supernatants were discarded, and cells were overlaid with media supplemented with certain concentrations of tested polymers. After a 30 min incubation at 37°C, cells were infected with the virus at TCID50 = 400/ml or inoculated with mock in the presence of polymers. Following 2 h incubation at 37°C, cells were washed thrice with phosphate buffered saline (PBS) to remove unbound virions and overlaid with fresh media supplemented with the compounds. Samples were collected and analyzed 2 days p.i.

Plaque assay Vero E6 cells were seeded in 24-well plates 24 h before the experiment. On the day of the assay, 10-fold serial dilutions of the virus stock were prepared and the cells were

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infected. Following 1 h incubation at 37°C, the cultures were washed with PBS, overlaid with DMEM media containing 10% heat-inactivated fetal bovine serum (FBS) and 1% methylcellulose (Sigma-Aldrich, Poland) and cultured for next 3 days. Subsequently, the media was discarded, and the cells were fixed and stained using 0.1% crystal violet solution in 50:50 water/ethanol mixture for 10 min at room temperature. Subsequently, cells were washed with tap water and plaques were counted.

Interaction between HSV-1 and cationic block copolymers Interaction between HSV-1 and cationic block copolymers was measured using a Malvern Nano ZS apparatus (Malvern Instrument, Worcestershire, UK) for dynamic light scattering (DLS) and zeta potential measurements. The samples were illuminated with a 633 nm laser, and the intensities of scattered light at an angle of 173° were measured using an avalanche photodiode. The z-average diameter, dispersity index (DI), and distribution profiles of the samples were automatically calculated using the software provided by Malvern.

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The virus was inactivated by exposure to UV radiation with a wavelength of 254 nm before the experiments. The solutions containing copolymers (0.1 mg/ml in 0.015 M NaCl) and HSV-1 were prepared. All measurements were carried out in triplicate at 25°C.

Results Cytotoxicity To evaluate the influence of tested compounds on cell viability, XTT assay was performed. Vero E6 cell line was used in this assay as a commonly accepted model for herpesvirus infection along with primary human skin fibroblasts (HSFs). Cell monolayers were cultured for 2 days in the presence of block copolymers and their viability was tested. Results are presented in Figure 1. Most of the tested compounds was not toxic to Vero E6 cells at tested concentrations. Only PEGx-b-PMAPTACy polymers with the longest PMAPTAC blocks exhibited ~50% cytotoxicity at the highest concentration (500 μg/ml). For HSFs, both tested compounds, i.e. PEG45-b-PMAPTAC52 and PEG45-b-PMAPTAC74 (see Inhibition of HSV-1 replication by cationic block copolymers section below) were toxic in concentrations above 100 μg/ml.

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Figure 1. Cytotoxicity of cationic block copolymers. Cell viability was determined by XTT. (A) A wide library of PEGx-b-PMAPTACy polymers was tested in the range of concentrations 50 - 500 μg/ml in Vero E6 cell line. (B) Two selected copolymers were tested for cytotoxicity in primary HSFs. Cell viability was calculated in reference to untreated cells (control). Results are presented as an average ± SEM. Similar results were obtained in at least three replicates in three independent experiments.

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Inhibition of HSV-1 replication by cationic block copolymers To determine, whether PEGx-b-PMAPTACy polymers possess the ability to hamper viral infection, virus replication assay (Assay 0) was performed for all tested compounds at 10 μg/ml. Results presented in Figure 2A show that all tested copolymers inhibited HSV-1 replication, as assessed using quantitative polymerase chain reaction (qPCR) test. No obvious correlation between DPPEG, DPPMAPTAC, DPPEG/DPPMAPTAC ratio, and molecular weight was noted. The strongest inhibitory effect was observed for PEG45-b-PMAPTAC52, while PEG45-b-PMAPTAC74 exhibited the weakest antiviral activity. Therefore, these two compounds were selected for further comparative studies. To determine their effectiveness in primary cells, Assay 0 was performed on HSF cultures (Figure 2B). For HSV-2 no statistically significant inhibitory effect was observed (data not shown).

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Figure 2. PEGx-b-PMAPTACy polymers inhibit HSV-1 replication. PEGx-b-PMAPTACy polymers at 10 μg/ml were present before, during and after HSV-1 infection. Vero E6 (A) and primary HSF (B) cell culture supernatants were collected two days p.i. and the amount of viral DNA was determined by qPCR. The difference in viral yield is presented on the y-axis as log reduction value (LRV), showing the relative decrease in the amount of virus in cell culture media compared to the control. Results are presented as average ± SEM. Similar results were obtained in at least three replicates in three independent experiments.

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Next, effective concentrations of the two selected compounds were assessed using qPCR and a standard plaque assay. The dose-response results are presented in Figure 3. Calculated IC50 values were 0.36 ± 0.08 µg/ml for PEG45-b-PMAPTAC52 and 0.84 ± 1.24 µg/ml for PEG45-b-PMAPTAC74, as determined by qPCR, and 0.59 ± 0.12 µg/ml for PEG45-b-PMAPTAC52 and 1.06 ± 0.12 µg/ml for PEG45-b-PMAPTAC74, as determined by plaque assay.

Figure 3. Dose-dependent inhibition of HSV-1 replication by cationic block copolymers. Viral yield in samples was quantified by qPCR (A) or plaque assay (B). For qPCR, the virus quantity is presented as log reduction value (LRV) of viral DNA copies number per milliliter and for plaque assay as LRV of plaque forming units (PFU). Results are presented as average ± SEM. Similar results were obtained in at least three replicates in three independent experiments.

Mechanism of antiviral activity A set of functional assays was carried out to delineate the mechanism of anti-HSV-1 activity of cationic block copolymers, as described before35. PEG45-b-PMAPTAC52 and PEG45-b-PMAPTAC74 were selected for mechanistic studies, as they both inhibit HSV-1 but to a different extent. Page 17 of 36



First, virus particles were pre-treated with polymers to test whether they inactivate HSV-1 virions (Assay I). No decrease in viral titers and yields was observed for both polymers, although interactions between HSV-1 and polymers was observed using dynamic light scattering (Figure S1 in Supporting Information) and capillary electrophoresis (data not shown). Subsequently, the ability of the compound to interact with the cell to prevent the infection was examined (Assay II). Almost complete inhibition of HSV-1 infection was recorded. Next, the influence of cationic block copolymers on viral adhesion to the cell was verified (Assay III). A reduction in viral yields and titers reaching over 99% and 90%, respectively, was observed. Similar inhibition was observed when polymers were present during virus replication, assembly, and release (Assay IV). The results are presented in Figure 4.

Figure 4. Mechanism of HSV-1 infection inhibition by cationic block copolymers. Tested compounds were added on different stages of viral infection. Samples were analyzed by qPCR (A) and plaque assay (B). Obtained results were normalized to the control sample not treated with polymers. Results are presented as average ± SEM. assay I: inactivation of virion; assay II:

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cell protection; assay III: virus attachment; assay IV: inhibition of virus replication, assembly, and release. Similar results were obtained in at least three replicates in three independent experiments.

Obtained results show that the polymers inhibit the infection already during the early stages of the replication cycle, interacting with the cell and blocking the virus entry and subsequent replication. However, polymers 2 h p.i. also hampered HSV-1 replication and progeny production. Apparent enhancement of infection by incubation of the virus with the copolymers visible in qPCR (Figure 4, Assay I) was not confirmed by plaque assay. To delineate the exact mechanism of polymer action, confocal imaging was performed to visualize single virus particles and to track their fate in the presence or absence of the polymers. Samples were prepared as described for Assay II and Assay III. The cells were then fixed and stained for HSV-1 and F-actin. Maximal projections of XY stacks are presented in Figure 5A. The number of HSV-1 particles attached to the cell surface or internalized within the cell was calculated using ImageJ FiJi version. The normalized data is presented in Figure 5B.

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Figure 5. Cationic block copolymers inhibit early stages of HSV-1 infection. Cells prepared as described for Assay II and Assay III. Blue – DNA, red – f-actin, green – HSV-1. Representative maximal projections of collected XY stacks are presented (A). Scale bar: 10 μm. The number of virus particles (counts) per cell was assessed using ImageJ FiJi software and is presented as % of control (untreated cells) (B). The data is presented as mean ± SEM and was calculated from at least 10 different cells. To determine the significance of differences between compared groups, one-way ANOVA with post hoc Tukey HSD test was used. Values statistically

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significant are indicated by asterisks: **p

Highly Effective and Safe Polymeric Inhibitors of Herpes Simplex Virus

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