Water-Soluble Fullerene C60 Derivatives Are Effective Inhibitors of Influenza Virus Replication

The influenza virus genome features a very high mutation rate leading to the rapid selection of drug-resistant strains. Due to the emergence of drug-resistant strains, there is a need for the further development of new potent antivirals against influenza with a broad activity spectrum. Thus, the search for a novel, effective broad-spectrum antiviral agent is a top priority of medical science and healthcare systems. In this paper, derivatives based on fullerenes with broad virus inhibiting activities in vitro against a panel of influenza viruses were described. The antiviral properties of water-soluble fullerene derivatives were studied. It was demonstrated that the library of compounds based on fullerenes has cytoprotective activity. Maximum virus-inhibiting activity and minimum toxicity were found with compound 2, containing residues of salts of 2-amino-3-cyclopropylpropanoic acid (CC50 > 300 µg/mL, IC50 = 4.73 µg/mL, SI = 64). This study represents the initial stage in a study of fullerenes as anti-influenza drugs. The results of the study lead us conclude that five leading compounds (1–5) have pharmacological prospects.


Introduction
Influenza viruses are highly contagious pathogens posing a major threat to human health. Circulating influenza viruses are estimated by the World Health Organization (WHO) to infect 5-10% of the population every year, causing a morbidity rate of 3 to 5 million and mortality rate of 250,000-500,000 deaths annually [1]. Vaccination remains the most effective tool to protect humans against influenza infection. However, vaccination does not always provide complete protection against drifted or, more noticeably, shifted influenza viruses [2]. Antivirals will also play a central role in the treatment and prophylaxis of influenza infections in a pandemic situation, as specific vaccines will take many months to produce. Although there are approved drugs for the treatment of influenza infections, influenza viruses resistant to current antivirals have been reported and continue to emerge. Therefore, there is an urgent need for novel antivirals for the treatment of influenza viral infections in humans.
Three classes of antiviral compounds (M2 inhibitors, neuraminidase inhibitors, viral polymerase complex inhibitors) have been developed and are proven effective in preventing and treating infections caused by susceptible influenza viruses. A range of novel antiviral were obtained in the form of powders. Weighed portions of these were dissolved in α-MEM medium.

Culture Medium
Alpha MEM growth medium (Corning, Corning, NY, USA) containing 4 mM L-glutamine, 5% fetal bovine serum FBS (Gibco, Waltham, MA, USA), and penicillin/streptomycin antibiotic (Gibco, Waltham, MA, USA) was used for cell cultivation. For experiments, growth culture medium was replaced with a serum-free maintenance medium with the addition of trypsin (1 µg/mL) for the cultivation of influenza viruses.

Synthesis of Water-Soluble Fullerenes
Compounds 1-3 were synthesized from chlorofullerene C 60 Cl 6 and amino acids using the approach reported earlier [15,16]. Fullerene derivative 4 was obtained by direct arylation of C 60 Cl 6 with 3-(3-phenylpropanamido)propanoic acid [17]. Reaction of chlorofullerene C 60 Cl 6 with methyl 2-(thiophen-2-yl)acetate, followed by the treatment with PPh 3 /H 2 O and hydrolysis of the ester groups, allowed us to obtain compound 5, as described in our recent work [18]. Synthesis and characterization of fullerene derivatives 1 [16], 2 [19], 4 [19], and 5 [18] was reported earlier. Compound 3 was obtained as described in the Supplementary Materials and characterized in the form of a tert-butyl ester . Information on the synthesis and characterization of compounds 37 and 39 is also available in the Supplementary Materials. References for the synthesis and characterization of compounds 1, 2, 4-36, 38, and 40-45 are provided in Table 1. Oseltamivir carboxylate was used as a reference compound [20,21].

Cytotoxicity Assay
The cytotoxicity of fullerene derivatives was studied using the microtetrazolium test (MTT) [22]. MDCK cells were seeded into 96-well plates for 24 h until a 90% monolayer was formed. Next, cells were incubated with the test substances in a range of concentrations (4-300 µg/mL) for 72 h. The plate was then washed, and 0.1 mL of MTT solution (0.5 µg/mL) was added to each well and incubated for 2 h in 37 • C (humidified atmosphere at 5% CO 2 ). Next, the medium was removed, 0.1 mL of dimethyl sulfoxide (DMSO) solution was added to each well, and the resulting formazan was extracted. Optical densities in the wells were measured on a Thermo Multiskan FC spectrophotometer (Thermo Scientific, USA) at a wavelength of 535 nm. The optical density of wells without fullerene derivatives was designated as the negative control (100% viability). Determination of 50% cytotoxic concentration (CC 50 ), causing the loss of viability of 50% of cells, was carried out using the GraphPad Prism 7.0.

CPE Reduction Assay
MDCK cells were seeded into 96-well plates for 24 h until a 90% monolayer was formed. Test substances were added in a range of concentrations (3-300 µg/mL) into the wells of the plates with a cellular monolayer in a volume of 0.1 mL. Plates were incubated for 30 min at 36 • C (5% CO 2 ). Next, virus (MOI 0.01) was added to the corresponding wells in maintenance medium with the addition of trypsin and incubated for 1 h. Further, the medium was removed from the wells and replaced with 200 µL of fresh medium with the test compounds at appropriate concentrations. The plates were incubated in 5% CO 2 at 36 • C for 72 h. Next, cell viability was determined using MTT assay as described above. The 50% inhibitory concentration (IC 50 ), causing protection of 50% of infected cells, was calculated using GraphPad Prism 7.0 (USA). Based on the data, the selectivity index (SI) was calculated as the ratio of CC 50 to IC 50 for each substance.

Time-of-Addition Experiments
To determine the stage of the influenza viral life cycle that is affected by the compounds, MDCK cells were seeded in 24-well plates and incubated in 5% CO 2 at 36 • C for 24 h until a confluent monolayer was formed. The cells were washed to remove growth medium. Influenza virus (MOI 0.1) A/Puerto Rico/8/34 (H1N1) was then introduced into the wells. Plates with cells were incubated for 1 h at +4 • C. The virus was then removed, and the plates were washed with clean maintenance medium. Next, at point 0, the substances were introduced into the wells and the plates were placed in a CO 2 incubator at 37 • C. The compounds were added at the following times relative to the addition of the virus: (-2)-the drug was introduced one hour before the addition of the virus, (-1)-0-simultaneously with the virus, and also 2, 4, and 6 h after infection. The interval (-2)-8 was used as a positive control, where the compounds were present throughout the experiment.
Subsequently, a series of ten-fold dilutions (10 −1 -10 −7 ) was prepared from the supernatant and added to 96-well plates with a MDCK cell monolayer. The plates were incubated for 72 h at 37 • C (5% CO 2 ). The results of the experiment were ascertained using the hemagglutination assay. For this purpose, 0.1 mL of culture medium was transferred into round-bottom wells and mixed with a 0.1 mL suspension of 1% chicken erythrocytes. The results were checked after 1 h incubation at room temperature. Viral titers were expressed as lgTCID 50 /0.2 mL.

Virus Yield Reduction Assay
MDCK cells were infected with influenza A/Puerto Rico/8/34 (H1N1) virus (MOI of 0.1). Infected cells were incubated at 36 • C for 72 h in a maintenance medium containing antibiotic and trypsin in the presence of the most promising compounds. At the indicated time point (8, 12, 24, 48, 72 h), the culture medium of the infected cells was harvested, and the titer of the progeny virus in the medium was determined by end-point titration, followed by hemagglutination assay, as previously described.

Statistical Analysis
Viral titers were calculated according to the method of Reed and Mench and expressed as log of the 50% infectious dose. Calculation of 50% cytotoxic concentration (CC 50 ) and 50% inhibitory concentration (IC 50 ) values was performed using GraphPad Prism 7.0 (USA). A 4-parameter equation of the log curve was taken as a working model for analysis (menu items "Nonlinear regression"-"logarithm of the inhibitor-response"). Based on the obtained data, the selectivity index (SI) was calculated for each compound, i.e., the ratio of CC 50 to IC 50 . Comparison of multiple unrelated samples was performed using the Kruskal-Wallis test (p < 0.05).

Anti-Influenza Activity of Fullerene Derivatives
A total of 45 fullerene derivatives (Table 1), differing in structure of solubilizing addends, were studied. Compounds containing thiophene fragments exhibited antiviral activity but were more toxic than other active compounds. It should be noted that fullerene derivatives wherein the sixth addend is an alkyl group (methyl and ethyl) were less active, and more toxic, than their analogues with a hydrogen atom.
Taking into account that C 60 fullerene itself has low toxicity, the obtained data on low CC 50 values (<300 µg/mL) for more than 64.4% of derivatives (29 out of 45) are due to the cytotoxic activity of addends. A total of 4 out of the 45 studied compounds (17.7%) had a pronounced antiviral effect and minimal toxicity, among which 5 leading compounds (1-5, Figure 1) were found to have the maximum inhibitory effect against the influenza virus ( Table 1). The highest activity and minimal toxicity (CC 50 > 300 µg/mL, IC 50 = 4.73 µg/mL, SI = 64) was demonstrated by compound 2 (containing residues of salts of 2-amino-3-cyclopropylpropanoicacid). Compound 38 (with five attached residues of potassium salt of 2-([1,1′-biphenyl]-2yloxy)acetic acid and a chlorine atom) combines the properties of the most active and toxic functionalized fullerene derivative (CC50 = 1.47 µg/mL, IC50 = 0.24 µg/mL), while having low SI = 6. Taking into account that C60 fullerene itself has low toxicity, the obtained data on low CC50 values (<300 µg/mL) for more than 64.4% of derivatives (29 out of 45) are due to the cytotoxic activity of addends. A total of 4 out of the 45 studied compounds (17.7%) had a pronounced antiviral effect and minimal toxicity, among which 5 leading compounds (1-5, Figure 1) were found to have the maximum inhibitory effect against the influenza virus ( Table 1). The highest activity and minimal toxicity (CC50 > 300 µg/mL, IC50 = 4.73 µg/mL, SI = 64) was demonstrated by compound 2 (containing residues of salts of 2-amino-3-cyclopropylpropanoicacid). In order to evaluate the activity spectrum of test compounds, we studied their virusinhibiting properties against a panel of influenza virus subtypes of different origin in vitro. The results are presented in Figure 2. The most promising compounds demonstrated a pronounced inhibitory effect against different influenza virus subtypes. In order to evaluate the activity spectrum of test compounds, we studied their virusinhibiting properties against a panel of influenza virus subtypes of different origin in vitro. The results are presented in Figure 2. The most promising compounds demonstrated a pronounced inhibitory effect against different influenza virus subtypes. Compound 38 (with five attached residues of potassium salt of 2-([1,1′-biphenyl]-2yloxy)acetic acid and a chlorine atom) combines the properties of the most active and toxic functionalized fullerene derivative (CC50 = 1.47 µg/mL, IC50 = 0.24 µg/mL), while having low SI = 6. Taking into account that C60 fullerene itself has low toxicity, the obtained data on low CC50 values (<300 µg/mL) for more than 64.4% of derivatives (29 out of 45) are due to the cytotoxic activity of addends. A total of 4 out of the 45 studied compounds (17.7%) had a pronounced antiviral effect and minimal toxicity, among which 5 leading compounds (1-5, Figure 1) were found to have the maximum inhibitory effect against the influenza virus ( Table 1). The highest activity and minimal toxicity (CC50 > 300 µg/mL, IC50 = 4.73 µg/mL, SI = 64) was demonstrated by compound 2 (containing residues of salts of 2-amino-3-cyclopropylpropanoicacid). In order to evaluate the activity spectrum of test compounds, we studied their virusinhibiting properties against a panel of influenza virus subtypes of different origin in vitro. The results are presented in Figure 2. The most promising compounds demonstrated a pronounced inhibitory effect against different influenza virus subtypes. The fullerene derivative with attached residues of the potassium salt of 3-aminopropanoic acid (1) suppressed the replication of influenza viruses of strains A/Puerto Rico/8/34 (H1N1) and A/California/07/09 (H1N1)pdm09. At the same time, compounds containing residues of salts of 2-amino-3-cyclopropylpropanoic (2), 2-amino-3-(benzo[b]thiophen-3-yl)propanoic (3), and 3-(3-phenylpropanamido)propanoic acids (4) in their structure were also active against the oseltamivir-resistant A/Vladivostok/2/09 (H1N1) strain and the A/mallard/Pennsylvania/1984 (H5N2) avian influenza strain.

Time-of-Addition Experiments
The mechanisms of inhibitory action of the lead compounds 1-5 ( Figure 1) were studied in time-of-addition tests. Infected cells were incubated with leader compounds at different stages of the viral replication cycle. To determine the stage of the viral life cycle at which the leader compounds exhibit maximum antiviral activity, test substances were added to the infected cell culture at different time points relative to the moment of infection. Then, after passing through one viral cycle (8 h), the infectious activity of viral progeny was evaluated. The results of the experiment are shown in Figure 3 using compound 1 as an example. Herafter, 'hours post infection' will be abbreviated (hpi).

Time-of-Addition Experiments
The mechanisms of inhibitory action of the lead compounds 1-5 ( Figure 1) were studied in time-of-addition tests. Infected cells were incubated with leader compounds at different stages of the viral replication cycle. To determine the stage of the viral life cycle at which the leader compounds exhibit maximum antiviral activity, test substances were added to the infected cell culture at different time points relative to the moment of infection. Then, after passing through one viral cycle (8 h), the infectious activity of viral progeny was evaluated. The results of the experiment are shown in Figure 3 using compound 1 as an example. Herafter, 'hours post infection' will be abbreviated (hpi).  The life cycle of the influenza virus consists of seven stages, in which strictly defined viral components play a key role in the replicative process. Direct-acting drugs can display activity at specific stages of the viral life cycle, thus inhibiting the entire replicative cycle. Consequently, determination of the stage of the influenza virus life cycle affected by the studied compounds allows us to draw a conclusion about the drug's viral target. In accordance with the results, the viral titer in the control was 10 5 TCID50/0.2 mL. The presence of compound 1 at the early stages of the viral life cycle (with simultaneous incubation of cells with the virus (-1) for 8 hpi, in the first hours of the viral life cycle for 0-8 hpi) led to a decrease in the infectious activity of viral progeny. The inhibitory effect of the leader compounds weakened when they were added at later stages of the viral cycle. The obtained temporal characteristic (of leading compound activity) suggests that the mechanism of action of new fullerene derivatives is based on inhibition of the early stages of viral replication, including the process of virus adsorption onto cells upon contact of the drug with extracellular virions.

One-Step Growth Curve in the Presence of the Most Active Compounds 1-5
In order to characterize the mechanism of antiviral action of the fullerene derivatives on the replication of the influenza A/Puerto Rico/8/34 (H1N1) virus, a one-step growth curve was examined in the presence of the test compounds. In the absence of the test substances, the infectious titer of the virus was 3 lg TCID50/0.2 mL after just 8 hpi, reaching a plateau at 24 hpi (Figure 4). The addition of water-soluble fullerene derivatives at the beginning of viral replication caused a notable decrease in the number of infectious viruses produced during this period, followed by restoration of infectious virus formation from 12 to 48 hpi. This was likely due to non-specific decomposition or metabolic degradation of the compounds over time. The life cycle of the influenza virus consists of seven stages, in which strictly defined viral components play a key role in the replicative process. Direct-acting drugs can display activity at specific stages of the viral life cycle, thus inhibiting the entire replicative cycle. Consequently, determination of the stage of the influenza virus life cycle affected by the studied compounds allows us to draw a conclusion about the drug's viral target. In accordance with the results, the viral titer in the control was 10 5 TCID 50 /0.2 mL. The presence of compound 1 at the early stages of the viral life cycle (with simultaneous incubation of cells with the virus (-1) for 8 hpi, in the first hours of the viral life cycle for 0-8 hpi) led to a decrease in the infectious activity of viral progeny. The inhibitory effect of the leader compounds weakened when they were added at later stages of the viral cycle. The obtained temporal characteristic (of leading compound activity) suggests that the mechanism of action of new fullerene derivatives is based on inhibition of the early stages of viral replication, including the process of virus adsorption onto cells upon contact of the drug with extracellular virions.

One-Step Growth Curve in the Presence of the Most Active Compounds 1-5
In order to characterize the mechanism of antiviral action of the fullerene derivatives on the replication of the influenza A/Puerto Rico/8/34 (H1N1) virus, a one-step growth curve was examined in the presence of the test compounds. In the absence of the test substances, the infectious titer of the virus was 3 lg TCID 50 /0.2 mL after just 8 hpi, reaching a plateau at 24 hpi (Figure 4). The addition of water-soluble fullerene derivatives at the beginning of viral replication caused a notable decrease in the number of infectious viruses produced during this period, followed by restoration of infectious virus formation from 12 to 48 hpi. This was likely due to non-specific decomposition or metabolic degradation of the compounds over time. Data are presented as the mean ± SD of three independent experiments. *-differences are significant relative to "no drug" (Kruskal-Wallis test, p < 0.05).

Discussion
Over the past few years, many research groups have begun to investigate biological application of fullerenes. The fullerene molecule is highly active due to a large amount of double bonds that can react with various radicals, including biologically active ones. It shows high and, in some cases, even unique antioxidant properties. Fullerene can penetrate cell membranes, modulate ion transport, cross the blood-brain barrier, and has adhesive potential. These properties can be used for synthesis of new immunomodulators, adjuvants, and vaccine holders, for example for HIV-antigens. As reported earlier, watersoluble fullerene adducts have photodynamic, antibacterial, as well as pro-and

Discussion
Over the past few years, many research groups have begun to investigate biological application of fullerenes. The fullerene molecule is highly active due to a large amount of double bonds that can react with various radicals, including biologically active ones. It shows high and, in some cases, even unique antioxidant properties. Fullerene can penetrate cell membranes, modulate ion transport, cross the blood-brain barrier, and has adhesive potential. These properties can be used for synthesis of new immunomodulators, adjuvants, and vaccine holders, for example for HIV-antigens. As reported earlier, water-soluble fullerene adducts have photodynamic, antibacterial, as well as pro-and antioxidant capacity. They can be used as scaffolds for the synthesis of anticancer, neuroprotective, and antiviral drugs [10,34]. We have previously reported on high antiherpetic activity with water-soluble fullerene derivatives [18].
The antiviral properties of novel, water-soluble fullerene derivatives, and their mechanismof-action against influenza were studied in vitro. It was shown that compounds from the library of water-soluble fullerenes have cytoprotective activity against the influenza virus. The maximum virus-inhibiting activity and minimum toxicity was shown by the derivative with attached residues of the potassium salt of β-alanine. In the present work, we observed a wide range of activity of water-soluble fullerene derivatives against influenza viruses of differing antigenic subtype. The mechanism of antiviral activity of the most promising compounds is the inhibition of early stages of viral replication (0-2 hpi).
The main issue for biomedical application of fullerenes concerns insolubility in water and aqueous solutions [34]. The current search for the most active fullerene derivative is promising, especially taking into account the possibilities of increasing the bioavailability of a potential drug using liposomal drug delivery complexes [35]. Hydroxylation is one of the cheapest and easiest ways to dissolve fullerenes in water and does not require extensive purification of the resultant product. Many studies are currently focused on non-functionalized (pristine) fullerenes in aqueous dispersions since they do not participate in metabolic processes due to their open surface [36][37][38][39]. However, even slight surface derivatization can increase the activity of fullerenes. In our work, fullerenes with solubilizing addends were studied. This certainly increases their bioavailability and potential medicinal application accordingly.
Thus, this research represents the initial stage in a study of water-soluble fullerene derivatives as anti-influenza drugs. It allows us to conclude that five leading compounds (1-5, Figure 1) have pharmacological prospects. These data are a key advancement that could be used in future strategies to refine fullerene-based drug design. Our analysis provides valuable new information for the design of novel, anti-influenza drugs. We believe the information presented here will be beneficial for the future development of novel fullerene-based drugs against influenza A.
An important issue in the search for antiviral drugs is assessment of the risk of drug resistance development. Further study should evaluate the ability of water-soluble fullerene derivatives to develop viral resistance, potentially leading to the emergence of strains with reduced drug susceptibility. Consequently, amino acid substitutions in the influenza virus proteins which lead to drug resistance should be mapped. The data obtained in this paper serve as a basis for continuing research on water-soluble fullerene derivatives as potential anti-influenza drugs.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author, E.O.S., upon reasonable request.