Anti-Influenza Activity of C60 Fullerene Derivatives

The H1N1 influenza A virus, which originated in swine, caused a global pandemic in 2009, and the highly pathogenic H5N1 avian influenza virus has also caused epidemics in Southeast Asia in recent years. Thus, the threat from influenza A remains a serious global health issue, and novel drugs that target these viruses are highly desirable. Influenza A RNA polymerase consists of the PA, PB1, and PB2 subunits, and the N-terminal domain of the PA subunit demonstrates endonuclease activity. Fullerene (C60) is a unique carbon molecule that forms a sphere. To identify potential new anti-influenza compounds, we screened 12 fullerene derivatives using an in vitro PA endonuclease inhibition assay. We identified 8 fullerene derivatives that inhibited the endonuclease activity of the PA N-terminal domain or full-length PA protein in vitro. We also performed in silico docking simulation analysis of the C60 fullerene and PA endonuclease, which suggested that fullerenes can bind to the active pocket of PA endonuclease. In a cell culture system, we found that several fullerene derivatives inhibit influenza A viral infection and the expression of influenza A nucleoprotein and nonstructural protein 1. These results indicate that fullerene derivatives are possible candidates for the development of novel anti-influenza drugs.


Introduction
In 1918, an influenza A pandemic caused 50 million deaths worldwide [1], and the development of strategies that can be used to prevent future expansions of this virus continues to be an important endeavor [2]. The avian H5N1 influenza A virus is highly pathogenic to humans [3], and the emergence of a new strain of this virus in 2009, the swine-originating A/H1N1 pdm influenza virus, further emphasizes that this issue is a serious global health problem [4,5]. Although inhibitors of influenza A, e.g., the neuraminidase-like compound oseltamivir, are widely used as antiviral drugs [6,7], the adverse effects of these agents and the emergence of viral strains that are resistant to these drugs have now been reported [8,9].
To prevent and control influenza outbreaks, the development of novel antiviral drugs that are not based on neuraminidase inhibition is now regarded as critical. The influenza A genome consists of segmented single-stranded RNA (-), and its transcription and replication require the activity of a highly conserved RNAdependent RNA polymerase [10,11]. This polymerase is essential for the propagation of the influenza A virus and is a very promising target for the development of antiviral drugs. The influenza A virus RNA polymerase is composed of three subunits-PA, PB1, and PB2 [12]-and synthesizes viral mRNA using short capped primers that are cleaved from the host's cellular pre-mRNAs by the viral endonuclease [13,14]. Yuan et al. and Dias et al. have shown that the N-terminal domain of the PA subunit contains the active site of the endonuclease, and that this domain also harbors RNA/DNA endonuclease activity [13,14]. Hence, we speculate that PA endonuclease would contain very effective targets for the development of novel anti-influenza A drugs, as we have shown that several chemicals, e.g., catechins, phenethylphenyl phthalimide analogs, and marchantin analogs, inhibit this endonuclease and possess antiviral activity [15][16][17].
Fullerene (C 60 ), a carbon buckyball, was discovered by Harold Kroto, James R. Heath, Sean O'Brien, Robert Curl, and Richard Smalley in 1985 [18]. It has since been utilized in electronic and mechanical applications [19]. In physiological studies, the biological effects of water-soluble fullerene derivatives containing several hydrophilic groups are noteworthy because fullerene itself is water-insoluble. Water-soluble fullerene derivatives are known to possess various biological and pharmacological properties, including antioxidant activity and inhibitory effects against human immunodeficiency virus (HIV) proteases and DNA photocleavage [20][21][22][23]. Mashino et al. also demonstrated that pyrrolidinium fullerene derivative 6 ( Fig. 1) has antiproliferative and antibacterial activity [24], malonic acid fullerene derivative 2 ( Fig. 1) has excellent antioxidant activity [25], and proline-modified fullerene derivative 3 ( Fig. 1) inhibits HIV-reverse transcriptase [22]. Thus, fullerene derivatives are expected to become a novel type of medication because of their unique skeleton.
In our current study, we used an in vitro influenza PA endonuclease assay to analyze the effects of 12 different fullerene derivatives on the endonuclease activity of the PA N-terminal domain and full-length PA. We found that the fullerene derivatives inhibit influenza PA endonuclease activity and viral infection. Our results indicate the possibility of developing fullerene derivatives into novel anti-influenza A drugs in the future.

Inhibition of PA Endonuclease by Fullerene Derivatives
For the in vitro PA endonuclease assay, we expressed and purified a recombinant influenza PA endonuclease domain (1-220 residues; Fig. 2A, B) using bacteria as described previously [15][16][17]. For the assay, we incubated 0.1 mM recombinant PA endonuclease domain with 1 or 10 mM of each fullerene derivative (Fig. 2C). The PA endonuclease domain digested M13 mp18 circular single-stranded DNA in vitro (Fig. 2C, lanes 1 and 2) [12][13][14][15][16], and we investigated whether any of the fullerene derivatives could inhibit this activity. The fullerene derivatives 2-5, 7, 8, 10, and 11 significantly inhibited the digestion of M13 mp18 at a dose of 10 mM, no. 12 slightly inhibited digestion, and no. 1, 6, and 9 had no or weak inhibitory activity (Fig. 2C). This is the first study to report that fullerene derivatives can inhibit the activity of influenza enzymes. Fullerene derivative no. 6 caused a mobility shift of M13 mp18, possibly because it has a cationic amine group (Fig. 2C, lane 13). The solubility of the fullerene derivatives 1 and 6 in water was relatively low, which might have been the cause of their decreased activity levels.
To investigate the effects of full-length PA protein on PA endonuclease activity and the inhibitory activity of the fullerene derivatives, we examined whether the fullerene derivatives also inhibit the endonuclease activity of full-length PA protein. We expressed the recombinant full-length PA protein by using a baculovirus to infect Sf9 insect cells (Fig. 3A) and purified it using a Ni-agarose and HiTrap-Q column (Fig. 3B). The recombinant full-length PA protein demonstrated endonuclease activity (Fig. 3C, lanes 1 and 2) [26,27]. Then, we tested the 12 fullerene derivatives using this same assay. The fullerene derivatives 2-5, 7, 8, 10, and 11 inhibited the endonuclease activity of full-length PA (Fig. 3C), which is consistent with the results for the PA endonuclease domain (Fig. 2C). The M13 mp18 band in the no. 12-treated lane was degraded (Fig. 3C lane 14), the band in the no. 1 and 9treated lanes slightly remained (Fig. 3C, lanes 3 and 11), suggesting that fullerene derivatives no. 1 and 9 also slightly inhibited the PA endonuclease activity of the full length PA protein.
As shown in Figs. 2C and 3C, the M13 mp18 band in the no. 6treated lanes shifted and was clear ( Fig. 3C lane 8), respectively, suggesting that no. 6 has the ability to cleave DNA. Thus, we examined the nuclease activity of the fullerene derivatives (Fig. 4). The result showed that fullerene derivative no. 6 by itself has  significant nuclease activity in the absence of PA endonuclease (Fig. 4). No. 12 also showed weaker nuclease activity by itself (Fig. 4).

Docking Simulation of Influenza A Endonuclease and C 60 Fullerene
Previously, we reported that 3 of 34 phthalimide chemicals and 5 of 33 phytochemicals inhibited PA endonuclease activity [12,15,16]. In the case of the fullerenes, 8 of the 12 fullerene derivatives inhibited PA endonuclease activity; thus, we thought that the fullerene skeleton itself could fit into the active pocket of the influenza PA endonuclease domain. To investigate how the fullerene molecule binds to and fits in the active pocket of the PA endonuclease domain, we performed in silico docking simulation analysis of this interaction at the level of the tertiary structure using Molecular Operating Environment software (MOE; Chemical Computing Group, Quebec, Canada) [28]. The results show that fullerene fits into and fills the active pocket of the endonuclease domain of the influenza RNA polymerase (Fig. 5A), suggesting that this may be the major cause of the inhibitory mechanism. The two divalent ions of manganese in the active pocket are reportedly necessary for influenza endonuclease activity [13,14]. Fullerene binds to manganese ions by arene-cation interactions at the back of the active pocket ( Fig. 5B and 5C), suggesting that this binding is also one of the inhibitory mechanisms.

Toxicity of the Fullerene Derivatives Against the Madin-Darby Canine Kidney Cell Line
We evaluated the toxicity of the fullerene derivatives against Madin-Darby canine kidney (MDCK) cells before examining their antiviral activity against the influenza A virus. Various concentrations (12.5-100 mM) of the fullerene derivatives were added to cultures of MDCK cells. Marchantin E (ME) was used as the positive controls for anti-influenza activity [16]. At 24 h postincubation, the cell viability of the treated-cells was determined using an MTT cell proliferation assay (Fig. 6A). The viability of the cells treated with the fullerene derivatives 1-12 and ME was not significantly different to that of the cells treated with dimethyl sulfoxide (DMSO) at a concentration of 12.5 to 100 mM. We also performed naphthol blue black assay for cytotoxicity of fullerene derivatives (Fig. 6B). At 24 h post-incubation, the viable cells were stained using a blue dye. The wells treated with 0.8-100 mM of the fullerene derivatives 1-12 and DMSO were stained blue (Fig. 6B). Taken together, these data show that the fullerene derivatives (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) are not toxic to MDCK cells up to a concentration of 100 mM.    (Table 1). Taken together, it indicated that several fullerene derivatives have stronger antiinfluenza activity than ME.
Moreover, we examined the expression levels of viral proteins by western blotting of treated-cell lysates in A/PR8/34 (H1N1)infected wells at 4, 8, 12 (Fig. 8A), and 24 h (Fig. 8B) postinfection. The expression levels of influenza A NP and nonstructural protein 1 (NS1) proteins in the cells treated with the fullerene derivatives 5, 6, and 11, and ME were reduced as compared with that of the DMSO-treated cells, but slightly reduced in the wells  treated with the fullerene derivatives 2-5, 7, 8, 10 and 12 ( Fig. 8A  and 8B). Conversely, the expression levels of influenza A NP and NS1 proteins in cells treated with the fullerene derivatives 1 and 9 were comparable to those in the DMSO-treated cells (Fig. 8A  and 8B). Taken together, these data show that the fullerene derivatives 2-8 and 10-12 possess antiviral effects against the influenza A virus, and their mechanism of action may be by the inhibition of PA endonuclease activity (no. 2-5, 7, 8, and 11) or their ability to cleave viral RNA (no. 6 and 12).

Discussion
In this study, we showed that the fullerene derivatives 2-5, 7, 8, 10, and 11 or 6 and 12 possess inhibitory activity against influenza PA endonuclease or the ability to cleave DNA, respectively. Moreover, we showed that the fullerene derivatives 2-8 and 10-12 inhibit the infection of the influenza A virus. Above all, no. 6 showed the strongest antiviral activity. A previous report showed that certain fullerene derivatives have DNA and RNA cleavage activity [20,30]. As shown in Figs. 2C and 3C, the M13 mp18 band in the no. 6-treated lanes shifted and was clear, respectively. Since no. 6 has the activity to cleave DNA, the antiviral activity of no. 6 may be caused by its cleavage of viral RNA. Therefore, fullerene derivatives are promising novel anti-influenza chemicals. These data are an important advance that could be used in future strategies to refine fullerene-based drug designs. Our analysis provides valuable new information for the design of novel antiinfluenza drugs. There was no correlation between the PA endonuclease and antiviral activity of fullerene derivative no. 12. This may be because it targets influenza A virus attachment/entry or growth in cells and also because of differences in its permeability into the cells. When we performed the anti-viral experiment without pre-incubation, we could not find an experimental condition under which the fullerene derivatives showed anti-virus activity. This suggests that the fullerene derivatives may have virucidal activity or they may enter cells by associating with the virus.
Other groups also have reported novel anti-influenza polymerase inhibitors such as T-705 and L-742,001 [27,[31][32][33][34][35][36], which are substituted pyrazine and piperidine compounds, respectively. Since the chemical structures of fullerene derivatives are completely different from those of them, indicating that fullerene derivatives are quite novel anti-influenza compounds.
Finally, we conclude that the chemical and biochemical information presented here will be very useful for the future development of novel fullerene-based drugs against influenza A.

Bacterial Expression and Purification of the PA Endonuclease Domain
The influenza A virus (A/PR/8/34 (H1N1)) RNA polymerase PA plasmid, pBMSA-PA, was obtained from the DNA bank at Riken BioResource Center (Tsukuba, Japan; originally deposited by Susumu Nakada) [37]. The cDNA fragment corresponding to the PA N-terminal endonuclease domain (residues 1-220; Fig. 2A) was amplified by polymerase chain reaction (PCR) [38] from pBMSA-PA. The amplified product was subcloned into the pET28a (+) plasmid (Novagen, Madison, WI, USA). The induction of recombinant protein expression was achieved by the addition of isopropyl-D-thiogalactopyranoside [39], and this was followed by purification using Ni 2+ -agarose [40]. The recombinant PA endonuclease domain protein was further purified to near homogeneity (Fig. 2B) using a HiTrapQ-FF column (GE Healthcare, Buckinghamshire, UK).

Baculoviral Expression and Purification of the Full-length PA Subunit
The cDNA fragment corresponding to the full-length proteincoding region of PA was amplified by PCR [38] from pBMSA-PA using the primers PA_start_TOPO (CAC CAT GGA AGA TTT TGT GCG AC), PA stop (CTA ACT CAA TGC ATG TGT AAG), PA_mid_anti (TCT TTG GAC ATT TGA GAC AG), and PA_mid_TOPO (CAC CAA TTG AAG AAA GGT TTG). The amplified product was then subcloned into the pENTR/D-TOPO plasmid (GatewayH, Life Technologies TM , Carlsbad, CA, USA) using topoisomerase I cloning. The resulting construct was then converted to pDEST10-PA using the clonase (Fig. 3A). The plasmid was transfected into Sf9 insect cells using the helper virus. The resultant baculovirus was again transfected into Sf9 cells [41]. The expressed full-length PA protein was purified using Ni 2+agarose and a HiTrap Q FF column (GE Healthcare) with the AKTA prime plus system (Fig. 3B).

PA Endonuclease Activity and Fullerene Derivatives Nuclease Assays
Influenza A RNA polymerase PA endonuclease activity assays were performed as described by Dias et al. with some modifications [12][13][14][15][16]. Briefly, the pH was lowered from 8.0 to 7.3, and 1 mg M13 mp18 single-stranded circular phage DNA was used as the substrate. A total of 0.25 mg recombinant PA endonuclease domain or full-length PA protein were added to 100 mL assay buffer for each reaction (the final concentration of the protein was approximately 0.1 mM). For the fullerene derivative nuclease assay, no PA protein was added at this step. The fullerene derivatives (summarized in Fig. 1) were then added to the reaction, and the products were analyzed by agarose electrophoresis and stained with ethidium bromide.
In Silico Docking Simulation Analysis of Fullerene C 60 and the PA Endonuclease Domain of Influenza RNA Polymerase All molecular modeling studies were performed using MOE software (Chemical Computing Group) [26,42,43]. Information regarding the tertiary structure of the influenza PA endonuclease domain (PDB ID: 3HW6) was obtained from a protein data bank [29]. This enzyme was prepared for docking studies in which (1)  the ligand molecule was removed from the active site of the enzyme; (2) hydrogen atoms were added to the structure using standard geometry; (3) the structure was minimized using an MMFF94s force-field; (4) MOE Alpha Site Finder was used to search for active sites within the enzyme structure and dummy atoms were created from the obtained alpha spheres; and (5) the obtained model was then used in the Dock program (Ryoka Systems Inc., Tokyo, Japan). The conformation of the fullerene was generated by systematic, stochastic searches and Low Mode MD (molecular dynamics).

MTT Cell Proliferation Assay
The cytotoxicity of the fullerene derivatives in MDCK cells was determined with an MTT cell proliferation assay kit according to the manufacturer's instructions (Cayman, Arbor, MI, USA). Briefly, MDCK cells were cultured in Dulbecco's modified Eagle medium (DMEM; Gibco/Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 4 mM L-glutamine at 37uC under 5% CO 2 . A confluent monolayer of MDCK cells was prepared in each well of a 96-well plate. Various concentrations (12.5-100 mM) of the fullerene derivatives and ME in DMSO (100 mM chemicals: 1%, 50 mM: 0.5%, 25 mM: 0.25%, 12.5 mM: 0.125%.), which were used as the anti-influenza activity [16], were mixed in an infection medium (DMEM supplemented with 1% bovine serum albumin, 1% penicillin-streptomycin, and 4 mM L-glutamine). The mixture was added to the cells, and the treated cells were incubated for 24 h at 37uC under 5% CO 2 . After incubation, the cells were treated with the MTT reagent and incubated for 4 h at 37uC under 5% CO 2. The wells were treated with the crystal dissolving solution to lyse the formazan produced in the cells, and the absorbance of each well was measured at 570 nm using a microplate reader.

Cytotoxicity Assay by Naphthol Blue Black
A confluent monolayer of MDCK cells was prepared in each well of a 96-well plate. Various concentrations (0.8-100 mM) of the fullerene derivatives in DMSO were mixed with an infection medium (DMEM supplemented with 1% bovine serum albumin, 1% penicillin-streptomycin, and 4 mM L-glutamine) and incubated for 30 min at 37uC under 5% CO 2 [43]. The mixture was added to the cells, and the treated cells were incubated for 24 h at 37uC under 5% CO 2 . After incubation, the cells were fixed using a 10% formaldehyde solution. Viable cells were stained with a naphthol blue black solution (0.1% naphthol blue black, 0.1% sodium acetate, and 9% acetic acid) [43].

Immunostaining of Influenza A Virus-infected Cells
MDCK cells were prepared in each well of a 96-well plate. Various concentrations (25-100 mM) of the fullerene derivatives and ME were mixed at a multiplicity of infection (MOI) of 1 influenza A virus (A/PR/8/34 (H1N1) or A/Aichi/2/68 (H3N2)) in the infection medium and incubated for 30 min at 37uC under 5% CO 2 . The mixture was added to the cells, and the treated cells were incubated for 24 h at 37uC under 5% CO 2 . After incubation, the cells were fixed with 4% paraformaldehyde in phosphatebuffered saline (-) for 30 min at 4uC and then permeabilized with 0.3% Triton X-100 for 20 min at room temperature. A mouse anti-influenza A NP antibody (FluA-NP 4F1; SouthernBiotech, Birmingham, AL, USA) and horseradish peroxidase-conjugated goat anti-mouse IgG antibody (SouthernBiotech) were used as primary and secondary antibodies, respectively [44]. To visualize the infected cells, TrueBlue peroxidase substrate (KPL, Gaithersburg, MD, USA) was added, and color development was terminated after 15 min of incubation by washing with H 2 O. The wells were photographed under a microscope, and the stained cells were counted.

Western Blotting
MDCK cells were prepared in each well of a 24-well plate. We mixed 100 mM of the fullerene derivatives and ME at an MOI of 1 influenza A virus (A/PR/8/34 (H1N1)) in the infection medium and incubated the solution for 30 min at 37uC under 5% CO 2 . The mixture was added to the cells, and the treated cells were incubated for 4, 8, 12, and 24 h at 37uC under 5% CO 2 . After incubation, the cells were lysed with a sodium dodecyl sulfate buffer (125 mM Tris-HCl, pH 6.8, 5% sodium dodecyl sulfate, 25% glycerol, 0.1% bromophenol blue, and 10% b-mercaptoethanol) and boiled for 5 min. The cell lysates were then loaded onto a 10% polyacrylamide gel. The proteins were transferred to a polyvinylidene fluoride microporous membrane (Millipore, Billerica, MA, USA). For primary antibodies, mouse anti-influenza A NP antibody (FluA-NP 4F1; SouthernBiotech) and goat antiinfluenza A NS1 antibody (vC-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used to detect NP and NS1, respectively. A rabbit anti-b-actin antibody (13E5; Cell Signaling, Danvers, MA, USA) was used as an internal control. Horseradish peroxidase-conjugated goat anti-mouse IgG antibody (South-ernBiotech), donkey anti-goat IgG antibody (sc-2020; Santa Cruz Biotechnology) and goat anti-rabbit IgG antibody (KPL) were used as secondary Abs. The blots were developed by using Western Lightning ECL Pro (PerkinElmer, Waltham, MA, USA).

Statistical Analysis
All results were expressed as the mean 6 standard error of the mean. Differences were analyzed for statistical significance by oneway analysis of variance (ANOVA) for comparison among the DMSO, ME and fullerene derivative-treated groups. The results were considered significantly different at *p,0.05 and **p,0.01 when comparing the number of stained cells in the ME or fullerene derivatives-treated groups to that of the DMSOtreated group.