N-stearoylethanolamine — a new inhibitor of the hepatitis C virus reproduction

Aim. The study of the effect of endogenous cannabimimetic compound - N-stearoylethanolamine (NSE) on the hepatitis C virus (HCV) reproduction. Methods. The model of the surrogate HCV is a bovine diarrhea virus; cell culture model is cells transfected with cDNA of the human HCV and molecular docking has been used. Results. In vitro studies showed that NSE effectively inhibited the reproduction of a surrogate HCV in both MDBK cells and transfected Jurkat cells. Molecular docking suggested that NSE can bind to the active centers of both NS3 serine protease and HCV NS5B-polymerase and has an inhibitory effect on their activity. Conclusions. The obtained data confirm that using NSE is promising for the develop-ment


Introduction
Hepatitis C (HCV) is a viral infection, which according to WHO affects today more than 170 million people worldwide [1].
Nowadays, new antiviral drugs for hepatitis C virus (HCV) infection, known as oral directacting antiviral agents (ODAs) are available on the market. They are more effective, safer and better-tolerated than existing therapies: 90 % of people are cured [2]. A significant disadvantage of ODAs is their high cost. According to the information above, there is an urgent need to develop and improve additional and alternative strategies in the hepatitis C treatment, in particular, targeting the main metabolic pathways of reproduction/replication and biological action of the HCV. Recent N. M. Hula, A. A. Chumak, A. G. Berdyshev ISSN 1993-6842 (on- research has established an essential role of lipids metabolism in the life cycle of HCV. Therefore, great attention has been paid to the interaction between HCV and cell lipid metabolism, considering the widespread development of steatosis in patients with chronic hepatitis C complications [3]. However, this issue remains poorly investigated. It has been shown that HCV replication was inhibited by statins and fatty acids [4,5]. Additionally, it has been shown that HCVs are secreted by the cell together with low density lipoproteins [6,7] and such products can be metabolically modulated by the addition of insulin or appropriate fatty acids. Some liver lipid modulators, originally developed for the treatment of atherosclerosis, are also potentially capable of preventing the replication of the hepatitis C virus. Target intervention in the metabolic pathway of virus reproduction may be significantly more effective than the direct action on the virus and, in turn, can prevent the formation of strains resistant to antiviral drugs [8,9]. Despite a high potential of this approach, the primary studies conducted with the use of atorvastin and bezafibrat did not meet the expectations -no significant effect of these drugs on the titer of the virus was shown [10,11].

Viruses and Cell
We early reported that N-stearo yletha no lami ne (NSE), which belongs to the class of minor lipids named N-acylethanolamines, showed the antiviral action against the influenza virus (H1N1 strain) [12]. The results obtained in our in vitro experiments, showed that the selectivity index of NSE is 100. This finding allows us to classify NSE as an active antiviral drug. Additionally, in vitro evaluation of NSE cytotoxicity (by determining its effect on mitotic index of cell proliferation) did not reveal any significant effect on cell mitosis. Thus, NSE is a potential non-toxic drug that does not cause the proliferative and mutagenic activities in vitro. The aim of this study was to investigate antiviral activity of the endogenous cannabimimetic compound NSE towards the hepatitis C virus.
NSE was synthesized in the Department of Lipid Biochemistry at Palladin Institute of Biochemistry as described previously [13]. Briefly, ethanolamine and stearic acid were co-condensed in argon atmosphere and upon defined temperature regime and NSE obtained was purified by re-crystallization in ethanol. The end product was verified by Agilent 7890A/5977B GC/MS system analysis.

Viruses and cells cultures
About 10 4 MDBK cells per well were seeded in wells of 96 plates and 24 hours later infected with a 10-fold dilution series of BVDV samples. The medium was replaced after 1 hour of incubation. The infectious titer BVDV in MDBK cells was 5.0 lg TCID 50 /ml.
The antiviral activity of NSE was studied in the MDBK cell culture, to which NSE in various concentrations (10 -10 -10 -6 M) and 100 TCD 50 dose of BVDV were added. The cells were incubated in a thermostat until the specific cytopathogenic action of the virus, and then the infectious titer in the culture medium was determined.
Maximum nontoxic concentration (MNTC) of NSE. The MDBK cells were cultured in 96well plates at the temperature of 37 °C in humidified 5 % CO 2 environment for 5 days. Control cells and cells treated with NSE were examined daily. Cytopathogenic effects (CPE) were estimated by cell morphology (rounding and shrinkage of cells, detachment of degenerative cells from the surface).
Minimum inhibitory concentration (MIC) of NSE. MDBK cells in 96-well plates were infected with 100 TCD 50 /0.1 ml BVDV. Following 1-h absorption at 37 °C, virus-containing medium was washed out and the maintaining medium (RPMI-1640 with 2 % of fetal calf serum) containing NSE within the range of 10 -10 -10 -6 М was added. BVDV in control and NSE-treated cells were then titrated in MDBK cells by CPE. MIC was evaluated by the reduction of the infectious titer by at least 2 lg TCD 50 .
Selectivity index (SI) of NSE. Selectivity index (SI) of NSE as anti-BVDV agent was calculated as [the] MNTC/MIC ratio.

Model of transfected HCV-producing Jurkat cell line
Jurkat cell line from the established cell line of human origin was obtained from Institute of Immunology RAMS (Russian Federation). These cells grew in suspension at 37 °C in the RPMI-1640 medium supplemented with 2 % [of] glutamine and 10 % of heated fetal serum, the CO 2 concentration in the incubator was 5 %.
HCV-producing Jurkat cell line culture model. Jurkat cells were transfected with HCV. As the HCV source we used non-diluted blood plasma samples of HCV-infected patients with different virus load; such plasma contained HCV RNA. All viral RNA preparations were isolated using "RIBO-prep" kit (Russian Federation).
Measurement of [the] HCV content in clinical samples. Quantitative estimation of the HCV mRNA levels in clinical samples was carried out using the real-time PCR with hybridization and fluorescent detection. For this aim, a reagent kit "AmpliSens HCV-Monitor-FRT" (Russia) and an apparatus "Rotor-Gene 3000/6000" ("Corbett Research", Australia) were used. The HCV complementary DNA (cDNA) was synthesized by the reverse transcription reaction using «Reverta-L» (RF) kit. 10 μl of RNA samples were added to 10 μl of prepared reaction mixture (lyophilized plant preparation, 125 μl of the RT-mix solution and 6 μl of the murine leukemia virus (MMLv) revertase); the transcription was carried out at 37 °C during 30 min, and cDNA was obtained.
Transfection procedure using the Turbofect. The transfection was conducted according to the standard protocol for Turbofect (Thermo Scientific, Lithuania). The virus detection was carried out using the PCR on the second passage (9th day of cultivation) and on the fifth one (17th day of cultivation). All the cDNA of hepatitis C patients transfected Jurkat cultures produced HCV on the 9th and 17th days of cultivation [14,15].

Docking procedure
The structures of HCV NS3 serine protease (2A4Q, 4A92) and HCV NS5B polymerase (3PHE, 2IJN) are retrieved from the open access digital data resource Protein Data Bank. Using these structures, we can dock different compounds (Tabl. 1) into the active sites of the HCV proteases and polymerases. This docking is performed after the removal of water molecules, the addition of missing hydrogens and the removal of reference ligands from the protein structure using AutoDock 1.5.6 software tools (ADT) and docking protocol type A described in [16]. The same program determined the amino acid residues involved in binding test ligands to the active sites of NS3 and NS5 proteins. Docking simulations are performed using Autodock Vina 1.1.2 software (ADV) [17]. Both the ligands and proteins are prepared for docking using ADT. The number of points in x-, y-and z-box dimensions are set to 80 with grid spacing of 0.375Aº. Center grid box is set to the center active site of protein. The ligands (excluding NSE) are taken from the crystal structures and re-docked using the same protocol. The structure of NSE was obtained from ChemSpider database as mol file and converted to mol2 format by OpenBabel 2.4.1 software [18]. The lowest free binding energy (∆G) of the ligand to the

Data analysis
All experiments were performed in triplicates. The antiviral activity of the NSE was expressed as the log10 reduction of the viral titer by comparison with untreated controls (inhibition of infectious titer). The standard deviation in the reduction of virus titer was about 0.5 log10.
The NSE was considered active only when the virus yield decreases ≥ 2 log10, at the effective dose ED50.

Results and Discussion
Considering the fact that human HCV cannot be obtained enough in its pure form to perform the experiment, the surrogate bovine diarrhea virus (BVBD) was used in this study. BVBD is morphologically close to the human HCV and does not require special safety procedures to work with [19]. Therefore, this virus was used in our screening studies to test active compound against hepatitis C [13]. The results of the infectious titer of BVDV for MDBK cells with or without NSE addition are presented in Table 2. It is shown that NSE at concentrations of 10 -6 -10 -7 M inhibits BVDV reproduction by 10 times, herewith NSE at concentration of 10 -8 -10 -9 M -by 100 times. NSE in concentration 10 -10 M did not affect the BVDV reproduction.

Table 2. The infectious titer of BVDV in MDBK cells
As was shown the results of morphological test the minimum of toxic concentration of NSE was 10 -4 M and maximum nontoxic concentration of NSE was 10 -5 M.
Maximum nontoxic concentration (MNTC), minimum inhibitory concentration (MIC) and selectivity index (SI) for NSE in MDBK cells was shown in Table 3. The results of selectivity index ( Table 3) estimation indicate that NSE has high antiviral potential to BVDV (or HCV).

Table 3. Maximum nontoxic concentration (MNTC), minimum inhibitory concentration (MIC) and selectivity index (SI) for NSE in MDBK cells
Because BVDV is morphologically close to human HCV we have supposed that the inhibitory NSE action could be extrapolated on HCV.
To obtain the HCV-producing cell cultures, HCV RNA was isolated from infected patients. Subsequently, cDNA, obtained on the HCV RNA matrix, was used for the transfection of Jurkat cells and further PCR analysis (Table 4).
Therefore, as a result of transfection procedures with Turbofect, we obtained a cell culture transfected with cDNA of HCV that shows a stable reproduction of the HCV. The results of studying the NSE effect on the reproduction of HCV in transfected Jurkat cells line on the 5 th day of cultivation presented in table 5 show that NSE at concentrations from 10 -6 M to 10 -9 M effectively inhibits the reproduction of HCV. NSE at concentration 10 -10 M did not affect the HCV reproduction.  Molecular docking is an effective tool to study the mechanisms of biologic processes, compare the effectiveness of various drugs, and predict the molecular and physicochemical properties of molecules. Molecular docking is an important method for studying protein-ligand interactions and facilitating the creation of potent drugs. A dock is a computing tool that places a small molecule (ligand) at the binding site of its macromolecular target (receptor) and evaluates its binding affinity. In molecular docking based on protein structures, thousands of possible positions of association of a ligand with its receptor are tested and evaluated using the energy counting function. The position with the lowest energy index is predicted to be "the best match", that is, the binding mode [20]. In accordance with these considerations, we used molecular docking to study the binding mode of HCV-NS3 protease and HCV-NS5B polymerase known inhibitors and NSE (see Table 1) to various viral proteins.
From Fig. 1 and Table 6 it may be suggested that NSE can bind to the active centres of both the NS3 serine protease and hepatitis C virus NS5B-polymerase and has an inhibitory effect on their activity.
Nonstructural protein 5B (NS5B), the RNAdependent RNA polymerase of Hepatitis C Virus (HCV), plays a key role in viral amplification and is an attractive and most explored target for discovery of new therapeutic agents against Hepatitis C.
Docking of NS3 and NS5B with NSE showed that NSE has a rather high binding affinity (inhibitory constant Ki ≈ 10 -5 M) for the active sites of NS3 and NS5B. Based on this result, it can be assumed that NSE is able to inhibit the hepatitis C virus replication.
Thus, it can be assumed that the observed effect of inhibiting the reproduction of the hepatitis C virus in Jurcat cells (Tabl. 5) is due to the binding of NSE to the active center of the viral proteins (NS3 and NS5B) responsible for the replication of the virus.

Conclusions
The present study has shown the strong antiviral activity of NSE against the human hepa-titis C virus, which allows suggesting NSE as a novel active compound for the treatment of human hepatitis C.