STUDY OF INTERMOLECULAR INTERACTIONS OF ANTIVIRAL AGENT TILORONE WITH RNA AND NUCLEOSIDES

1 B. Verkin Institute for Low Temperature Physics and Engineering of the National Academy of Sciences of Ukraine, 47, Nauky Ave., Kharkov, 61103, Ukraine e-mail: pashynska@ilt.kharkov.ua 2 D.K. Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine, 154, Acad. Zabolotnoho str., 03680, Kyiv, Ukraine e-mail: n.zholobak@gmail.com 3 Institute of Organic Chemistry of Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Magyar tudosok korutja, 2, Budapest, H-1117, Hungary; 4 National University of Food Technologies, 68, Volodymyrska str., 01601, Kyiv, Ukraine Submitted January 29, 2017 Accepted February 23, 2018

Tilorone is known as an effective antiviral and interferon-inducing agent from the seventies of the last century [1][2][3]. Tilorone is a reactant of the national pharmaceutical preparation Amixin IC (InterChem SLC, Odessa, Ukraine) and some other preparations which V.A. Pashynska, N.M. Zholobak, M.V. Kosevich, A. Gomory, P.K. Holubiev, A.I. Marynin are widely used in the treatment of a number of viral infections and some other diseases [4][5][6][7][8]. The interferon-inducing action of tilorone and, in particular, stimulation of synthesis of all three types of interferon in a human body is considered as the basic mechanism of the preparation activity [9].
However, in spite of active usage of tilorone in medical practice in Ukraine and some others FSU countries, the discussions about its efficiency and investigations of its pharmacological activity as well as toxicity are currently continued [10][11][12]. For example, there is investigation testified to miscoordination of interferon-inducing and antiviral effects of tilorone [13]. The problem of molecular mechanisms of antiviral tilorone activity is remaining open since it is still not clear whether this activity is related just to interferoninducing activity or it is also connected with other intracellular cascade reactions and intermolecular interactions. That is why the molecular level model studies of interactions of tilorone with potential targeting biomolecules and their components are considered to be helpful in understanding the molecular mechanisms of the agent biological activity, which is necessary for development of more efficient and less toxic medicines. In particular, one of the existing hypotheses about nucleic acids and their constituents as potential molecular targets for tilorone binding requires experimental confirmation.
The current study is devoted to examining the mechanistic intermolecular interactions of tilorone with its possible molecular targets in the viral and host cells, which are believed to be RNA and nucleosides. An experimental investigation of biologically significant intermolecular interactions of tilorone with ssRNA and a number of nucleosides (adenosine (Ado), thymidine (Thd), and uridine (Urd)) has been performed by dynamic light scattering (DLS) and electrospray (ESI) ionization mass spectrometry methods.
Single-stranded RNA (ssRNA) for DLS experiments was obtained from Saccharomyces cerevisiae yeast as described in [14]. The following dilution buffer composition was used as a solvent: RNA-free phosphate buffered saline solution (PBS, Sigma, USA) and 10% (volume to volume) of fetal bovine serum (FBS, Sigma, USA), рН=7.4. After preparation of solutions of tilorone (10 g•L -1 or 2.07 . 10 -2 mol•L -1 ) and ssRNA (8 g•L -1 ) in the above mentioned solvent, they were ex tempore filtered through 0.2 μm syringe filter Minisart® NML with surfactant-free cellulose acetate (SFCA) (Sartorius AG, Germany). The samples of the model system (tilorone + ssRNA) were prepared just before DLS measurements by mixing of the two solutions to obtain the final molar ratio of tilorone to ssRNA as 1:10.
Adenosine (Ado), thymidine (Thd), and uridine (Urd) nucleosides and methanol (MeOH) for mass spectrometric experiments were purchased from the Sigma-Aldrich company (Germany). Initial solutions of tilorone and nucleosides (5 mM) were prepared in methanol (polar solvent which is commonly used in ESI mass spectrometry) and used for preparation of binary (tilorone + nucleoside) (1:10 molar ratio) and triple (tilorone + Ado + Urd) (1:10:10 molar ratio) model systems. In our study we did not investigate the model systems including guanosine, since from our previous experimental experience we know that guanine derivatives have less solubility in polar solvents in comparison with other nucleosides. It could result in distortion of mass spectrometric information about intermolecular interactions in the multicomponent model systems containing guanosine. Study of intermolecular interactions of antiviral agent tilorone with RNA and nucleosides.
The mixtures were kept at the room temperature for at least 10 minutes before the ESI mass spectrometric analysis. The spraying procedure required dilution of the solutions to be studied to the final 250 μM concentration of the diluted components of the model systems in each solution. Scheme 1. Tilorone dihydrochloride (Til•2HCl) chemical structure (adapted from the web site of the Sigma-Aldrich supplier https://www.sigmaaldrich.com/catalog/product/aldrich/220957?lang=en&region=UA).
Dynamic light scattering Dynamic light scattering (DLS) method, which is also known as photon correlation spectroscopy, is a powerful tool for studying the size distribution of molecular particles, and in particular their aggregates, basing on their diffusion behavior in solution. The diffusion coefficient, and hence the hydrodynamic radii calculated from it, depends on the size and shape of the particles present in solutions.
The particles sizes and distribution as well as polydispersity indexes (PdI) of solutions of ssRNA and (tilorone + ssRNA) mixture in the dilution buffer are measured using Malvern Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., Malvern, UK) and analyzed by Zetasizer software (Malvern Instruments). For each sample a separate disposable polystyrene cuvette (Sarstedt AG & Co., Germany) is used. Water is used as a dispersant. All DSL measurements are carried out at a standard temperature of +25°C, and three measurements with at least 10 sub-runs are performed for each sample.
The Zetasizer software supplied with the instrument provides a number of analysis tools to study aggregation by the DLS. "Size Distribution by Intensity" and "Size Distribution by Volume" are the most widely used tools. The Size Distribution by Intensity method is suitable for detection of high molecular weight particles including aggregates, which scatter light disproportionately relative to smaller particles, enabling detection despite their relatively low concentration in a sample [15]. The Size Distribution by Volume method is used in our study with the aim to investigate of characteristics of the particles of ssRNA and aggregates in (tilorone + ssRNA) system in the dilution buffer.

ESI mass spectrometry
Electrospray ionization (ESI) mass spectra of the systems studied are obtained in the positive ion mode using triple quadrupole (QqQ) Micromass Quattro Micro mass spectrometer (Waters, Manchester, UK) equipped with the electrospray ion source. This source is operated in the standard ESI mode. The ESI source temperature is set to 120 o C and the desolvation temperature is 200 o C. The spraying capillary is operated at 3.5 kV. The cone voltage (CV) value of 10 V is used. The analyzed solutions (20 μL) are injected into the mass spectrometer at a constant flow rate of 0.2 mL•min -1 of methanol solvent. The ESI spectra are recorded in the mass range of m/z 100-2000. Data acquisition and processing are performed using MassLynx 4.1 software (Waters, Manchester, UK).

RESULTS AND DISCUSSION
DSL experimental study DLS method was applied to examine the size characteristics of the particles present in solutions of ssRNA and (tilorone + ssRNA) system (1:10 molar ratio) in the dilution buffer. The obtained (PdI) and Size Distribution by Intensity values are summarized in Table 1. The summarized data on the particles Size Distribution by Volume are presented in Table 2. Statistic graphs of the particles size distributions in solutions of ssRNA and (tilorone + ssRNA) system by volume are presented in Fig. 1.
The experimental results demonstrate that ssRNA solution in the dilution buffer is quite monodisperse system, since it contains particles of similar sizes. Indeed, the mean diameter of these particles is between 150 and 154 nm and the size distribution is narrow, as is evidenced by the polydispersity index values (ranging from 0.18 to 0.20) ( Table 1). The data obtained for PdI and Size Distribution by Intensity for the ssRNA solution most probably point to creation of similar sizes molecules aggregates of ssRNA with Bovine Serum Albumin (BSА) and/or other molecules existing in the used buffer solution (Pk1 in Table 1, peak in the statistic graph, Fig. 1). Value of Z-average (also known as the "cumulants mean") testifies to the monodispersity of the ssRNA solution sample too.
Introduction of tilorone solution into the solution of ssRNA in the buffer resulted in almost twice reliable increase of PdI values (range of PdI value is 0.27 -0.39) comparing with the values for the ssRNA solution itself. In the statistic graph of (tilorone + ssRNA) system (Fig. 1) there are two peaks showing the presence of two different size particles populations in the system. The first peak (which input into the total scattering intensity is 75%) is most probably related to aggregates of tilorone with the initial particles of ssRNA, and mean diameter of the aggregates is more than 10 times exceeds the mean diameter of the particles in the ssRNA solution itself. The second peak (with input into the total scattering intensity of 25%) is related to bigger aggregates of the system components (obviously, including BSA and other serum components) with the mean diameter ranging between 4.62 and 5.57 μm. Study of intermolecular interactions of antiviral agent tilorone with RNA and nucleosides. It should be noted that in our investigation, with the purpose of obtaining reliable results for sizes distribution and taking into account the ranges of the equipment sensitivity, we take solutions which contained more high concentrations of tilorone and ssRNA than usually are used for biological objects in vitro [16,17].
Thus, the obtained DSL data demonstrate that under conditions similar to the physiological ones, the introduction of tilorone into the system of ssRNA solution in the buffer results in active aggregation of tilorone with the ssRNA particles and in enlargement of the aggregates likely contained tilorone, ssRNA and other components of the used dilution buffer. Earlier it was showed that (tilorone + ssRNA) complex with 1:10 components ratio demonstrated significant antiviral activity [16], and also induced interferon formation in vitro [17] as well as in vivo [18]. The data obtained in the current and earlier investigations are in a good agreement with the modern ideas about the effect of double-stranded allogenic RNA of different length on the formation of interferon-mediated or interferon independent antiviral resistance of the cells [19,20].
To confirm the (tilorone + ssRNA) noncovalent complexation at the monomer level and with the purpose to find the possible RNA components which can be considered as centers of tilorone binding to the nucleic acid molecules, the following ESI mass spectrometry study of interactions of tilorone with nucleosides was performed.

ESI mass spectrometry investigations
At the first stage of the ESI mass spectrometric experimental study solution of tilorone in methanol was investigated. The characteristic mass spectrum of tilorone is presented in Fig. 2   At the next stage the intermolecular interactions of tilorone with selected nucleosides Ado, Thd, and Urd were examined by the ESI mass spectrometry probing of methanol solutions of (tilorone + nucleoside) mixtures in 1:10 molar ratio. In the current measurements, we applied the ESI approach, which we developed and effectively harnessed in our previous investigations [21][22][23][24][25] for the study of intermolecular interactions of biologically active compounds, including drugs, with the targeting biological molecules. Study of intermolecular interactions of antiviral agent tilorone with RNA and nucleosides.
The mass spectra of all studied model systems (tilorone + nucleoside) contain ions characteristic of the individual components of the mixtures. At the same time, the most interesting result from the biophysical point of view relates to observation in the spectra of (tilorone + Urd) system (Fig. 3) the ions of stable ion-molecular clusters of uridine with tilorone dication. The presence of the peak of doubly charged ion Urd•Til•2H 2+ (m/z 328. 3) with relative intensity about 10% in the ESI mass spectrum testifies to the formation of stable noncovalent complexes of uridine with tilorone in solution. It is notable that in the other model systems examined the formation of such complexes of tilorone with adenosine or thymidine is not detected by the ESI method.
To check the idea about selectivity of tilorone interaction with uridine we examined a three-component model system of (tilorone+Ado+Urd) (1:10:10 molar ration). The ESI mass spectrum of the triple system (Fig. 4) contains characteristic peaks of tilorone, adenosine, and uridine. The signal of the cluster of tilorone with uridine the peak of Urd•Til•2H 2+ at m/z 328.3 has been detected too, while the peak of the complex Ado•Til•2H 2+ with expected m/z 339.4 or other peaks of any noncovalent complexes of adenosine with tilorone have not been found in the spectrum.
To determine the structural and energetic parameters of the registered in the mass spectrometry experiments noncovalent complexes of tilorone with uridine we are planning to perform quantum-mechanical calculations in our following study, similarly to approach developed in [23][24][25]. However, right now we can suggest that registered Urd•Til•2H 2+ complexes can be stabilized by electrostatic interactions of partially negatively charged two carbonyl groups of uridine and positively charged quaternary ammonium groups of tilorone. The partially negatively charged carbonyl groups of thymidine may be less sterically accessible, because of methylation of C 5 of pyrimidine cycle in thymidine, that can cause less stability of noncovalent complexes of tilorone with thymidine (the complexes are not recorded in the mass spectrum). As for adenosine, in its structure there are no carbonyl groups with significant partial negative charge, and therefore electrostatic interactions of adenosine with positively charged protonated groups of tilorone will be weaker in comparison with the ones for uridine.
Taking into account that uridine is affiliated just with RNA (but not DNA) the obtained data testifies to the possible specificity of interactions of tilorone with the RNA (not DNA) components, which can be important for revealing the mechanisms of the tilorone biological activity.

CONCLUSIONS
The performed DLS investigations reveal the formation of large-scale molecular aggregates of tilorone with ssRNA in the buffer solution contained RNA-free phosphate buffered saline solution and 10% of fetal bovine serum, which is similar to the physiological solution in physical and chemical characteristics. The addition of tilorone into the ssRNA solution in the buffer in the molar ratio of 1:10 results in the formation of complexes of ssRNA particles with tilorone with the mean diameter of 10 times larger than the diameter of the particles in the ssRNA solution itself. We suggest that similar complexation of tilorone with ssRNA could take place in real biological systems and could provoke the tilorone antiviral effect as well as induce activation of interferon-mediated or interferon independent pathways of formation of antiviral resistance of the host cells.
The ESI mass spectrometric study of the model systems of (tilorone + nucleosides) (Ado, Urd, or Thd) demonstrates the formation of stable noncovalent complexes Urd•Til•2H 2+ , while the complexes of tilorone with Ado and Thd are not detected in the experiments. It testifies to the possibility of formation of stable noncovalent complexes of tilorone with the RNA and their components in biological systems and pointed at Urd as one of the potential V.A. Pashynska, N.M. Zholobak, M.V. Kosevich, A. Gomory, P.K. Holubiev, A.I. Marynin centers of specific binding of tilorone to the RNA molecules. Such intermolecular interactions of tilorone with viral RNA and/or with RNA in the host cells could be considered as the molecular mechanism of antiviral activity of tilorone as well as the molecular basis of the possible drug toxicity for the host cells.