Decamethoxin viruciDal activity: in vitro anD in silico stuDies

the data on the representative of decamethoxin short-term action on infectious bronchitis virus (IBV) strain h120 used as a human-safe model of SarS-coV-2 virus are presented. the viral activity was estimated with the use of inverted microscope PrimoVert (Germany) by destructive effect on BHK21 fibroblastic cell line. in vitro results demonstrated that decamethoxin (100 μg/ml) completely inactivated iBV coronavirus strain at exposure of 30 sec and more. at the lowest decamethoxin exposure of 10 sec the antiseptic virucidal activity was 33% and 36% of control at 24 and 48 h of cultivation respectively. molecular docking analysis indicated the significant similarity of iBV and saRs-CoV-2 main protease (M pro ) structure. Docking studies of decamethoxin interaction with IBV m pro and SarS-coV-2 m pro active centers demonstrated the ligand-protein complexes formation with the estimated binding energy of -8.6, -8.4 kcal/mol and key amino acid residues aSN26, Gly141, GlU187, GlU164, thr24, thr25, aSN142, Gly143, cyS145, hIS164 and GlU166.

D isinfectants and antiseptics are important determinants in a pandemic, including coronavirus infection . Successful disinfection of SARS-CoV-2 is defined by the charac teristics of the virus, the properties of the disin fectant or antiseptic, and the environmental conditions in which the virus is present. SARS-CoV-2 is stable over a wide pH range (pH 3-10) at room temperature [1] and is very stable in a favorable environment [2] but is usually disinfected [3]. Conside ring viral load, persistence, stability, viability and environmental factors, disinfection of medical and other public facilities is a necessary part to prevent transmission and waves of COVID-19 infection.
Among the well-known disinfectants such as detergents, acids, oxidizing agents, alcohols, alkalis, aldehydes, biguanides, halogens, phenols, quaternary ammonium compounds (QAC) occupy a special place [4]. Most disinfectants target the outer lipid layer of coronaviruses [5]. Сross-linking, coagulation, structural and functional damage and oxidation appear to be the main mechanisms of the disinfectants virucidal activity [6]. In the case of corona viruses, disinfectants affect the protein and lipid structures of the coronavirus and limit the spread of the virus [7].
Quaternary ammonium compounds (QACs) as cationic surfactants contain the amphiphilic molecules and have a broad spectrum of antimicrobial activity [8]. Their chemical structure includes four aliphatic or aromatic radicals linked to a central nitrogen atom. The antibacterial and antifungal activity of QAC is associated with the presence of 12 to 16 carbon atoms in their alkyl chain [9].
Wherein, the antimicrobial mechanism of QAC action is based on the electrostatic interaction of a positively charged cationic element of QAC with a doi: https://doi.org/10.15407/ubj94.03.081 negative charge of the cytoplasmic membrane of bacteria or fungi, leading to membrane disorganization and its autolysis. In the case of SARS-CoV-2, disruption of the phospholipid bilayer by QAC occurs more easily due to the lack of a cell wall in the virus [10].
The basic/cationic structure of QAC is a quaternary nitrogen fragment (Fig. 1), which plays an important biological role in the living systems [11].
The negatively charged anionic moiety (X-) is usually chlorine or bromine and is bonded to nitrogen to form the QAC salt. This structural diversity makes it possible to significantly change/improve the QAСs efficiency of and expand the scope of application including the viral infections [12]. The existing variety of QAСs structural features allows to classify them into several subclasses: mono-, bis-, multi-and polyderivatives according to the number of the charged nitrogen atoms, including in heterocyclic compounds (piperidine, pyridine, imidazole, etc.) [13].
Since the beginning of the 20 th century, a significant amount of work has been devoted to the development of this class of biocides. Thus, according to modern literature, since 2021 more than 17000 articles about QACs were published [14]. Virucidal activity of QACs, including anti-SARS-CoV-2 activity of decamethoxin, confirmed by a number of authors and has the scientific and practical interest in a wide range of researchers, especially in terms of potential molecular mechanisms of their virucidal action [15][16][17][18][19][20][21].
Today, a number of viral proteins have been established as the main targets for SARS-CoV-2 in-

Fig. 1. Structural features of decamethoxin and Qacs
hibitors, which include the spike S-protein, RNAdepleted RNA polymerase (RdRp) and main protease (M pro ). M pro presented in many strains is a key enzyme in the coronavirus replication mechanism and is responsible for copying and reproducing the SARS-CoV-2 genetic material. Therefore, pharmacists often consider the M pro as the main target in the fight against SARS-CoV-2, because its blocking can be an effective approach to preventing virus replication. In addition, M pro is being intensively pursued as a main target not only for SARS-CoV-2, but also for SARS-CoV and MERS-CoV, as well as enteroviruses, rhinoviruses and noroviruses [22,23].
Cysteine protease M pro is virus encoded and contains a glutamine residue at position P1 [24]. This structural feature is absent in the related host proteases, indicating the high selectivity of M pro as a target. [25].
A characteristic structure feature of the M pro inhibitors is the presence of reactive functional groups (β-ketoamide, aldehyde, aldehyde bisulfite, Michael acceptors), forming the covalent bonds with the Cys145 residue in the catalytic center of the enzyme [26].
The currently known M pro inhibitors -calpain II and calpain XII (are in preclinical studies) [26], as well as Boceprevir (approved as an antiviral drug) are expensive. Therefore, the search and study of new protease inhibitors seems to be an actual task.
In this regard, calculation /computational methods as well as in vitro methods are of particular importance, which allows one to effectively analyze the potential mechanisms of molecular interactions of promising biologically active molecules [27][28][29][30].
This paper presents the in vitro and in silico studies results of the decamethoxin virucidal activity as a representative of bis-QAC compounds.

materials and methods
In vitro study materials and methods. Antiseptic Dekasan (Yuria-Farm, Ukraine) with decamethoxin content of 0.2 mg was used as QAC. Non-pathogenic to human the strain H-120 virus IBV with an infectious titer by 3.0 lg TCID 50 /0.1 ml was used [31].
As cell culture was used a transplant culture of BHK-21 cells obtained from the cell cultures collection of R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiology of National Academy of Sciences of Ukraine. The RPMI-1640 and DMEM mediums with low glucose and glutamine and fetal blood sera of cows (Sigma, USA) were used. Cell Proliferation Kit I (MTT) (Roche Diagnostics, Germany) was used for cell viability analysis by colorimetric method.
The volume of the cell monolayer was 100 μl. The study was conducted fourfold. Standart culture flasks (Nunc, Denmark) for 96 wells microplates with an adhesive surface (Cellstar Greiner Bio-One, Austria) were used as laboratory equipment. Inverted microscope PrimoVert (Karl Zeiss, Germany) with a video camera and compatible software was used for in vitro cell culture monolayer fixation and visualization of microscopy results.
In silico study materials and methods. The crystal structures of the IBV and SARS-CoV-2 main proteases are obtained from the RCSB Protein Data Bank PDB ID:2Q6F [32] and PDB ID: 7L0D [33]. The enzyme has been prepared by AutoDock Tools (ADT) 1.5.6 [34] and saved in PDBQT format. The structure of decamethoxin was created and saved in Mol format using ChemAxon Marvin Sketch 5.3.735 [35]. The structure of decamethoxin was optimized and the energy was minimized by the MOPAC2016 program [36]. For the main proteases and decametho xin were computed of partial charges using ADT and the Gasteiger method and saved in PDBQT format. AutoDock Vina 1.1.2 [37] program was applied for the molecular docking. The docking center has been set with coordinates x = 24.017; y = -63.765; z = 10.463 and the grid map 30*30*30 points with a grid spacing of 1 Å. The presentation of the results and the ligand-protein complex analysis were conducted by Accelrys DS [38]. The N3 inhibitor was used as a co-crystal structure in the active site of the main IBV protease [39].

results and Discussion
In vitro study. Table presents the in vitro study results of decamethoxin virucidal activity under the conditions of an extended time experiment based on some previously obtained data [40].
The presented in vitro results (Table) show that decametoxin completely inactivates IBV virus in BHK21 cell culture starting from 30 sec of exposure. At the same time, under the conditions of the lowest decamethoxin exposure (10 and 20 sec) a partial virucidal activity of the antiseptic is observed as 1 and 2 lg (TCID 50 /0.1 ml) at 24 and 48 h of cultivation, respectively. At the same time (without decamethoxin treatment) the control IBV infectious titer was 4.5 and 5.5 lg (TCID 50 /0.1 ml at 24 and 48 h of cultivation respectively (Fig. 2). Further, IBV M pro substrate-binding site was used for the docking procedure based on the structural analysis data. Visual demonstration of the molecular docking and intermolecular interactions of decamethoxin are presented in Fig. 7.
The docking results show that the formation of the ligand-protein complex (Fig. 7) was accompanied by an estimated binding energy of -8.6 kcal/ mol. This ligand-protein complex is stabilized by the six hydrogen bonds (2.22-3.66 Ǻ) with amino acids Sequences alignment. To confirm the potential complexation of decamethoxin in the SARS-CoV-2 M pro active site, a comparative analysis (Fig. 8) of the primary structures of IBV M pro (2Q6F) and SARS-CoV-2 M pro (7C8B) [41] was performed using the NCBI Protein BLAST server [42].
The received results (Fig. 8) indicate the significant similarity of M pro IBV and M pro SARS-CoV-2sequence identities and sequence similarity indicators were calculated as 41 and 55%, respectively.
The M pro active sites of IBV and SARS-CoV-2 were compared using the Universal Protein Resource (UniProt) the "Align" tool for multiple sequence alignment (Fig. 9) [43]. Fig. 8 as well as Fig. 9 demonstrates not only a high degree of studied enzymes similarity, but also the structural similarity of their active centers. Next, molecular docking of decamethoxin into the active site of the M pro SARS-CoV-2 was performed.
The docking results demonstrate the formation of the ligand-protein complex (Fig. 10) by the estimated binding energy of -8.4 kcal/mol. This ligandprotein complex is stabilized by the seven hydrogen bonds (1.94-3.68 Ǻ) with amino acids THR24, THR25, ASN142, GLY143, CYS145, HIS164, GLU166, the one electrostatic interaction (4.84 Ǻ) with HIS41 and the five hydrophobic interactions (3.81-4.81 Ǻ) with the amino acid residues HIS41, CYS145, HIS163. It is necessary to emphasize the formation of hydrogen, electrostatic and hydrophobic bonds between decamethoxine and amino acids of the catalytic dyad HIS41 -CYS145 of the M pro active site.  Conflict of interest. Authors have completed the Unified Conflicts of Interest form at http://ukrbiochemjournal.org/wp-content/uploads/2018/12/ coi_disclosure.pdf and declare no conflict of interest.