Molecular Docking Analysis of Some Bioactive Molecules of Cistus incanus Against SARS CoV-2

Coronavirus disease affects all the world with the pandemic way that we are still living. The fight against the disease continues with vaccination all over the world. Considering the protection time and the difficulties in attaining the vaccine, in order to be successful in fighting against the disease, we need drugs that enable to kill or hinder replication of the viruses. In drug studies, after analyzing the effect of phytochemicals on the viruses, isolated phytochemical is modified in order to synthesize a more effective molecule. It is not possible to analyze the anti - viral activity of each isolated molecule by in - vitro methods, and in - silico methods can help to overcome this problem. Cistus incanus is a plant whose anti - viral activity has been confirmed by previous trials on many viruses. In this study, the interaction of myricetin 3-O-hexoside, myricitrin, quercitrin and kaempferol 3 -O- rutinocide which were detected in the Cistus incanus , were analyzed by molecular docking methods with papain - like prote ase and main protease crystal. Strong H - bonds were detected between the investigated molecules and papain - like protease and main protease .


Introduction
Bioactive compounds in plants have been used against viral diseases for a long time.Some of these phytochemicals have antioxidant, anti-allergic and anti-inflammatory properties (Bhushan et al., 2020) and also modulate the functions of the receptors and enzymes (Carbonell-Capella et al., 2015).Plant extracts can be used for the preparation of peptides/proteins for medical purpose (Adhikari et al., 2020).Mediterranean people use Cistus species for the treatment of skin diseases, digestive problems (Papaefthimiou et al., 2014) and also as anti-inflammatory (Attaguile et al., 2000).
Humanity's struggle with coronavirus is continued by vaccination.The total of 6.495.672.032vaccine doses as of 13 October 2021 have been administered (https://covid19.who.int/).However, the studies have to focus on the detection of new drug molecules without any retardation.The SARS-CoV-2 vaccines obtained by encoding the virus will not be suitable for the treatment of coronavirus disease in the future, since the limited efficiency duration of the vaccines and the problems about the virus mutations (Lipsitch and Dean, 2020;Chen et al., 2021).
Drug studies begin with analyzing the activity of natural products (Behl et al., 2021).Later, the isolated active molecules are modified by organic methods and more effective molecules are synthesized (Adhikari et al., 2020).If money, labor, and time opportunities are considered, it does not possible to examine the various bioactivities of each active molecule and modified active molecules.In addition, it is very important to have foresights about more active molecules in the pandemic process we are experiencing.The molecular docking is the most commonly used method for analyzing the activity of structure-based drugs (Contreras-Puentes and Alvíz-Amador, 2020; Wua et   al., 2020; Shawky et al., 2020).It provides to obtain important information about the binding regions of target molecules with ligands.In the fight against COVID-19, the analysis of molecules with some known activities has been primarily evaluated.Kandeel and Al-Nazawi (2020) analyzed many molecules which approved by the FDA with PLP in SARS-CoV-2.In addition, the anti-covid activity of many newly synthesized molecules has been frequently examined by molecular docking studies (Liang et al., 2021).Also, many studies have been examined the effects of phytochemicals in various plants against coronavirus (Ali and Kunugi, 2021;Antonio et al., 2021;Alfaro, 2020;Gori, 2016).
In the drug research, it is important to examine the protein-ligand binding properties of viral proteins.In this study, the papain-like protease and main protease inhibition activity of quercitin, myricitrin, myricetin-3-O-hexoside, and kaempferol-3-O-rutinoside molecules that can be obtained from Cistus incanus were investigated by molecular docking method.To the best of our knowledge, the activity of the myricetin-3-O-hexoside molecule against COVID-19 disease hasn't been studied.

Content analysis of the plant Cistus incanus was performed by Gori et al. HPLC-DAD-MS /
MS was used for detailed analysis of components obtained from crude ethanolic leaf extract.The most common components according to the peak length were determined as 3-O-hexoside, myricitrin, quercitrin and kaempferol 3-O-rutinocide (Gori et al., 2016).Papain-like protease inhibition capacity of these active molecules has been in-silico determined.The 3d structures of the active molecules were downloaded from the National Library of Medicine (National Center of Biotecnology Information; https://pubchem.ncbi.nlm.nih.gov/).The crystal structure of Papain-like proteas with PDB ID: 6w9c and main protease with PDB ID: 6lu7 were taken from Protein Data Bank (https://www.rcsb.org/ ) (Osipiuk et al., 2021;Jin et al., 2020;Trott and Olson, 2010).Water molecules were removed from the crystal and only polar hydrogens were added.The protein target molecule was saved as pdbqt and imported into the AutoDockTools.Kollman Charges and Lamarkian Genetic Algorithms were used in calculations.BIOVIA Discovery Studio Visualizer was used to visualize molecules and binding interactions (https://discover.3ds.com/discovery-studio-visualizer ) (Holt et al., 2008;Li et al., 2021).

Findings and Discussion
PLP is a proteolytic enzyme effective in host innate immunity and viral replication since it can be used as a target molecule as a coronavirus inhibitor (Su et al., 2021;Reddy et al., 2021;Banerjee et al., 2020).There is no FDA-approved drug as a PLP inhibitor.Maiti (2020) recorded that the PLP which is encoded from SARS-CoV-2 have active sites such as the labile region consist of Cys189, Cys192, Cys224 and Cys226, and catalytic region consist of Cys111, His272 and Asp286 amino acids and these sites have key functions in viral replication (Banerjee et al., 2020).These regions can be considered as important region for candidate molecules.It has been interpreted that the inhibition of the Cys amino acid in the catalytic region by sulfur-based molecules can provide dysfunction of PLP and stop viral replication.The 6w9c coded PLP crystal structure was released in April 2020 (Osipiuk et al., 2021).In molecular docking studies with this crystal structure, the interactions with the region formed by amino acids such as Leu162, Val202, Met206 and Met208 were recorded mostly.
However, the interactions with polar amino acids such as Ser170 and Asn156 are also remarkable (Saakre et al., 2021;Banerjee et al., 2021).Many H-bonds were identified for this study.H-bonds with Asp76, Glu167, Thr74, Cys155, and Tyr171 amino acids for kaempferol 3-O-rutinoside were recorded with the binding energy of -4.83 kcal/mol.In addition, it is also expected of contribution of many Van der Waals interactions and Pi-pi T-shaped interaction with His175 to the binding energy calculated for kaempferol 3-Orutinoside.To the best of our knowledge, the activity of the myricetin-3-O-hexoside against COVID-19 has never been studied before.Although the binding energy of the myricetin-3-O-hexoside is relatively low, H-bonds with Asp76, Arg82, Ala153, Asn156, Gln174, Tyr171, and His175 are noteworthy besides many Van der Waals interactions.Myricitrin interacted with approximately the same region of the target molecule with -3.46 kcal/mol binding affinity.H-bonds of myricitrin were recorded with Asp76, Arg82, Asn156, and Tyr171.The interactions of the quercitrin with nearly similar amino acids can be examined in Table 1 and Fig   of citrus limon and garlic and suggested some phytochemicals such as ellagic acid and narirutin as an inhibitor of SARS CoV-2 (Chebaibi et al., 2021).Many other phytochemicals also analyzed for main protease inhibition activities by Sisakht et al (Sisakht et al., 2021).Chhetri et al. synthesized and characterized new azo imidazole derivatives and searched for their inhibitor activity against COVID-19 main protease (Chhetri et al., 2021).Co(II), Ni(II), and Cu(II) deoxycholate complexes also analyzed wit molecular docking methods by Refat et al. and high binding affinities were recorded that could be a signal for possible anti-viral activity (Refat at al., 2021).
All the active molecules studied in this research interacted nearly the same region of the main protease crystal.Relatively higher binding affinities were calculated than the that of PLP.Quercitrin has the best binding affinity as -5.50 kcal/mol, and kaempferol 3-O-rutinoside, myricetin-3-O-hexoside, and myricitrin have -3.95 kcal/mol, -4.79 kcal/mol, and -5.34 kcal/mol binding energies, respectively.
Quercitrin has four H-bonds with Lys5, Trp207, Leu282, and Glu288 while myricitrin interacted with Gln127, Lys137, Gly138, and Asp289 for H-bonds.All other interaction types and details can be examined in Table1 and Figure 3.

Figure 1 .
Figure 1.Cistus incanus and four active bio-structures encoded by Gori et al.
2. The docking score of the quercitrin was calculated as -3.91 kcal/mol.