Isolation and In Silico Anti-SARS-CoV-2 Papain-Like Protease Potentialities of Two Rare 2-Phenoxychromone Derivatives from Artemisia spp.

Two rare 2-phenoxychromone derivatives, 6-demethoxy-4`-O-capillarsine (1) and tenuflorin C (2), were isolated from the areal parts of Artemisia commutata and A. glauca, respectively, for the first time. Being rare in nature, the inhibition potentialities of 1 and 2 against SARS-CoV-2 was investigated using multistage in silico techniques. At first, molecular similarity and fingerprint studies were conducted for 1 and 2 against co-crystallized ligands of eight different COVID-19 enzymes. The carried-out studies indicated the similarity of 1 and 2 with TTT, the co-crystallized ligand of COVID-19 Papain-Like Protease (PLP), (PDB ID: 3E9S). Therefore, molecular docking studies of 1 and 2 against the PLP were carried out and revealed correct binding inside the active site exhibiting binding energies of −18.86 and −18.37 Kcal/mol, respectively. Further, in silico ADMET in addition to toxicity evaluation of 1 and 2 against seven models indicated the general safety and the likeness of 1 and 2 to be drugs. Lastly, to authenticate the binding and to investigate the thermodynamic characters, molecular dynamics (MD) simulation studies were conducted on 1 and PLP.

A. commutata [36] and A. glauca [37] are perennial herbs that are cultivated in northern and eastern Kazakhstan [38]. The lack of scientific records about the phytochemistry and the biological effects of these two species encouraged us to examine their phytochemical properties aiming at the isolation of promising secondary metabolites.
The WHO declared on December 28, 2021, that the confirmed cases of COVID-19 infections on a global base are 280,119,931. Sadly, 5,403,662 of them are dead [39]. In regard to these bad numbers, massive work is demanded from scientists all over the world to find a cure.
The field of computer-aided/based (in silico or computational) drug design and discovery is a fast-growing approach that has shown several successes and advancements during the last decade [40]. The structural informatics explosion allowed the identification of the exact chemical and physical properties of molecules. Consequently, the ability to compare different structures and predict a specific biological activity became much easier [41]. Also, the huge advancement in the field of proteomic enabled scientists to expect the biding and hence the activity of a molecule and target enzyme [42]. Our teamwork reported the utilization of the in silico methods to suggest a treatment that could be effective against COVID-19 in many reports [43][44][45][46][47].
We here in this manuscript, report the isolation and structure elucidation of two rare 2-phenoxychromone derivatives, 6-demethoxy-4'-O-capillarsine (1) and tenuflorin C (2) from the areal parts of A. commutata and A. glauca, respectively, for the first time. Because of the lack of research on the isolated compounds and the severe need to find a cure against COVID-19, the potential inhibitory effects of 1 and 2 against COVID-19 were investigated depending on a multiphase in silico screening method. The potentialities of 1 and 2 against COVID-19 Papain-Like Protease were confirmed according to molecular similarity, structure fingerprint, molecular docking, and molecular dynamics simulations studies. Additionally, ADMET and toxicity properties of 1 and 2 were studied to predict their likeness to be used as drugs.

Isolation of Compounds
The areal parts of A. commutata, (940 g) and A. glauca (1.02 kg) were collected from the western part of the Altai Mountains. The dried plants were extracted with chloroform. The chloroform extract of A. commutata was subjected to chromatographic purifications steps to afford the amorphous powder of 1. Compound 1 was identified to be 6-demethoxy-4'-O-capillarsine ( Figure 1). Identification of 1 was done depending on 1 H and 13 C NMR (Table S1, in the Supplementary Materials), comparing the published data. Compound 1 was previously isolated from the leaves of Mimosa tenuiflora [48], A. capillaris [49], and A. rupestris [50]. The chloroform extract of A. glauca was subjected to chromatographic purification steps to give the amorphous powder of 2. Compound 2 was identified to be 5,7,4 -trihydroxy, 3 -methoxy-2-phenoxychromone ( Figure 1). Compound 2 was identified depending on 1 H and 13 C NMR (Table S2, in the Supplementary Materials) compared to the published data. Compound 2 was previously isolated from Mimosa tenuiflora [48]. previously isolated from the leaves of Mimosa tenuiflora [48], A. capillaris [49], and A. rupestris [50]. The chloroform extract of A. glauca was subjected to chromatographic purification steps to give the amorphous powder of 2. Compound 2 was identified to be 5,7,4′trihydroxy, 3′-methoxy-2-phenoxychromone ( Figure 1). Compound 2 was identified depending on 1 H and 13 C NMR (Table S2, in the supporting data) compared to the published data. Compound 2 was previously isolated from Mimosa tenuiflora [48]. .

Molecular Similarity Studies
To understand the principle of molecular similarity, we have to understand that the bioactivity of any compound is a result of some well-known interactions with a specific protein target. These protein-ligand interactions depend on specific physical and chemical interactions such as hydrogen bonding and hydrophobic interactions. Consequently, the similarity in the chemical structures of two compounds will give a similarity in the number and position of hydrogen bond donors, acceptors, and hydrophobic centers in addition to the steric configuration. This similarity could cause a noticeable similarity in the bioactivity too [51].

Molecular Similarity Studies
To understand the principle of molecular similarity, we have to understand that the bioactivity of any compound is a result of some well-known interactions with a specific protein target. These protein-ligand interactions depend on specific physical and chemical interactions such as hydrogen bonding and hydrophobic interactions. Consequently, the similarity in the chemical structures of two compounds will give a similarity in the number and position of hydrogen bond donors, acceptors, and hydrophobic centers in addition to the steric configuration. This similarity could cause a noticeable similarity in the bioactivity too [51].

Fingerprints Studies
The molecular fingerprint is an in silico method that analyzes the similarities of two molecules or more. Molecular fingerprint explores the property profiles of a certain compound, the technique computes these properties in the forms of bits vectors examining the existence, absence, and frequencies it in the reference and target compounds.

Fingerprints Studies
The molecular fingerprint is an in silico method that analyzes the similarities of two molecules or more. Molecular fingerprint explores the property profiles of a certain compound, the technique computes these properties in the forms of bits vectors examining the existence, absence, and frequencies it in the reference and target compounds.

Pharmacophoric Features and Flexible Alignment Studies
The reported main pharmacophoric features of SARS-CoV-2 Papain-Like protease inhibitors (PLPIs) are two hydrophobic systems separated be a linker [67]. As shown in Figure 4, the co-crystallized ligand (TTT) has naphthyl and phenyl groups as hydrophobic centers. The two hydrophobic groups are separated by hydrophilic linker (amide group). In addition, the terminal phenyl group is substituted by amino group as a hydrogen bonding group.
Similarly, compounds 1 and 2 have coumarin moiety as the first hydrophobic center in addition to phenyl group as the second hydrophobic moiety. In each compound, the two hydrophobic centers were separated by oxygen atom as a hydrophilic linker. This linker facilitates these compounds to have the same configuration of TTT. Additionally, the two phenyl rings were substituted by hydrophilic groups as which can form hydrogen bonds with the target receptors as TTT. From medicinal chemistry point of view, this investigation revealed that there is a great similarity between the two tested molecules and the co-crystallized ligand (TTT).
centers. The two hydrophobic groups are separated by hydrophilic linker (amide group). In addition, the terminal phenyl group is substituted by amino group as a hydrogen bonding group.
Similarly, compounds 1 and 2 have coumarin moiety as the first hydrophobic center in addition to phenyl group as the second hydrophobic moiety. In each compound, the two hydrophobic centers were separated by oxygen atom as a hydrophilic linker. This linker facilitates these compounds to have the same configuration of TTT. Additionally, the two phenyl rings were substituted by hydrophilic groups as which can form hydrogen bonds with the target receptors as TTT. From medicinal chemistry point of view, this investigation revealed that there is a great similarity between the two tested molecules and the co-crystallized ligand (TTT).  3D-Flexible alignment of the compounds 1 and 2 with TTT was presented in Figure 5. From the figure, it is possible to observe that, in general, the structure of compounds 1 and 2 have a good overlap with the reference molecule (TTT). In addition, the two tested compounds showed the same spatial orientation of TTT due to the presence of the flexible linker at the center of these compounds. 3D-Flexible alignment of the compounds 1 and 2 with TTT was presented in Figure  5. From the figure, it is possible to observe that, in general, the structure of compounds 1 and 2 have a good overlap with the reference molecule (TTT). In addition, the two tested compounds showed the same spatial orientation of TTT due to the presence of the flexible linker at the center of these compounds.

Docking Studies
To examine the obtained results from structural similarity and fingerprint studies, molecular docking experiments were achieved for 6-demethoxy-4`-O-capillarsine (1) and tenuflorin C (2) against PLP (PDB ID: 3E9S) using TTT, the co-crystallized ligand, as a reference. We considered the binding free energies (∆G) besides the binding modes as the bases of evaluation.
Firstly, a validation process has proceeded via the re-docking of TTT against PLP.

Docking Studies
To examine the obtained results from structural similarity and fingerprint studies, molecular docking experiments were achieved for 6-demethoxy-4'-O-capillarsine (1) and tenuflorin C (2) against PLP (PDB ID: 3E9S) using TTT, the co-crystallized ligand, as a reference. We considered the binding free energies (∆G) besides the binding modes as the bases of evaluation.
Firstly, a validation process has proceeded via the re-docking of TTT against PLP. The obtained RMSD value between the two poses was 2.1 • A confirming the validity of the docking method ( Figure 6).

Docking Studies
To examine the obtained results from structural similarity and fingerprint studies, molecular docking experiments were achieved for 6-demethoxy-4`-O-capillarsine (1) and tenuflorin C (2) against PLP (PDB ID: 3E9S) using TTT, the co-crystallized ligand, as a reference. We considered the binding free energies (∆G) besides the binding modes as the bases of evaluation.
Firstly, a validation process has proceeded via the re-docking of TTT against PLP. The obtained RMSD value between the two poses was 2.1 ᵒ A confirming the validity of the docking method ( Figure 6).  The binding mode of TTT showed a free energy of −20.32 kcal/mol. The p-toluidine part occupied the first pocket of PLP forming one H-bond with the amino acid, Leu163. The same moiety was engaged hydrophobically in two interactions with the amino acids Asp165 and Tyr269. In addition, the amide linker formed two H-bonds with the amino acids Asp165 and Gln270. The naphthalene moiety was buried in the second pocket making five hydrophobic interactions with Pro249, Pro248, and Tyr269 ( Figure 7A  The binding mode of 6-demethoxy-4'-O-capillarsine (1) exhibited a binding free energy of -18.86 kcal/mol. The anisole moiety was directed into the first pocket of PLP forming one H-bond with the amino acid, Gln270, in addition to two hydrophobic interactions with the amino acids, Tyr269 and Asp165. The 5,7-dihydroxy-4H-chromen-4-one part occupied the second pocket of PLP forming a H-bond with the amino acid Pro249 besides two hydrophobic bonds with the amino acids Pro248 and Pro249 ( Figure 8A-C).
Tenuflorin C (2) bonded to PLP exhibiting a binding energy of −18.37 kcal/mol. The 2-methoxyphenol part was directed into the first pocket of PLP with two H-bonds with the amino acids Gln270 and Leu163. Additionally, the same moiety was engaged hydrophobically in two interactions with the amino acids Tyr269 and Asp165. Further, the 5,7-dihydroxy-4H-chromen-4-one part occupied the second pocket of PLP forming two H-  The binding mode of 6-demethoxy-4`-O-capillarsine (1) exhibited a binding free energy of -18.86 kcal/mol. The anisole moiety was directed into the first pocket of PLP forming one H-bond with the amino acid, Gln270, in addition to two hydrophobic interactions with the amino acids, Tyr269 and Asp165. The 5,7-dihydroxy-4H-chromen-4-one part occupied the second pocket of PLP forming a H-bond with the amino acid Pro249 besides two hydrophobic bonds with the amino acids Pro248 and Pro249 ( Figure 8A  Tenuflorin C (2) bonded to PLP exhibiting a binding energy of −18.37 kcal/mol. The 2-methoxyphenol part was directed into the first pocket of PLP with two H-bonds with the amino acids Gln270 and Leu163. Additionally, the same moiety was engaged hydrophobically in two interactions with the amino acids Tyr269 and Asp165. Further, the 5,7dihydroxy-4H-chromen-4-one part occupied the second pocket of PLP forming two Hbonds with Tyr274 and Ala247. Finally, it was incorporated hydrophobically in three bonds with the amino acids Pro248, Asp165 and Met209 ( Figure 9A-C).

In Silico ADMET Study
There is always a need to examine the ADMET properties of new molecules especially in the stage of designing to decrease the possibility of late-stage attrition. Also, the identification of ADMET properties provides good information regarding the amount and frequency of dosing as well as the toxicity [68].
The in silico ADMET parameters were determined for 6-demethoxy-4`-O-capillarsine (1) and tenuflorin C (2) using Discovery Studio software and utilizing Remdesivir as a reference. The results were illustrated in Figure 10 and Table 3.

In Silico ADMET Study
There is always a need to examine the ADMET properties of new molecules especially in the stage of designing to decrease the possibility of late-stage attrition. Also, the identification of ADMET properties provides good information regarding the amount and frequency of dosing as well as the toxicity [68].

Molecular Dynamics (MD) Simulation Studies
The main advantage of molecular dynamics (MD) simulation is its ability to recognize the flexibility of the protein-ligand complex. This advantage enables the MD studies to estimate accurately the thermodynamics and kinetics that take place during the drugenzyme binding [69].
MD simulations can explore the dynamic structural information of the protein-ligand complex and provide plentiful information on the energetic changes that resulted from the interactions between protein and ligand. Such information could be very useful to understand the structure-function relationship of the protein-ligand complex interactions [70]. In order to authenticate the binding and to investigate the thermodynamic characters of the isolated compounds, molecular dynamics (MD) simulation studies were conducted to 6-demethoxy-4'-O-capillarsine, 1, as it showed a bitter binding energy, against PLP.
The atomical dynamic movements and conformational variations of backbone atoms of the 6-demethoxy-4'-O-capillarsine -PLP complex were calculated by RMSD to detect their stability upon ligand bonded and apo states. It was observed that the protein and ligand exhibit lower RMSD with no major fluctuations indicating their greater stability. The 6-demethoxy-4'-O-capillarsine -PLP complex was slightly fluctuating till 70 ns~and stabled later ( Figure 11A). The flexibility of PLP was evaluated in terms of RMSF ( Figure 11B) to get more information about the protein regions that had been fluctuating during the simulation. It can be understood that the binding of 6-demethoxy-4'-O-capillarsine does not make PLP very flexible. The compactness of the 6-demethoxy-4'-O-capillarsine -PLP complex was evaluated through the examination of the radius of gyration (Rg) ( Figure 11C). Decreasing fluctuation during the simulation study is an indication of the higher compactness of the system. The Rg of the 6-demethoxy-4'-O-capillarsine -PLP complex was found to be similar throughout the simulation period. Interaction between 6-demethoxy-4'-O-capillarsine -PLP complexes and solvents was evaluated through the calculation of solvent accessible surface area (SASA) over the simulation period. So, SASA of the 6-demethoxy-4'-O-capillarsine -PLP complex was calculated to analyze the extent of the conformational changes that occurred during the interaction. Interestingly, PLP featured a reduction of the surface area showing a relatively lower SASA value than the starting period ( Figure 11D). Hydrogen bonding in the 6-demethoxy-4'-O-capillarsine -PLP complex is essential to stabilize the structure. It was noticed that the highest number of conformations of the PLP formed up to three H-bonds with the 6-demethoxy-4'-O-capillarsine ( Figure 11E).

Isolation and Structure Elucidation of Compounds
A total of 0.94 kg of Artemisia commutata and 1.04 kg of Artemisia glauca areal parts were extracted with chloroform. Successive chromatographic methods led to the isolation of compounds (1 and 2). Detailed description in the method part in supporting data.

Isolation and Structure Elucidation of Compounds
A total of 0.94 kg of Artemisia commutata and 1.04 kg of Artemisia glauca areal parts were extracted with chloroform. Successive chromatographic methods led to the isolation of compounds (1 and 2). Detailed description in the method part in Supplementary Materials.

Molecular Dynamics Simulations
The system was prepared using the web-based CHARMM-GUI [80][81][82] interface utilizing CHARMM36 force field [83] and NAMD 2.13 [84] package. The TIP3P explicit solvation model was used (See the method part in Supplementary Materials).