Comparative Antibacterial Analysis of the Anthraquinone Compounds Based on the AIM Theory and Molecular Docking Analysis

Hydroxyanthraquinones and anthraquinone glucoside derivatives are always considered as the active antibacterial components. In the present text, a comprehensive comparison and analysis of these compounds were performed for their structure characteristics and antibacterial effect by applying quantum chemical calculations, atoms in molecules theory and molecular docking procedure. The molecular geometric conguration, electrostatic potential, the frontier orbital energies and topological properties were analyzed. Once glucose ring is introduced into the hydroxyanthraquinone rings, almost all of the positive molecular potentials are distributed among the hydroxyl hydrogen atoms of the glucose rings. The anthraquinone glucoside compounds have generally higher intermolecular binding energies than the corresponding aglycones due to the strong interaction between the glucose rings and the surrounding amino acids. Once glucoside ring is introduced into the emodin, low electron density ρ(r) and positive Laplacian value of the O-H bond are the evidences of the highly polarized and covalently decreased bonding interactions. The type of carboxyl, hydroxyl, hydroxylmethyl groups on phenyl ring and the substituent glucose rings are important to the interactions with the topoisomerase type II enzyme DNA gyrase B.


Introduction
With the widespread use of antibiotics and the emergence of resistant bacteria, there is a need for developing new antibacterial compounds that have new mechanisms of action with activity against bacterial strains. Therefore, the search for new antibacterial agents remains a signi cant challenge for the future. Natural products are structurally complex and pharmacologically valuable products, the separation, synthesis and the structure-activity relationship attract extensive attention for scienti c research. There are three authorised Rhubarbs in Chinese Pharmacopoeia, namely Rheum palmatum L., Rheum tanguticum Maxim. ex Balf and Rheum o cinale Bail with the roots and rhizomes generally called o cial rhubarbs. Rhubarb contains a lot of active compounds, such as anthraquinone, anthrone, buty-rophenone, tannin and some glycoside derivatives, and it also has the functions of purging heat, clearing the intestines, cooling blood, lowering blood sugar, antibacterial, antiviral, anti-tumor and lipid-lowering, etc. (Zhu et al., 2016). The hydroxyanthraquinones are considered as the active antibacterial components, such as aloe-emodin, rhein, emodin and chrysophanol (Wang et al., 2010). In addition, some anthraquinone glucoside derivatives are also attracting more and more attentions due to their good microbicidal activities (Li et al., 2019;Zhang et al., 2021). It was reported that the anthraquinone glucosides were much more potent than their corresponding aglycones. For example, emodin has lower bacterial neuraminidase inhibitory activity than the corresponding glucoside derivative (Uddin et al., 2016). However, there are few comparative researches about the anthraquinone and its glycosides.
DNA gyrase is a member of topoisomerase type II enzyme, which controls the topology of DNA in the time of transcription, replication and recombination processes (Tomašić and Mašič, 2014). DNA gyrase is composed of two A subunits (GyrA) and two B subunits (GyrB), where the GyrA is involved in DNA transit and the GyrB subunit contains an ATP-binding domain (Champoux, 2001). DNA gyrase has proven to be a worthy target for antimicrobial agents (Gellert et al., 1976 Jogula et al., 2020). Due to the absence of detailed report about the relationship of the structureantibacterial activity for these aglycones and their anthraquinone glucoside derivatives by now, the aim of present review is to give a comprehensive comparison and analysis of these compounds derived from rhubarb by applying standard quantum chemical calculations (Gonçalves et al., 2014), Bader's theory (Bader, 1990) of atoms in molecules (AIM) and molecular docking procedure (Guisasola et al., 2016;Jerome et al., 2016;Singh et al., 2012) selected the DNA gyrase as a target.

Material And Methods
The molecular structure optimization of the compounds and corresponding energies are calculated by using GAUSSIAN 09W program package (Frisch et al., 2009). The DFT with Becke-3-Yang-Parr (B3LYP) combined with standard basis sets 6-31G (d, p) was applied. Topology analysis of electron density and the molecular electrostatic potential surfaces on van der Waals (vdW) surface in both forms were performed by using the Multiwfn program . The protein of the template structure DNA gyrase B (PDBID: 4DUH) was downloaded from PDB website (http://www.rcsb.org). In target DNA gyrase B preparation, the missing atoms were identi ed and repaired. The crystal water (H 2 O) molecules and other ligands were deleted. The polar hydrogen atoms and Gasteiger charge were added. The protein molecule was also assigned AD4 type. The ligand was docked into the catalytic pocket of the DNA gyrase B based on the AutoDock 4.2 program (Morris et al., 2009). Genetic algorithm with default settings was set in the search parameter, and the number of runs was set at 100. The con guration le with binding site information was generated with grid coordinates 3.024, 2.008 and 36.865 for x-, y-and z-axis respectively. The docking grid box was set at 60⋅60⋅60 points with a spacing value of 0.375 Å. Topological analysis has been performed by using AIMALL package (Parameswari and Kumaradhas, 2013), and the corresponding wave functions were also generated in order to gain further insights into the bonding characters.

Molecular geometry
These anthraquinone compounds were subjected to geometry optimization in the ground state. The optimized molecular structure of the anthraquinone compounds with atom numbering scheme adopted in the present study is presented in Fig. 1. On compared with the optimized phase, geometric conformations of these compounds changed obviously after interacting with the DNA gyrase B at the active site. There are obvious changes about the bond angles and dihedral angles rather than the bond lengths, which suggested strong inter-molecular interactions. The corresponding structural parameters (bond length and dihedral angle) are listed in Supplementary Table S1. Compared with the optimized structure, it can be seen from the Table S1 that there is almost no change in the length of each bond of compounds at the active site. The glucose ring that binds to the aromatic ring ketone is obviously bent at the active site, ascribing to the more serious intermolecular interaction with surrounding amino acids (Wang et al., 2010). In general, almost all of the anthraquinone skeleton planes are stable, whereas the angles between the glucose groups and the phenyl ring are more or less changed (Fig. 1). This can be concluded that the interactions of these anthraquinone compounds might be related with the type and number of substituent groups on the molecular structure, as well as the substituted glucose rings.

Molecular docking
According to the literature (Devipriya and Kumaradhas, 2013;Zhu et al., 2021), the binding modes and binding a nities play important roles in the inhibition of the small molecules to the enzyme. Results indicated that the selected binding a nities for these complexes of molecules-DNA gyrase B are from -7.27 to -10.24 Kcal/mol. It is worth noting that the bonding energies of the anthraquinones are -8.50, -8.24, -7.27, -7.91 and -7.66 kcal/mol for molecules M1-M5, respectively. However, the corresponding anthraquinone glucoside compounds have generally higher intermolecular binding energy due to the strong interaction between the glucose ring and the surrounding amino acids Asn46, Arg76, Pro79, Gly101, and Lys103 (-10.24 -9.47 -9.74 -9.31 and -9.11 kcal/mol for molecules M6-M10, respectively). This is consisted with the experience result that the anthraquinone glucosides have much more antibacterial effect than their corresponding aglycones (Wang et al., 2010). At the same time, the structure of the glucose ring has also undergone signi cant changes (Table S1). Among them, the O1 of the glucose ring of the lowest binding energy molecule M6 (-10.24Kcal/mol) forms a hydrogen bond with the hydrogen atom HD21 of ASN46 at a length of 2.197Å, and the side hydroxyl group of the glucose ring forms hydrogen bond interactions with LYS103 and ASN46 at length of 2.091 Å and 2.062 Å, respectively (Fig. 2f).
Compared with the compound M6, the glucose ring at the active site of M7 rotates clearly clockwise, resulting in a smaller angle with the anthraquinone ring plane. The dihedral angle of C11-C12-O18-C26 is changed from 16.23º at the optimized state to -116.95º, which can be ascribed to the strong hydrogen bonding and electrostatic interactions with surrounding amino acids Asn46, Ala100, Phe104, Gly117 and Val120 (Fig. 2g). Besides the multiple hydroxyl groups on the anthraquinone skeleton, hydroxyl groups of the glucose ring are also important to the antimicrobial effect, which may be ascribed as the hydrogen bonding interactions at the active site. In fact, the combined anthraquinone chrysophanol 8-O-beta-Dglucoside shows good bioactivity on compared with the free chrysophanol (Li et al., 2007). Therefore, the in uence of substituent groups on anti-DNA gyrase B activity might not only be related with the type and number of substituent carboxyl, hydroxyl and hydroxylmethyl on phenyl ring groups, but also the combined glucose rings. Compared with the hydroxyanthraquinones, the interact area of the whole anthraquinone glucosides are larger (the part shown by the purple circle in Fig. 2k), between which the hydrophobic interaction plays a very important role. The nearest neighbors and short contact distances (Å) of anthraquinone compounds with the amino acid residues of DNA gyrase B are shown in Table 1. Usually, MEP is also related to the electronic density (Vijayalakshmi and Suresh, 2008), thus it is always used to anticipate reactive sites that electrophilic and nucleophilic reagents attack (Selvaraju et al., 2012). In this text, the negative (green) and positive (red) regions are regarded as electrophilic and nucleophilic reactivity regions, respectively (Fig. 5), while the white regions shows zero potential. In general, the electrostatic potential distribution of anthraquinone compounds is more uniform than that of the anthraquinone glucosides. The main positive charge is distributed near the hydrogen atom of the hydroxyl group on the side chain of the aromatic ring plane. For the molecule M3, the oxygen atom O18 in the carboxyl group acts as an electron donor to form a hydrogen bonding interaction with the HH11 of the amino acid Arg136, and thence the region at vicinity of the connected hydrogen atom H28 remains the highest electrostatic potential value of 66.73Kcal/mol. Due to the strong interaction between the side chain hydroxyl oxygen atoms (O17 and O19) of molecule M1 and the surrounding amino acid Gly77, and the electrostatic interaction between the oxygen atom O15 of the anthraquinone ring and amino acid Arg76, the electrostatic potential has reached the lower value of -58.25 Kcal/mol (Fig. 3a).
Once glucose ring is introduced into the hydroxyanthraquinone plane, it was shown that almost all of the positive molecular potentials of anthraquinone glucosides are distributed among the hydroxyl hydrogen atoms of the glucose structures except the molecule M9. These substituent hydroxyl groups on the molecular aromatic ring interact with the amino acids Glu42, Asn46 and Gly11 by hydrogen bonding and electrostatic interaction, which might lead to imbalance of distribution of electrons (the value of the global surface maximum is 75.40 Kcal/mol, Fig. 3i). In general, due to the presence of the hydroxyl group in the glucose ring, it can interact with surrounding amino acids Lys103, Asn46, Ala100, Phe104 and Val120 by intermolecular hydrogen bonds, electrostatic attraction and Van der Waals forces, resulting in major changes in the molecular structure and the molecular electrostatic potential distribution. In general, the positive molecular potentials at the vicinity of the molecular hydrogen atom are increased obviously when glucose ring is introduced into the hydroxyanthraquinone ring. Where, A and I are the ionization potential and electron a nity of the compounds respectively.
Commonly, the atom occupied by more densities of HOMO should have stronger ability to detach an electron whereas the atom with more occupation of LUMO should have ability to gain an electron. In the present study, the HOMO and LUMO energies are predicted at B3LYP method with 6-31G (d, p) basis sets. It is clear from Fig. 4 Table 2. Generally, the combined anthraquinone glucosides have larger dipole moments than that of the free anthraquinones.  However, the electron densities of the M1 at active site are higher than that of the other status. The electron densities (ρ bcp ) of the C-C bonds on aromatic ring for these molecules in both forms are ranged from 1.583 to 2.159 eÅ -3 , implying strong covalent interactions (Luis, 2016). Due to the strong electrostatic interactions between the carbonyl O7 atom and amino acids Asn46, His99, Phe104 and Val118, the electron density of the bond C6-O7 remains the highest value. In short, almost all of the electron densities of M1 at the active site are reduced, while the M6 remains constant (Fig. 5).

Conclusion
Rhubarb is a widely used traditional medicine and has been reported to elicit a number of biological effects, such as antibacterial effects. There are a lot of active compounds that were isolated from Rhubarb including some anthraquinone and anthraquinone glucosides. It was concluded from this text that almost all of the anthraquinone skeleton planes are constant when interacting with the target protein DNA gyrase B.
Compared with the multiple hydroxyl groups on the anthraquinone skeleton, hydroxyl groups of the glucose ring are also important to the antimicrobial effect, which may be ascribed as the hydrogen bonding interactions at the active site. In contrast to the average distribution of anthraquinone compounds, MEP analysis indicated that the almost all of the positive molecular potentials of anthraquinone glucosides are distributed among the hydroxyl hydrogen atoms of the glucose structures. In fact, the electron densities of the hydroxyanthraquinone are generally higher than that of the corresponding anthraquinone glucosides.
The electron densities become more depleted after docking with the DNA gyrase B, especially the hydroxyl groups. Although the anthraquinone glucosides were much more potent antibacterial effects than their corresponding aglycones, the raw Rhubarb is traditionally converted to processed Rhubarb by specialized heating to alleviate its side effects most probably caused by anthraquinones glucosides (Qin et al., 2011). It may be concluded that not only the functional groups carboxyl, hydroxyl and hydroxylmethyl, but also the glucose ring on phenyl ring can improve the antimicrobial activity. The structure-antibacterial effect of the hydroxyanthraquinone and anthraquinone glucosides can provide more references and insights for studying the antibacterial action mechanism of these natural components of Rhubarb.