Potential of hydroxybenzoic acids from Graptopetalum paraguayense for inhibiting of herpes simplex virus DNA polymerase – metabolome profiling, molecular docking and quantum-chemical analysis

: According to our previous investigation the total methanol extract from Graptopetalum paraguayense E. Walther demonstrates a significant inhibitory effect on herpes simplex virus type 1 (HSV-1). To clarify what causes this inhibitory activity on HSV-1, a metabolic profile of the plant was performed. Three main fractions: non-polar substances, polar metabolites and phenolic compounds were obtained and gas chromatography–mass spectrometry (GC-MS) analysis was carried out. Since it is well known that phenolic compounds show a significant anti-herpes effect and that viral DNA polymerase (DNApol) appears to play a key role in HSV virus replication, we present a docking and quantum-chemical analysis of the binding of these compounds to viral DNApol amino acids. Fourteen different phenolic acids found by GC-MS analyses, were used in molecular docking simulations. According to the interaction energies of all fourteen ligands in the DNApol pockets based on docking results, density functional theory (DFT) calculations were performed on the five optimally interacting with the receptor acids. It was found that hydroxybenzoic acids from phenolic fraction of Graptopetalum paraguayense E. Walther show a good binding affinity to the amino acids from the active site of the HSV DNApol, but significantly lower than that of acyclovir. The mode of action on virus replication of acyclovir (by DNApol) is different from that of the plant phenolic acids one, probably.


Introduction
Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are members of the Herpesviridae family and are among the most common human pathogens, infecting about 90% of the world population [1]. Generally speaking, the main treatments available include several selective drugs that are classified as virucidal, immunomodulators, and chemotherapeutic agents. Common therapies for herpes infections employ nucleoside analogs, such as acyclovir (ACV), and target the viral DNA polymerase (DNApol), essential for viral DNA replication. However, the widespread use of such drugs has led to undesirable side effects and drug-resistant strains [2]. A better understanding of the herpes virus replication will help the development of new safe and effective broadspectrum anti-herpetic drugs that fill an unmet need.
When searching for potent bioactive compounds, it is especially important to understand the nature of the interaction between biological macromolecules (enzymes, receptors) and small ligands (inhibitors, drugs). Theoretical chemistry can be used for the prediction of chemical structures and reactions, which would support biological testing, save time and resources. A promising way is to combine the complete three-dimensional structure of the biomacromolecule-ligand complex with theoretical energy calculations [3]. A very important methodology for elucidating the reaction mechanisms and other properties of enzymes is the so-called quantum chemical cluster: a small part of the enzyme around the active site is treated with relatively accurate quantum chemical methods [4]. Quantum-chemical calculations, most commonly density functional theory be included in the following groups: alkaloids and nitrogenated compounds, coumarins, flavonoids, lignans, miscellaneous compounds, monoterpenoids, diterpenoids and sesquiterpenoids, phenolic acids, phenylpropanoids, quinones, tannins, thiophenes, triterpenoids and polyacetylenes [5][6][7]. Phenolic compounds attract great interest due to their antioxidant activity and beneficial effects on human health [8]. Results from preliminary antiviral plate assay of the green solvent (hydro-ethanolic) shoot extract of Limonium densiflorum showed that gallic acid and epigallocatechin gallate have strong activity, while pinoresinol and N-trans-ferulolyl tyramine have moderate activity [9]. It was found that phenolic compounds from acetonic and methanolic extract of apple pomace inhibit both HSV-1 and HSV-2 replication in Vero cells by more than 50%, at noncytotoxic concentrations [10]. Viral replication is not the only therapeutic goal studied, as several in vitro studies have shown that many plant extracts (eg aqueous root and bark extract of Rhus aromatica L. [11] as well as aqueous and hydroalcoholic extracts of propolis [12]) and their isolated compounds can inhibit the penetration of a virus. The authors attribute the anti-herpetic activity to the interference of the extracts with the viral envelope structures required for adsorption. Phenyl carboxylic acids, as well as polyphenols, have been identified as responsible compounds, although the synergistic activity of the plant complex has been suggested.
Graptopetalum paraguayense E. Walther (GP) is a species of succulent plant that belongs to the jade family Crassulaceae, originating from Tamaulipas, Mexico. In Taiwan, Graptopetalum paraguayense is a medicinal plant that is considered a vegetable with health benefits. As early as ancient China GP was traditionally used to treat a number of diseases: modulating blood pressure, relieving liver disease, relieving pain and infections, detoxification. Reported biological effects of GP include tyrosinase inhibition [13], antioxidants [14], regulation of hypertension [15,16], hepatoprotective effect [17,18], antitumor activity [19], anti-inflammatory [20] and Alzheimer's disease therapy [21].
There are no data in the literature on the antiviral activity (including anti-herpes activity) of GP. Recently we found that the total methanol extract from the plant demonstrates a significant inhibitory effect on HSV-1 [22]. Therefore, it is important to investigate the metabolic profile of the plant and which metabolites are responsible for this activity. Since virus-encoded DNA polymerase appears to be a key feature in the replication of large DNA viruses such as HSV, we present metabolome analysis of the plant Graptopetalum paraguayense E. Walther as well as theoretical investigations on the binding expedient of the compounds from phenolic fraction to viral DNA polymerase amino acids.

Extract preparation
Leaf extract was prepared as described in our previous study [22]. Briefly, 1.

GC-MS sample preparation
Prior to the gas chromatography-mass spectrometry (GC-MS) analysis, fractions "A", "B" and "C" were derivatized by the following procedures. 100.0 μL pyridine and 100.0 μL BSTFA were added to the dried residues (fractions "A" and "C"), then heated on  and Born solvation water model. The site finder algorithm [24] was used to identify sites for the docking procedure and two of them, that lie on the contact surface between protein and DNA and are close to amino acids in active site of the DNA polymerase, were selected for further work. Conformation library for the docking study was generated using The best 50 poses for every ligand for every pocket were further optimized with Induced Fit methodology using MMFF94 force field and optimization cutoff of 6A from the ligand [25]. The GBVI/WSA ΔG [24] was used as a rescoring function and the best 30 poses were collected for the next coming analysis.

Quantum-chemical calculations
According to the interaction energies of all fourteen ligands (phenolic acids from fraction "C") in the pockets based on the GBVI/WSA ΔG scoring function, DFT calculations were performed on the five optimally interacting with the receptor acids. The B3LYP [26,27] functional has been widely used over the years as it has been found to provide a good balance between speed and accuracy. Thus, geometric optimization of the computational level without symmetry constraints. Hydroxybenzoic acids and some of the amino acids that interact in the active center pocket of the enzyme are deprotonated.
The same applies to acyclovir and its interacting amino acids. Acyclovir as an inhibitor of viral DNA synthesis is monophosphorylated by virus-encoded thymidine kinase (TK).
Subsequent diphosphorylation and triphosphorylation are catalyzed by host cell enzymes, resulting in acyclovir triphosphate [28]. Therefore, we use in our model was set to 1 and we chose the argon as medium to mimic the nature of the enzyme inside based on the previous studies [30,31]. The nature of the local minima on the potential energy surface was checked again by the absence of imaginary frequency. The DFT calculations were performed using Gaussian09 software package [32].

Results and discussion
In our pilot study [22] on the anti-herpes activity of Graptopetalum paraguayense E.
Walther (Figure 1), we showed that the total GP methanol extract demonstrated a significant inhibitory effect on HSV-1 (97.5% cell protection), compared to ACV (with total protection of the cells 100%). The in vitro cytotoxicity assays of the GP extract indicate that it is characterized by a high cell tolerable concentration range. The results showed that the GP extract could be administrated in a concentration range (maximal nontoxic concentration, MNC, and lower) that avoids significant cell damage. The present study aims to further clarify both the organic composition of the plant and the mechanism of action of its main phytochemical components. The amount of phenolic components was 1701.50 μg/g DW ( Overall, β-amyrin (2080.86 μg/g DW), β-sitosterol (2010.41 μg/g DW), and α-tocopherol (1447.18 μg/g DW) were the predominant compounds in the fraction of sterols, terpenoids, and tocopherols (Table 3S).      triphosphate-amino acids complex were performed at the same theoretical level. This is necessary because the available treatments are based on several selective drugs such as acyclovir, which are able to inhibit DNA polymerase [2]. In this way, the inhibitory activity of phenolic acids from GP to viral DNApol could be compared with that of acyclovir triphosphate (AcvTP). The surroundings in the active enzyme pocket of AcvTP were simulated similarly to phenolic acids, according to molecular docking results. The three hydroxyl groups from the phosphate residue in the acyclovir triphosphate molecule are deprotonated and interact with three protonated lysine molecules (at amino groups) in the enzyme active site (Fig. 8). AcvTP binds by hydrogen bonds to Glu, Tyr and two water molecules forming AcvTP-3pLys-Glu-Tyr-2H2O complex.
All species (PA, AcvTP, AA and water molecules), as well as all complexes, were optimized at the B3LYP/6-31+G(d,p) level of theory.
In order to evaluate the possibility of intermolecular hydrogen bonds formation between PA and AA, the energies of interaction (Eint) and interaction free Gibbs energies (Gint) were calculated by eq. (1) and (2): EPA, EAA, Ewater, and Ecomplex are the Et energies; GPA, GAA, 2Gwater, and Gcomplex are the G298 energies, calculated at B3LYP/6-31+G(d,p) level, for each phenolic acid, amino acids, AcvTP, water molecules and its complex, respectively. Eint and G298 of all complexes are presented in Table 2. can be seen from the results received, the interaction energy of acyclovir triphosphate complex is more than four times higher than that of tFA and GntA complexes (with highest Eint). The reason could be in the larger number of deprotonated -OH groups in the AcvTP molecule than in the phenolic acids ones. This leads to stronger binding of AcvTP to the active site of the enzyme compared to PA from GP. The picture is slightly different when the free Gibbs energies of interaction were considered: the AcvTP complex again has the highest interaction energy (477.28 kcal mol -1 ), but the energy differences with hydroxybenzoic acids complexes decrease by an order of magnitude ( interactions that are not accounted for by standard DFT functionalities [36]; ii). Despite the good results for the binding expedient of the phenolic fraction of GP, it is likely that the mechanism of action of these compounds includes inhibition of HSV-1 replication not only through DNA polymerase but probably by other enzymes. It is possible that the manner of the inhibitory activity on virus replication of AcvTP (by DNA polymerase) is different from that of the plant hydroxybenzoic acids one. Further investigations in this direction would help to clarify the antiviral activity of Graptopetalum paraguayense E. Walther.

Conclusions
Current treatment for HSV infection relies mainly on the use of acyclovir and related synthetic nucleoside analogs. The widespread use of these drugs has led to the establishment of side effects and drug-resistant strains, which has increased the need for new natural antiviral agents. Total methanol extract from the succulent plant Graptopetalum paraguayense E. Walther demonstrates a significant inhibitory effect on HSV-1, according to our recent study [22]. In order to explain this strong antiviral activity, metabolic analysis of the plant was performed. Three main fractions: non-polar substances, polar metabolites, and phenolic compounds were obtained and GC-MS analysis was carried out. Fourteen different phenolic acids such as gallic, trans-ferulic, syringic acids, and others found by GS-MS analyses, were used in molecular docking simulations. The optimally interacting with the receptor amino acids were used for quantum-chemical calculations at B3LYP/6-31+G(d,p) computational level to establish the binding affinity of hydroxybenzoic acids from GP to HSV DNApol active site. According to the interaction energies of all five ligand-amino acid complexes, the hydrogen bonding in all of them is strong but significantly weaker than that in the acyclovir complex. The reason could be in DFT methods used as well as in the mechanism of antiviral effect. It is possible the mode of the inhibitory activity on the virus replication of AcvTP (by DNA polymerase) is different from that of the plant hydroxybenzoic acids one. Further investigations in this sense would help to clarify the antiviral activity of Graptopetalum paraguayense E. Walther.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1: Polar metabolites fraction from G. paraguayense determined by GC-MS analysis, Table S2: Main saturated fatty acids fraction from G. paraguayense determined by GC-MS analysis, Table S3: Sterol fraction from G.paraguayense determined by GC-MS analysis. Figure S1: Gallic acid and its vicinity after docking procedure, Figure S2: Gentisic acid and its vicinity after docking procedure, Figure S3: Syringic acid and its vicinity after docking procedure and Figure S4: Vanillic acid and its vicinity after docking procedure.