The Search for Herbal Antibiotics: An In-Silico Investigation of Antibacterial Phytochemicals

Recently, the emergence and spread of pathogenic bacterial resistance to many antibiotics (multidrug-resistant strains) have been increasing throughout the world. This phenomenon is of great concern and there is a need to find alternative chemotherapeutic agents to combat these antibiotic-resistant microorganisms. Higher plants may serve as a resource for new antimicrobials to replace or augment current therapeutic options. In this work, we have carried out a molecular docking study of a total of 561 antibacterial phytochemicals listed in the Dictionary of Natural Products, including 77 alkaloids (17 indole alkaloids, 27 isoquinoline alkaloids, 4 steroidal alkaloids, and 28 miscellaneous alkaloids), 99 terpenoids (5 monoterpenoids, 31 sesquiterpenoids, 52 diterpenoids, and 11 triterpenoids), 309 polyphenolics (87 flavonoids, 25 chalcones, 41 isoflavonoids, 5 neoflavonoids, 12 pterocarpans, 10 chromones, 7 condensed tannins, 11 coumarins, 30 stilbenoids, 2 lignans, 5 phenylpropanoids, 13 xanthones, 5 hydrolyzable tannins, and 56 miscellaneous phenolics), 30 quinones, and 46 miscellaneous phytochemicals, with six bacterial protein targets (peptide deformylase, DNA gyrase/topoisomerase IV, UDP-galactose mutase, protein tyrosine phosphatase, cytochrome P450 CYP121, and NAD+-dependent DNA ligase). In addition, 35 known inhibitors were docked with their respective targets for comparison purposes. Prenylated polyphenolics showed the best docking profiles, while terpenoids had the poorest. The most susceptible protein targets were peptide deformylases and NAD+-dependent DNA ligases.


Introduction
Recently, established antibiotics have become less effective against numerous infectious organisms, and the Centers for Disease Control and Prevention (CDC) has warned of a "post-antibiotic era" [1]. This concern is heightened by our tenuous ability to detect, contain, and prevent emerging infectious diseases. The emergence of pathogenic microbes with increased resistance to existing antibiotics provides a major incentive for the discovery of new antimicrobial agents. The problems of drug-resistant pathogens have been reviewed recently [2][3][4][5]; there is a pressing need for more effective antibacterial therapy. Based on several recent reports, pathogens of immediate concern are methicillin-resistant Staphylococcus aureus (MRSA), a common cause of hospital-acquired infections, and which is evolving a resistance to vancomycin [6]; Pseudomonas aeruginosa in which multidrug resistance has become problematic [7]; Streptococcus pneumoniae, a common respiratory pathogen in which multidrug resistance is spreading [8]; multidrug-resistant strains of Mycobacterium tuberculosis [9], which are causing an alarming increase in the incidence of tuberculosis; and virulent strains of Escherichia coli, which continue to emerge [10][11][12].
Virtual screening using cheminformatics, pharmacophore, or ligand-and structure-based target prediction methods [13] has emerged as an advantageous alternative to high-throughput screening for identification of potential lead structures or biological targets for anti-infective drug discovery. For example, Bernal and Coy-Barrera have used a combination of molecular docking and multivariate analysis to identify antifungal and antiviral xanthone lead compounds [14]. Rahimi and co-workers have used a structural similarity search along with molecular docking to identify potential Shigella flexneri DNA gyrase inhibitors [15]. Molecular docking has been used to identify bacterial peptidyl-tRNA hydrolase as an additional alternative target for known antibiotic drugs [16].
Until the beginning of the twentieth century, virtually all medicines were derived from natural sources, most often from plants, and plants continue to serve as sources of new medicines and provide lead compounds for drug development. These antimicrobial agents derived from higher plants have been reviewed recently [17,18]. In the discovery of new and complementary antibacterial agents, phytochemicals that show antibacterial activity can be examined for potential inhibition of bacterial target proteins such as peptide deformylase (PDF), topoisomerase IV (TopoIV), DNA gyrase B (GyrB), protein tyrosine phosphatase (Ptp), UDP-galactopyranose mutase (UGM), cytochrome P450 (CYP121), and NAD + -dependent DNA ligase, as well as phytochemical inhibitors of bacterial efflux pumps or quorum sensing proteins, or agents that enhance the immune system. In this work, we have carried out an in-silico screening of phytochemicals identified in the Dictionary of Natural Products [19] as showing antibacterial activity against several potential bacterial protein targets.

Peptide Deformylase
The process of bacterial protein synthesis is initiated with N-formylmethionine (f-Met-tRNAi), which is generated through the enzymatic transformylation of methionyl-tRNA (Met-tRNAi) by formyl methionyl transferase (f-Mett). The N-formyl group of the polypeptide that emerges from the ribosome after completion of the elongation process is removed by the sequential action of peptide deformylase (PDF) [20,21]. Methionine amino peptidase (MAP) then removes the N-terminal methionine depending on the nature of the second amino acid in the peptide chain [22]. Therefore, deformylation plays a pivotal role in bacterial protein maturation, growth, and survival; PDF is vital in a variety of pathogenic bacteria but it is not required for cytoplasmic protein synthesis in the eukaryotes. Hence, PDF has been identified as a potential antibacterial drug target [23]. Bacterial PDFs are metallohydrolases that use Fe 2+ as the catalytic metal ion (which can be replaced with Ni 2+ or Zn 2+ ) that is tetrahedrally coordinated to two histidine residues, a cysteine residue, and a water molecule [24].

DNA Gyrase/Topoisomerase IV
Topoisomerase enzymes control the topological state of DNA within cells and are important for the essential process of protein translation and cell replication. Much attention in antibacterial drug discovery has been focused on the DNA gyrase (a type II topoisomerase) and topoisomerase IV. These types of topoisomerases are present in bacteria and plants, but not animals. DNA gyrase and topoisomerase IV share high structural and sequence similarity, yet play different necessary roles in the replication of DNA. Because of their vital nature and mechanisms of action, topoisomerases have become key drug targets for antibacterial drug discovery [25,26].

Bacterial Peptide Deformylase
MolDock docking energies (E dock ) and normalized docking scores (DS norm ) of antibacterial phytochemical ligands with bacterial peptide deformylase enzyme structures are summarized in Table 2. There were very few alkaloids docking to the bacterial peptide deformylase protein targets with notable docking scores. Those alkaloids that had large exothermic docking energies usually violated Lipinski's rule of five [66], with molecular weights >500 or hydrogen-bond acceptor atoms >10. Tuberine (76), however, did show excellent docking to Escherichia coli peptide deformylase (EcPDF) (E dock = −136.7 kJ/mol; DS norm = −126.5) compared to docking of the ligand with human PDF (HsPDF, E dock = −121.7 kJ/mol) or compared with the docking energy of the co-crystallized ligand actinonin (E dock = −111.8 kJ/mol). (+)-Tuberine (76), isolated from Haplophyllum tuberculatum, has shown antibacterial properties against Staphylococcus aureus and Bacillus subtilis, as well as E. coli [67,68].
Condensed and hydrolyzable tannins showed strong docking to bacterial PDFs, but these compounds violate Lipinski's rule of five [66], and are generally known to be non-selective protein complexing agents [91].
For comparison, several synthetic bacterial PDF inhibitors were also investigated in this docking study.  [86], showed docking energies with EcPDF of −101.6, −117.5, and −112.6 kJ/mol, respectively (i.e., they do not correlate). However, the docking energies of these compounds with BcPDF, MtPDF, and SaPDF do correlate with EcPDF inhibition as well as with Bacillus subtilis antibacterial MIC values [86].

Bacterial Topoisomerase IV/Gyrase B
The MolDock docking energies for the phytochemical ligands with E. coli topoisomerase IV, E. coli gyrase B, and M. tuberculosis gyrase B are summarized in Table 3. The co-crystalized ligand for EcTopoIV and MtGyrB was phosphoaminophosphonic acid-adenylate ester, which crystallized in the ATP binding site of the proteins (E dock~− 176 kJ/mol). The co-crystallized ligand for EcGyrB was novobiocin (E dock = −114.2 kJ/mol). (−)-Epicatechin gallate and (−)-epigallocatechin 3-gallate are known inhibitors of EcGyrB [93] and these compounds had docking energies of approximately −140 kJ/mol for EcTopoIV and MtGyrB (Table 3). There is a slight correlation between the docking energies of quercetin, epicatechin, epicatechin gallate, epigallocatechin, and epigallocatechin 3-gallate (−90.6, −87.6, −91.8, −90.8, and −94.1 kJ/mol, respectively) and the experimental dissociation constants (K d ) with EcGyrB (54, 36, 34, 23, and 15 µM, respectively) [93]. Similarly, there is a correlation between the experimental IC 50 values for quercetin (0.14 µM), norfloxacin (0.09 µM), and novobiocin (0.05 µM) [94] and the docking energies with EcGyrB (−90.6, −94.0, and −114.2 kJ/mol, respectively). Plaper and co-workers have carried out a binding study of quercetin with E. coli DNA gyrase [95]. These researchers found that quercetin (272) binds to EcGyrB with a K d of 15 µM. Furthermore, they carried out a molecular modeling analysis using InsightII v. 97. The final orientation of quercetin in the binding site of EcGyrB is very different from the orientation of the lowest-energy docked pose in this MolDock study (Figure 26).  [95]. These researchers found that quercetin (272) binds to EcGyrB with a Kd of 15 μM. Furthermore, they carried out a molecular modeling analysis using InsightII v. 97. The final orientation of quercetin in the binding site of EcGyrB is very different from the orientation of the lowest-energy docked pose in this MolDock study ( Figure 26).                      Wu and co-workers have examined the E. coli gyrase B inhibitory activity of several flavonoids [98]. Although none of the flavonoids were strong inhibitors, kaempferol (242) was the best with IC 50 = 0.037 mg/mL, followed by quercetin (272) (IC 50 = 0.076 mg/mL), chrysin (233) (IC 50 = 0.18 mg/mL), and myricetin (261) (IC 50 = 1.18 mg/mL). There is no correlation between these gyrase inhibitions and the docking energies to EcgyrB (E dock = −90.1, −94.6, −87.1, and −99.3 kJ/mol, respectively), except that these compounds are all relatively poor docking flavonoids and are also weak EcGyrB inhibitors.

Conclusions
This docking study of 561 known antibacterial phytochemicals helps to elucidate the possible biochemical targets for these compounds and there are some notable trends. The poorest docking ligands to the bacterial protein targets in this investigation were the terpenoids, while the best docking ligands, those with large negative (exothermic) docking energies, were generally phenolics. The most susceptible protein targets, based upon docking energies, for phytochemical ligands were E. coli peptide deformylase (EcPDF), E. coli topoisomerase IV (EcTopoIV), and E. coli DNA ligase (EcLigA). As a class, the alkaloids showed excellent docking to EcPDF, as did the diterpenoids and miscellaneous phenolics. S. aureus DNA ligase (SaLigA) was a good target for chalcones, flavonoids, and especially stilbenoids, while flavonoids and isoflavonoids docked well to EcTopoIV. Prenylated chalcones and flavonoids generally showed excellent docking properties to bacterial peptide deformylases and to bacterial DNA ligases. In evaluating the ligand docking in this work, we considered the criteria of docking selectivity (promiscuous binding compounds are unlikely to be useful therapeutic agents) and whether the docking characteristics of the ligand were noticeably better than known inhibitors. In this analysis, we have also considered drug likeness. That is, we have generally overlooked those phytochemical ligands that violate Lipinski's rule of five [66] (ligands with MW > 500 g/mol, hydrogen-bond-donating atoms > 5, hydrogen-bond-accepting atoms > 10, or ClogP > 5), even though they may have strong docking energies.
There are several limitations to in-silico docking results that should also be considered. Some of the phytochemicals examined may not be bioavailable due to limited solubility or poor bacterial cell wall permeability. In this study, we have examined the docking of the natural ligands (or their aglycones) and we did not take into account in vivo hydrolysis or other metabolic derivatization. The compounds examined have not been filtered for potential mammalian toxicity [109]. The docking studies also do not account for synergism in enzyme inhibition or antibacterial activity. The molecular docking method itself suffers from inherent limitations (e.g., the protein is modeled as a rigid structure without flexibility, solvation of the binding site and the ligand is excluded, and free-energy estimation of the protein-ligand complexes is largely ignored) [110,111]. Nevertheless, the results of this current study underscore the importance of natural products from higher plants in antibacterial drug discovery, and may provide potential avenues for the development of chemotherapeutic agents for the replacement of current antibiotic regimens or complementary management for bacterial infections.

Conclusions
This docking study of 561 known antibacterial phytochemicals helps to elucidate the possible biochemical targets for these compounds and there are some notable trends. The poorest docking ligands to the bacterial protein targets in this investigation were the terpenoids, while the best docking ligands, those with large negative (exothermic) docking energies, were generally phenolics. The most susceptible protein targets, based upon docking energies, for phytochemical ligands were E. coli peptide deformylase (EcPDF), E. coli topoisomerase IV (EcTopoIV), and E. coli DNA ligase (EcLigA). As a class, the alkaloids showed excellent docking to EcPDF, as did the diterpenoids and miscellaneous phenolics. S. aureus DNA ligase (SaLigA) was a good target for chalcones, flavonoids, and especially stilbenoids, while flavonoids and isoflavonoids docked well to EcTopoIV. Prenylated chalcones and flavonoids generally showed excellent docking properties to bacterial peptide deformylases and to bacterial DNA ligases. In evaluating the ligand docking in this work, we considered the criteria of docking selectivity (promiscuous binding compounds are unlikely to be useful therapeutic agents) and whether the docking characteristics of the ligand were noticeably better than known inhibitors. In this analysis, we have also considered drug likeness. That is, we have generally overlooked those phytochemical ligands that violate Lipinski's rule of five [66] (ligands with MW > 500 g/mol, hydrogen-bond-donating atoms > 5, hydrogen-bond-accepting atoms > 10, or ClogP > 5), even though they may have strong docking energies.
There are several limitations to in-silico docking results that should also be considered. Some of the phytochemicals examined may not be bioavailable due to limited solubility or poor bacterial cell wall permeability. In this study, we have examined the docking of the natural ligands (or their aglycones) and we did not take into account in vivo hydrolysis or other metabolic derivatization. The compounds examined have not been filtered for potential mammalian toxicity [109]. The docking studies also do not account for synergism in enzyme inhibition or antibacterial activity. The molecular docking method itself suffers from inherent limitations (e.g., the protein is modeled as a rigid structure without flexibility, solvation of the binding site and the ligand is excluded, and free-energy estimation of the protein-ligand complexes is largely ignored) [110,111]. Nevertheless, the results of this current study underscore the importance of natural products from higher plants in antibacterial drug discovery, and may provide potential avenues for the development of chemotherapeutic agents for the replacement of current antibiotic regimens or complementary management for bacterial infections.