IN-SILICO SCREENING OF POTENTIAL PHYTOCOMPOUNDS AGAINST STAPHYLOCOCCUS AUREUS AND AN IN-VITRO ANTIBACTERIAL EVALUATION

IN-SILICO SCREENING OF POTENTIAL PHYTOCOMPOUNDS AGAINST STAPHYLOCOCCUS AUREUS AND AN IN-VITRO ANTIBACTERIAL EVALUATION

Vidya and Sathish Kumar *

Department of Biotechnology, Kongunadu Arts and Science College, Coimbatore, Tamil Nadu, India.

ABSTRACT: Staphylococcus aureusis a Gram-positive facultative pathogenic bacterium responsible for a wide range of infections ranging from skin to life-threatening infections. Antibiotic resistance of Staphylococcus aureus is an emerging global concern. Thus, developing viable antibiotics are in high demand. This study identified novel lead compounds from traditionally used medicinal plants via in-silico molecular docking and in-vitro antibacterial analysis. Thus, we have derived literature-evident phytocompounds from numerous traditional medicinal plants such as Boerhavia diffusa, Clerodendrum infortunatum, Sida rhombifolia, Tephrosia purpurea, Scoparia dulcis, Breynia retusa, Euphorbia heterophylla, Hemigraphis alternata, Hedyotis corymbose, Imperata cylindrica and their structures were retrieved from PubChem. Lipinski’s rule in ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) profiles were used to screen derived phytocompounds, followed by in-silico docking to the target protein. Clumping factor A (ClfA)-fibrinogen, a key virulence factor in S. aureus, was taken as a target protein. ClfA is a cell-wall-anchored protein that causes bacterial adherence to the blood plasma protein fibrinogen, which causes a variety of infections. Thus, an appealing strategy is to discover a novel lead compound with antiadhesive properties to prevent cell adherence. After performing molecular docking, Eupalitin 3-o-galactoside, a natural compound derived from Boerhavia diffusa, exhibited strong binding affinity with the least glide score of -8.56 kcal/mol. Antibacterial investigations were carried out using different solvent extractions of plants with phytocompounds that exhibited a significant glide score. Leaf extract of Boerhavia diffusa, Clerodendrum infortunatum and Sida rhombifolia shows the strongest activity against Staphylococcus aureus.

Keywords: Staphylococcus aureus, Antibiotic resistance, Multidrug Resistance, ClfA-Fibrinogen, Phytocompounds, ADME, Molecular docking, Antibacterial

INTRODUCTION: Staphylococcus aureus is a Gram-positive spherical bacterium belonging to the Staphylococcaceae family. It is one of the most harmful bacteria, causing diseases ranging from minor skin infections like folliculitis and impetigo to life-threatening infections including bloodstream infection, endocarditis, and pneumonia ¹.

S. aureus can create a broad spectrum of virulence factors connected to the cell wall and play a major role in invading microbes into the host tissue. It can also release exotoxins, which promote staphylococcal infections. The adherence of bacteria to host extracellular matrix proteins such as fibrinogen, fibronectin, and collagen triggers the molecular pathogenesis of infections ².

Antibiotics have been used to treat bacterial illnesses since the early 20^th century. Antibiotic exploitation is assumed to be responsible for antibiotic-resistant strains' widespread growth. Most bacteria are now resistant to multiple treatments, making this scenario difficult to treat. Multidrug-resistant bacteria cause a massive range of bacterial diseases, with Staphylococcus aureus being one of the most threatening MDR strains. The emergence of resistant strains poses a persistent hazard to human health, which is now a serious challenge ³. To combat multidrug resistance strain, a variety of techniques have been implemented. Currently, antibiotics such as ceftaroloine, ceftobiprole, co-trimoxazole, cephalosporin, dalfopristin, tedizolid and linezolid are available but they are not employed in clinical procedures because of their high cost and safety concerns ⁴; therefore, there is a compelling need for the development of novel drugs. The conventional drug development method is complex and time-consuming so computer-assisted docking can be utilized to find new lead molecules. Molecular docking is a time-saving method for docking a large number of molecules to a target protein ⁵.

In the current scenario, virtual screening is employed to develop an efficient therapeutic compound in which structure-based virtual screening will be carried out where screening is based on receptor structure ⁶. In this study plant, phytocompounds were used as ligands since plants produce a vast range of secondary metabolites with diverse pharmaceutical properties like antimicrobial, anti-fungal, anti-inflammatory, and antitumor activity and are mostly preferred due to their fewer no side effects ⁷. Developing drugs from herbal plant samples would be cost-effective and biocompatible ⁸.

And as target protein ClfA- fibrinogen was utilized. Clumping factor A (ClfA) is a cell adhesion protein anchored to the surface of Staphylococcus aureus that allows bacteria to adhere to fibrinogen in host tissue, thus it is known to be a fibrinogen binding protein. Fibrinogen is a glycoprotein found in the blood that consists of a polypeptide chain (2α, 2β and 2γ chains) ⁹. A- region of ClfA binds to fibrinogen by interacting C-terminal of two γ- chains of fibrinogen. ClfA protects S. aureus against macrophage phagocytosis, as a result, bacteria become more virulent ¹⁰.

Therefore, we have considered ClfA- fibrinogen as a potential target protein and phytocompounds from medicinal plants such as Boerhavia diffusa, Clerodendrum infortunatum and Sida rhombifolia, Scoparia dulcis, Breynia retusa, Euphorbia heterophylla, Hemigraphis alternata, Imperata cylindrica, Hedyotis corymbosa and Tephrosia purpurea were used as ligand. Boerhavia diffusais a well-known Ayurvedic plant and the aerial part of B. diffusa are reported to have notable antioxidant and antibacterial activity ¹¹. The entire plant, including the roots, leaves, and stem of Clerodendrum infortunatum, exhibits a variety of biological functions. According to studies, leaf extracts of C. infortunatum poses significant antibacterial and antifungal activities than root and stem ¹². Similarly, the aerial part of Sida rhombifolia is rich source of phytoconstituents and is known to have antibacterial properties ¹³. Therefore, the current study focuses on in-silico analyses to determine the phytocompound with the best binding efficiency against the target protein and in-vitro antibacterial screening was used to determine the plant extract with optimum antibacterial activity against Staphylococcus aureus.

METHODS:

In-silico Studies: Molecular docking is an efficient and expanding method for developing prospective lead drugs. Molecular docking involves various computational procedures, including preparing receptors and ligands, docking and post-docking analyses, etc. ¹⁴. The computational software maestro Schrodinger version 9.0.211 was utilized for ADME profiling, LigPrep, Protein preparation, Glide grid generation, and G scoring function.

Structure Retrieval: This study aimed to evaluate the antibacterial activity of phytocompounds from different natural plants. After conducting a literature survey, GC-MS identified 125 phytocompounds from 10 different plants considered ligands. The PubChem database (http://pubchem.ncbi.nlm.nih.gov) was used to derive the chemical structures of phytocompounds ¹⁵. The three-dimensional structure of the protein to be targeted, ClfA- Fibrinogen was retrieved from the PDB- Protein Data Bank database (http://www.rcsb.org/pdb). The target protein's active site region was determined using the LigSite online tool (http://projrct.biotec.tudresden.de/pocket/), which predicts the amino acids with binding pockets ¹⁶.

ADME Profiling of Phytocompounds: To assess the drug-likeness of particular ligands, the ADME properties, which include Absorption, Distribution, Metabolism, Excretion, and Toxicity of phytocompound were analyzed. The Schrodinger program's QikProp module version 4.4 was used to predict the ADME properties including, the number of rotatable bonds, molecular weight, number of donor hydrogen bonds, number of acceptor hydrogen bonds and octanal water partition coefficient logP, etc as per Lipinski rule of five ¹⁷ and the Pass online way2drug online tool (http://way2drug.com/PassOnline/predict.php)was used to further analyze the biological properties of the ligand ¹⁸.

Ligand and Receptor Preparation: Before molecular docking, ligands were prepared using the LigPrep module, optimized by bond ordering and angles. In contrast, GLIDE's Protein preparation wizard was used to prepare proteins. In which water molecules were removed from the structure for preparation, hydrogen bonds were optimized and energy was minimized, further structure-based virtual screening was performed ¹⁹.

Molecular Docking: Identification of new therapeutic compounds is a critical step in the in-silico investigation and is accomplished through molecular docking, where structure-based virtual screening was performed for each screened phytocompound against ClfA-Fibrinogen. The glide module of the Schrodinger program was used to simulate receptor-ligand interaction and binding affinities.

Effective ligands against the target protein will be identified due to molecular docking based on the least glide score value and by the formation of hydrogen bonds and hydrophobic interactions ²⁰. The PyMol visualization tool was further used to see the hydrogen bond interaction between the ligand and the target protein, where the interaction between the amino acid residues and the hydrogen bonds with bond length can be evaluated ²¹.

In-vitro Studies:

Sample Collection and Authentication: The three different plant species Boerhavia diffusa, Clerodendrum infortunatum and Sida rhombifolia were chosen for in-vitro antibacterial screening among the remaining ten plants as a result of in-silico studies. Those plants were harvested within the Kanyakumari district of Tamil Nadu and the Botanical Survey of India (BSI) in Coimbatore, Tamil Nadu, identified those plants as Boerhavia diffusa (BSI/SRC/5/23/2022/Tech/520), Clerodendrum infortunatum (BSI/SRC/5/23/2022/Tech/518) and Sida rhombifolia (BSI/SRC/5/23/2022/Tech/519).

Sample Preparation: Leaves, stem, root and flowers of Boerhavia diffusa, Clerodendrum infortunatum and Sida rhombifolia were washed, dried and crushed into fine powder. 50 grams of powdered samples were subjected to Soxhlet extraction using hexane, ethyl acetate and methanol at 40°C for roughly 6-8 hours ²². After extraction excess solvents were evaporated using rotary evaporator under lower pressure. Final extracts were collected and kept for future use in an airtight container.

Anti-Bacterial Screening: The Agar well diffusion method was used to determine the antibacterial activity of various extracts ²³. Staphylococcus aureus culture was purchased from MTCC (MTCC-96). 100 µl of bacterial culture were swabbed into MHA plates, followed by 6 mm wells that were created using a sterile cork borer.

50 µl of crude extract from each sample and 50 µl of neomycin sulphate as a positive control were introduced to the wells. Organic solvents hexane, ethyl acetate, and methanol were used as the negative control. Plates were further kept for overnight incubation at 37 °C.

After the incubation period, the sensitivity of the test plates was assessed using a zone of inhibition, with the diameter of the zone surrounding the well determined in millimeters.

RESULT:

Structure Retrieval and Active Site Prediction: 3D Structure of the target protein ClfA-Fibrinogen was retrieved from Protein Data Bank with a PDB ID of 2VR3 (Fig. 1), and its active site pockets ASN 525, ILE 384, GLU 526, ALA 528 was discovered via the LigSite online tool.

FIG. 1: 3D STRUCTURE OF TARGET PROTEIN 2VR3

ADME Screening Using QIKPROP Module: The bioavailability of selected phytocompounds was predicted using ADME profiling, which is a crucial step in the discovery of potential lead compounds. Out of 125 phytocompounds, only 24 compounds satisfy the Lipinski rule of five and are considered to be drug-likeness. Parameters like lipophilicity, permeability in octanol/ water partition coefficient and brain/ blood barrier along with these, properties like Number of rotatable bonds, Number of metabolic reactions, Molecular weight, Hydrogen bond donor, Hydrogen bond acceptor and Skin permeability were evaluated. The compounds that satisfied the Lipinski rule of five were tabulated in Table 1 and the pharmacological properties of ADME-cleared compounds were validated using PASSonline Way2Drugand are reported in Table 2.

TABLE 1: ANALYSIS OF ADME PROPERTIES FOR THE PLANT COMPOUNDS USING QIKPROP

TABLE 2: 2D STRUCTURE OF ADME CLEARED PHYTOCOMPOUNDS WITH PHARMACOLOGICAL PREDICTION

S. no.	Phytocompound with PubChem ID	2D structure	Pass prediction
1	3'4-dihydroxy-benzyl alcohol (101663520)
2	4-(2-methoxy phenyl) piperidine
3	5- Benzyloxypyrimidine (561874)
4	Acacetin (5280442)
5	Apigenin (5280443)
6	Benzofuran 2,3 dihydro (20209882)
7	Boeravinone B (14018348)
8	Caffeic acid (689043)
9	Desulphosinigrin (9601716)
10	Ellagic acid (5281855)
11	Epicatechin (72276)
12	Eupalitin 3-O- galactoside (44259727)
13	Ferulic acid (445858)
14	Furon- 2yl methanol (49962474)
15	Gallic acid monohydrate (24721416)
16	Kaempferol (5280863)
17	Malic acid (522155)
18	Purpurin (6683)
19	Quercetin (5280343)
20	Semiglabrin (156341)
21	Succinic acid (520988)
22	Ursolic acid (64945)
23	Vasicinol (442934)
24	Vasicinone (442935)

Molecular Docking Studies: Molecular docking investigation predicts the interaction of bioactive compounds against the target protein in Staphylococcus aureus. Molecular docking was carried out using the Glide module of maestro Schrodingerr software.

The docking result interprets the active site and binding efficiency of phytocompounds against the target protein 2VR3. Phytoconstituents from Boerhavia diffusa, Clerodendrum infortunatum and Sida rhombifolia were found to be potential lead compounds against 2VR3.

Each phytocompounds were allowed to dock with the target protein and the binding efficiency that is the formation of hydrogen bonds was visualized using the PyMol visualization tool. The bioactive compound Eupalitin 3-O- galactoside showed efficient binding interaction against the target protein with theleast Glide score is -8.56 Kcal/mol and the residues interacted were GLN 253 (O-H), GLN 253 (H-O), GLN 253 (H-O), HIS 252 (H-O), GLU 526 (H-O), GLU 526 (O-H), ASN 525 (O-H), ARG 506 (O-H) and ILE 384 (O-H) and with a bond length of 2.6Å, 2.0Å, 2.5Å, 2.1Å, 1.8Å, 1.8Å, 1.9Å and 2.3Å, respectively. Additionally, the compound quercetin and vasicinone from the plants Clerodendrum infortunatum and Sida rhombifolia also hada better binding ability with a G. score of -8.35 and -5.29 Kcal/mol respectively.

Apart from these phytocompounds from other plants such as Tephrosia purpurea, Scoparia dulcis, Breynia retusa and Euphorbia Herterophylla also had significant binding properties with the target protein. The phytocompound Ursolic acid from the plant Boerhavia diffusa has shown poor binding interaction with the target protein with a G score of -1.92 Kcal/mol.

The binding interactions with the glide score value was reported in Table 3. Fig. 2 represents the binding efficiency of Eupalitin 3-O-galactoside with the target protein 2VR3. Therefore, the molecular docking studies reveal that phytocompounds from Boerhavia diffusa, Clerodendrum infortunatum and Sida rhombifolia have significant inhibition against 2VR3 and further in-vitro antibacterial studies were carried out to investigate its antibacterial activity.

TABLE 3: MOLECULAR DOCKING OF PHYTOCOMPOUNDS AGAINST TARGET PROTEIN CLFA- FIBRINOGEN

FIG. 2: MOLECULAR INTERACTION OF EUPALITIN 3-O- GALACTOSIDE WITH THE TARGET PROTEIN (A) AND DOCKED COMPLEX (B). Note: The deep teal color represents the target protein and the deep salmon color indicates the Eupalitin 3-O-galactoside. Blue dots represent the hydrogen bond interaction of Eupalitin 3-O-galactoside with the active site region of the target protein.

Antibacterial Screening: As a result of in-silico studies, we conclude that phytocompounds from Boerhavia diffusa have shown significant binding interaction with the target protein, followed by the plants Clerodendrum infortunatum and Sida rhombifolia. Thus, the antibacterial activity of these pants was performed using the agar well diffusion method. Fig. 3 depicts the antibacterial activity of different plant extracts in which crude ethyl acetate leaf extract of all three plants showed efficient antibacterial activity. The zone of inhibition was used to determine the sensitivity of test plates. The maximum zone of inhibition was 24 mm observed in ethyl acetate leaf extract of Boerhavia diffusa and a minimum zone of inhibition was 10 mm observed for hexane root extract as illustrated in Table 4. Neomycin sulphate was used as a positive control and exhibited good antibacterial activity. Whereas all the negative controls hexane, ethyl acetate and methanol revealed no activity against Staphylococcus aureus.

FIG. 3: ANTIBACTERIAL ACTIVITY OF PLANTS EXTRACTS AGAINST STAPHYLOCOCCUS AUREUS. Note: Agar well diffusion method was used to evaluate the Antibacterial activity of leaf, stem, root, and flower extracts against Staphylococcus aureus. (a) represents the activity of the plant Clerodendrum infortunatum, followed by Boerhavia diffusa (b), and Sida rhombifolia (c)

TABLE 4: DIAMETER OF ZONE OF INHIBITION OF DIFFERENT PLANT EXTRACTS AGAINST STAPHYLOCOCCUS AUREUS

Note: (M): Methanol, (EA): Ethyl acetate, (H): Hexane, (NS): Neomycin sulphate. For each extract’s average zone of inhibition, the diameter was calculated from the triplicates.

DISCUSSION: Antibiotic resistance is a massive issue. Overuse or abuse of antibiotics leads bacteria to become more resistant to the inhibitory effects of antibiotics ²⁴. Every year 700,000 people die due to diseases caused by multidrug-resistant bacteria ²⁵. Staphylococcus aureus is one of the multidrug-resistant bacteria and is resistant to penicillin and methicillin. The lack of treatment options for MRSA infections is also a major global concern ²⁶. Thus, the current research mainly focuses on developing efficient drug molecules against Staphylococcus aureus from traditional medicinal plants. Here we used bioinformatics tools such as molecular docking to find efficient lead molecules and further validation was done using in-vitro antibacterial evaluation. S. aureus infections are usually caused by the adhesion of multiple surface-anchored virulence proteins, which interact with the host tissue ²⁷. Studies have revealed that ClfA is important in developing staphylococcal infections ²⁸. Additionally, ClfA is responsible for infective endocarditis, which is initiated by platelet aggregation in the host ²⁹. It was reported that the extracellular matrix protein ClfA found in Staphylococcus aureus protects the bacterium from phagocytosis, rendering them more virulent ¹⁰. Thus, targeting ClfA could be a promising way to identify the effective lead molecule. Hence, in this work, ClfA-Fibrinogen is considered the target protein. The literature identified that phytocompounds from different plants were considered ligands since phytochemicals have a wide range of biological activities ³⁰. These bioactive compounds were evaluated for ADME characteristics, and their drug-likeness was determined using the Lipinski rule of five (RO5) ³¹. This study used structure-based virtual screening to estimate phytocompound interaction and binding affinities with the target protein by which efficient drug molecules will be identified based on scoring function and glide energy ³².

As a result of docking studies, phytocompounds from the plants Boerhavia diffusa, Clerodendrum infortunatum, and Sida rhombifolia have shown excellent glide scores. And all these three plants are known for their biological properties. Docking studies revealed that the compound eupalitin 3-O- galactoside from the plant Boerhavia diffusa had remarkable binding interaction with the target protein. A study reported that the Boerhavia diffusa contains phytoconstituents with a wide range of therapeutic benefits, including antioxidant and anticancer properties. Methanolic extracts are found to have strong antioxidant activity ³³. According to reports, it was revealed that roots and aerial parts of B. diffusa contain methylated eupalitin in both free and glycoside forms ³⁴.

It has anti-inflammatory and immunosuppressive properties as a consequence of the high content of polyphenols in it. Additionally, Eupalitin was found to have improved cancer chemopreventive properties. it induces ROS levels which leads to apoptosis in prostate cancer ³⁵. In-silico studies have revealed that the anti-inflammatory compound eupalitin-3-O-galactoside has a dual effect on cancer, one by inhibiting the target protein aldose reductase enzyme (ALR2) and other by suppressing cancer-mediating pathways ³⁶. This suggests that eupalitin 3-O- galactoside can be used to develop efficient drug molecule against S. aureus. To confirm the in-silico studies we have evaluated invitro antibacterial activity of Boerhavia diffusa along with the other two plants whose bioactive compounds also showed efficient interaction with the target protein. In a study, various solvents were used to conduct an antibacterial study on the roots, stems and leaves of B. diffusa. where There was no indication of antibiotic resistance in the aqueous or chloroform extracts. The root extract shows a maximum level of inhibition. At 200 µg of extract concentration, the greatest inhibition of bacterial growth was discovered with a zone of roughly 8 mm³⁷. In this study, crude ethyl acetate leaf extract showed significant activity with a maximum zone of inhibition of 24mm, followed by stem and flower extract.

Another study observed that a low polar ethanolic extract of B. diffusa had a higher concentration of phytochemicals than an aqueous extract. Since, phytocompounds are more soluble in less polar solvents. In addition, leaf extracts of B. diffusa were found to have effective antibacterial efficacy against S. aureus³⁸. This supports our outcome that the mid-polar solvent, ethyl acetate leaf extract of B. diffusa has efficient antibacterial activity. Ethanolic and chloroform extracts of Clerodendrum infortunatum were studied to have efficient inhibitory efficacy against S. aureus when compared to the common drugs tetracycline and fluconazole ³⁹. Similarly, in our study, when compared to the standard antibiotic neomycin sulphate, the ethyl acetate leaf extract of C. infortunatum demonstrated effective inhibitory activity against S. aureus.

A study reported that the ethyl acetate and chloroform extracts had the strongest antibacterial activity against S. aureus, whereas the petroleum ether extract had the weakest. Furthermore, their findings indicate active compounds in plant extracts are more likely to be found in mid-polar solvents ⁴⁰. Likewise, in our study, when compared to other solvent extracts, the mid-polar ethyl acetate leaf extract demonstrated significant inhibition, thereby validating our result. Studies shows that, aerial part of Sida rhombifolia has been effective against a wide range of gram-positive and gram-negative bacteria⁴¹. Altogether, the in-silico molecular docking studies demonstrated the binding interaction of phytocompound against the target protein. From findings, it was identified that the bioactive compound eupalitin 3-O- galactoside can be usedto develop potential inhibitors against S, aureus by targeting ClfA- Fibrinogen protein. From in-vitro antibacterial studies, it was found that the leaf extracts of all three plants Boerhavia diffusa, Clerodendrum infortunatum and Sida rhombifolia have shown efficient antibacterial activity against the growth of Staphylococcus aureus.

CONCLUSION: The present study clearly indicates that phytocompounds from the plants Boerhavia diffusa, Clerodendrum infortunatum, and Sida rhombifolia are found to be potential inhibitors of the ClfA-fibrinogen protein. The phytocompound Eupalitin 3-O- galactoside from Boerhavia diffusa effectively binds to target protein 2VR3 with a least G score of -8.56 Kcal/mol. Our in-vitro research revealed that the crude ethyl acetate extract of Boerhavia diffusa exhibited significant antibacterial activity with a zone of inhibition of 24mm. Future studies will concentrate on the molecular dynamic study to understand the stability and structural behaviour of the identified compound. Further exploration into concentration-based antibacterial analyses and their mode of action on Staphylococcus aureus needs to be done.

Declarations:

Ethics Approval and Consent to Participate: This article does not contain any authors' studies involving animals and human participants.

Consent for Publications: Not applicable

Availability of Data and Materials: The Data used and/or analyzed in the present study will be available from the corresponding author upon reasonable request.

Funding: No funding was received for conducting this study.

Author Contribution: Vidya S. L- Performed the in-silico and in-vitro studies and drafted the manuscript. R. Sathishkumar- Designed the study, edited the manuscript and guided throughout the study. The final manuscript was read and approved by all authors

ACKNOWLEDGMENT: The authors are grateful to the Kongunadu Arts and Science College administration for supplying all software required to complete this project.

CONFLICTS OF INTEREST: The authors declared that they have no competing interest in the studies.

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    How to cite this article:

    Vidya SL and Kumar R: In-silico screening of potential phytocompounds against Staphylococcus aureus and an in-vitro antibacterial evaluation. Int J Pharm Sci & Res 2023; 14(8): 4128-41. doi: 10.13040/IJPSR.0975-8232.14(8).4128-41.

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