In silico screening of phytoconstituents of Cissus quadrangularis and Chromolaena odorata against proteins of antimicrobial resistance and wound healing

In silico screening is a methodological approach, which is invaluable for rational drug design and the identification of potential therapeutic agents. In the context of antibiotic - resistant infectious wounds, molecular docking can provide a deeper understanding of how phytocompounds might interfere with bacterial virulence and antibiotic resistance. In this study, proteins involved in antimicrobial resistance and wound healing were docked against major phytoconstituents of ethyl acetate extract of Cissus quadran-gularis (EACQ) and ethanol extract of Chromolaena odorata (EECO), two me-dicinal plants that have been traditionally used. Receptor structures for interleukin 6 (PDB id: 1n26) IL6, of human and mice, IL6 (Uniprot id p 20607) of rat, vascular endothelial growth factor (VEGFR, PDB id: 2ctw) for human, mice, rat and penicillin binding protein 2a (PBP2a, PDB id: 1vqq) of S. aureus were downloaded from the database of the RCSB protein data bank. The ligand structures were downloaded from PubChem compound database in structure data file (.SDF) format. The docking studies were conducted using Autodock4. and the results of the docking analysis were visualised using Discovery Studio Visualizer. The docking log (dlg) file, featuring an RMSD table, provides binding energy values in Kcal/mol for each molecule at its optimal docked postures, offering insights into structural accuracy and ligand - receptor interaction strength in molecular docking simulations. In silico analysis of ligands showed that squalene of EACQ and epilupeol of EECO had the least binding energy towards proteins of antimicrobial resistance and wound healing. Thus, these compounds could emerge as promising lead molecules against infectious wounds.


Introduction
Wound healing, as a normal biological process in the human body, is achieved through four precisely and highly programmed phases: hemostasis, inflammation, proliferation, and remodelling.For a wound to heal successfully, all four phases must occur in the proper sequence and time frame (1).However, the emergence and spread of antibiotic-resistant bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA) have posed significant challenges to successful wound healing.Integration of a staphylococcal cassette chromosome mec (SCCmec) element into the chro-mosome converts drug-sensitive S. aureus into the notorious hospital pathogen MRSA, which is resistant to practically all β-lactam antibiotics (2).In recent years, the search for alternative therapeutic approaches has intensified, with a focus on natural products and traditional remedies with potential wound healing properties.Cissus quadrangularis and Chromolaena odorata are two medicinal plants that have been traditionally used in various cultures for their purported wound healing effects.
C. quadrangularis (L.) commonly known as "Hadjod" or "Veldt Grape," belongs to the Vitaceae family and is native to tropical regions of Asia and Africa.It has been employed in traditional medicine for its antiinflammatory, analgesic, and bone-regenerating properties (3).Similarly, C. odorata, (L.) King and Rob. also known as "Siam Weed" or "Jack-in-the-Bush," is a plant native to Central and South America.It has a long history of use in traditional medicine as an antimicrobial and wound healing agent (4).
Given the widespread use of these two plant extracts in traditional medicine, there is a growing interest in evaluating their wound healing potential, particularly in the context of antibiotic-resistant infectious wounds.Thus, this study aims to investigate the antibiotic-resistant infectious wound healing activity of phytoconstituents detected in C. quadrangularis and C. odorata extracts using molecular docking.
Molecular docking is a computational technique that enables us to simulate and predict the binding interactions between molecules at the atomic level.In our context, it offers a powerful tool for elucidating the potential interactions between phytocompounds and MRSAassociated molecular targets, such as enzymes and proteins.By virtually "docking" these compounds into the target structures, we can gain insights into their binding affinity, binding modes, and the strength of their interactions.This methodological approach is invaluable for rational drug design and the identification of potential therapeutic agents.In the context of antibiotic-resistant infectious wounds, molecular docking can provide a deeper understanding of how phytocompounds might interfere with bacterial mechanisms of virulence and antibiotic resistance.Moreover, it can help us to pinpoint the most promising candidates for further experimental validation.This research will contribute to the growing body of knowledge on natural products and their potential therapeutic benefits in wound healing, especially in the context of antibiotic-resistant infections.The findings from this study may pave the way for the development of novel and effective treatments that can complement or even replace conventional therapies for antibiotic-resistant infectious wounds, thereby offering new hope for patients facing challenging wound healing scenarios.Ultimately, the exploration of these traditional remedies holds promising prospects for enhancing wound management strategies and improving patient outcomes in the face of antibiotic resistance.

Preparation of extracts
C. quadrangularis and C. odorata were collected, shadedried and extracted using ethyl acetate as solvent for C. quadrangularis (EACQ) and ethanol as solvent for C. odorata (EECO).The EACQ and EECO are reported to have antimicrobial activity against S. aureus (5,6).These extracts were used for gas chromatography-mass spectroscopy analysis.

Gas chromatography mass spectroscopy
Gas chromatography-mass spectroscopy analysis of extracts was done at Kerala Forests Research Institute, Peechi, Kerala, India.Gas chromatography Mass Spectrometer (Shimadzu Nexis GC-2030) with a mass range of 1.5-1000 m/z was used.Helium was used as the carrier gas at flow rate of 1 mL/min.The oven temperature was maintained at 60° C and then increased to 280° C in 5 min.The injector temperature was 260° C and total analysis time was 50 min.Aliquots of extracts (0.4 µL) were injected into the chromatographic column after a clear baseline was obtained.Major constituents were identified by using mass spectrum library (NIST 20).

Preparation of macromolecule
Receptor structures for interleukin 6 (PDB id: 1n26) IL6, of human and mice, IL6 (Uniprot id p 20607) of rat, vascular endothelial growth factor (VEGFR, PDB id: 2ctw) of human, mice, rat and penicillin binding protein 2a (PBP2a) (PDB id: 1vqq) (Fig. 1) were downloaded from the database of the RCSB protein data bank (http://www.rcsb.org) in PDB format.The structure was prepared using Accelrys Discovery Studio Visualizer 3.5.0.12158 (Copyright© 2005-12, Biovia Software Inc) for further processing and docking.Further, the macromolecule was processed in AutoDock 1.5.6 (Molecular Graphics Laboratory tools, www.mgltools.scripps.edu)according to the AutoDock Tools (ADT) tutorial's standard protocol and parameters.Phytoconstituents of C. quadrangularis and C. odorata obtained by GC-MS were the ligands used in this study.Indomethacin, allantoin and ceftriaxone were used as the standards (Fig. 2).The ligand structures (Fig. 3 and 4) were downloaded from PubChem compound database (National Center for Biotechnology Information; https:// pubchem ncbi.nlm.nih.gov/) in structure data file (.SDF) format.Then they were processed using Marvin View 17.25.0(www.chemaxon.com)and modified to Tripos Mol 2 format.The modifying tools of ADT for ligands were used for processing them in terms of detection of roots, root expansion and choosing the number of rotatable bonds.Following the preliminary preparations, the ligand molecules were transformed to PDBQT format for use in Auto-Dock4.

Methodology of docking
The docking studies were conducted using Autodock4 developed by the Scripps Research Institute (La Jolla, CA, www.autodock.scripps.edu).The grid map for the present study was computed with Autodock4.The grid centre was set as tabulated (Table 1).The processed file was saved in grid parameter file (gpf) format.Using the parameters optimised by ADT, the docking parameter file (dpf) was generated.The Lamarckian genetic algorithm was used for all docking runs.The docking log (dlg) file with RMSD table contained the binding energy (KCal/mol) for each molecule's optimal docked postures.Using Discovery Studio, the results of the docking analysis were visualised.After studying each ligand interactions with the protein and analysing their binding poses, the best and most energetically beneficial conformations of each ligand were selected (Laskowski and Swindells, 2011).

In silico analysis of compounds
In the process of wound healing, the transition from proinflammatory to a reparative microenvironment has to be precisely controlled (11).A crucial regulator of the inflammatory and reparative process is IL-6 (12).Interleukin-6 helps in leukocyte differentiation, endothelial activation, keratinocyte proliferation, and fibroblast proliferation (13).
Hence in silico docking studies of various ligands of EACQ and EECO were conducted against the receptor IL-6 of human, mice and rat.The in silico docking results of ligands of EACQ revealed that all the compounds except 1tridecene showed good binding affinity to IL-6 and the maximum binding affinity was exhibited by squalene whereas all the compounds of EECO exhibited good binding affinity for IL-6 with maximum binding affinity for epilupeol.This study first reports the interaction of IL-6 with epilupeol as there are no reported studies about the interaction of IL-6 with epilupeol.
One of the key proteins involved in antimicrobial resistance is pencillin binding protein (PBP)2a which confers resistance to beta lactam antibiotics in S. aureus (14).Compounds that inhibit PBP2a offer promising treatment against antibiotic resistant infections.In the present study, squalene from EACQ and all the compounds of EECO showed good binding affinity for PBP2a, comparable to the standard drug, ceftriaxone.Thus it could be inferred that EECO offered better inhibition of antimicrobial resistance than EACQ.Thus EECO could offer better promising treatment against MRSA infection.Among the various compounds of EACQ and EECO, squalene and epilupeol showed good binding affinity towards PBP2a.This study first reports the inhibition of antimicrobial resistance of these compounds against MRSA as there are no reported works.
Vascular endothelial growth factor (VEGF)-B is involved in the maintenance of newly formed blood vessels in pathological conditions and it indirectly influence the vascular growth by influencing VEGF-A action (15).In the present study, five compounds of EACQ and all the 11 compounds of EECO was found to have a good binding affinity when compared to the standard drug, allantoin.As observed for IL-6 and PBP2a, squalene and epilupeol showed maximum binding affinity for VEGF-B also.As there are no reported works related to the binding affinity of these compounds against VEGF-B, this study first reports this interaction.Thus it could be assumed that EECO could offer better maintenance of newly formed blood vessels in MRSA infected wound conditions and promote wound healing.
In the present study, different phytocompounds showed better affinity towards proteins of antimicrobial resistance and wound healing as compared to standard ligands used.Squalene is a bioactive triterpene that occurs naturally plays a key role in the process of making sterols and has good wound healing activity (16).Squalene promotes wound healing by increasing macrophage response in inflammation.Therefore, squalene could be useful at the resolution stage of wound healing (17).This is in accordance with the results of the present study, where binding energies of squalene with IL6 of and VEGF-B are -6.5 (human, mice), -7.1(rat) and -5.6 (human, mice and rat) Kcal/mol indicating good affinity towards proteins of wound healing.
Phytol showed a binding energy of -7.3 Kcal/mol towards PBP 2a indicating good affinity.Phytol showed a biphasic effect and appeared to have both bacterial growthinhibitory and growth-accelerating effects, and the net effect of each depended on its concentration (18).Methanolic extract of Phlomis russeliana exhibited in vitro and in vivo wound healing activity which was attributed to the presence of phytol and 1-heptadecanoic acid as active constituents (19).Thus it could be assumed that EACQ and EECO could be effective in antibiotic resistant infectious wound.
Gamma sitosterol showed lower binding energies compared to standards towards proteins of wound healing and penicillin resistance.Gamma sitosterol is shown to inhibit matrix metalloproteinase -1 (MMP-1), reduce collagen breakdown, and promote the synthesis of collagen in human keratinocytes (6).Moreover, they promote keratinocyte migration through the reduction of oxidative stress that effectively accelerates the healing process (20).Hemostatic and wound healing properties of C. odorata could be attributed to the presence of major phytoconsti-tuents scutellarin and stigmasterol (8).This corroborates with present study where stigmasterol from C. odorata showed binding energies of higher negativity as compared to standard drugs.No data is available on activity of other compounds on antibiotic resistant infectious wound healing.

Conclusion
Most phytoconstituents of plants C. quadrangularis exhibit higher negativity in binding energy when compared to standard compounds.In terms of affinity towards protein PBP 2a, compounds squalene and phytol of C. quadrangularis showed more affinity.Most of the constituents showed higher affinity towards VEGF and IL6 when compared to standards in which squalene being the highest.In case of phytoconstituents of C. odorata, epilupeol showed most affinity towards the proteins, followed by stigmasterol, gamma sitosterol and 2-(3,4-Dimethyloxyphenyl)-2,3-dihydro-5,7-dihydroxy-4H-1benzopyran-4-one.To summarize, both plants had phytoconstituents that have good affinity towards proteins of antibacterial resistance and wound healing.Among the plants, C. odorata showed higher affinity towards these proteins when compared C. quadrangularis.Further in vitro and in vivo studies are required to confirm the antibacterial and wound healing potential of these phytocompounds.

Table 3 .
Phytochemicals obtained on GC-MS analysis of EECO leaves

Table 4 .
Different ligands selected for in silico analysis

Table 5 .
Binding energy of different ligands of C. quadrangularis

Table 6 .
Binding energy of different ligands of C. odorata

Table 7 .
Binding energy of standards