IN SILICO INVESTIGATION OF ECHINACEA PURPUREA PHYTO LIGANDS TARGETING HUMAN PAPILLOMAVIRUS TYPE 18’S L1 PROTEIN: IMPLICATIONS FOR CERVICAL CANCER MANAGEMENT

Objectives: Human papillomavirus (HPV) is a highly oncogenic virus responsible for the majority of intraepithelial lesions and cervical cancer. Among various HPV types, 16 and 18 contribute to approximately 70% of cervical cancer cases globally, making them the most prevalent high-risk oncogenic variants associated with this disease. Numerous vaccines (Gardasil 9, Gardasil, and Cervarix) have been approved by FDA to combat HPV infections; however, their widespread implementation faces challenges due to their limited cost-effectiveness. Methods: Echinacea purpurea’s components have already been studied for in silico analysis against HPV Type 16’s L1 protein. In the present analysis, we aimed to explore the potential interaction between E. purpurea phytoligands (curcumin, echinacoside, and chicoric acid) and the major capsid protein L1 of HPV type 18 (2R5I) through molecular docking analysis. Results: Molecular docking analysis revealed that the echinacoside, one of the components of E. purpurea , has the best binding affinity (−7.9 kcaL/moL) against the L1 protein of the HPV type 18. Conclusion: The molecular docking analysis indicates that


INTRODUCTION
Human papillomavirus (HPV) is a widespread group of viruses that commonly infect the skin and mucous membranes in humans.One particular variety, HPV type 18 is recognized as a high-risk oncogenic variant.HPV type 18 is mainly transmitted through sexual contact and primarily affects the genital and anal regions.Cervical cancer, as well as other vaginal and anogenital malignancies, are all highly associated with HPV type 18 infection that persists over time.HPV type 18 is widely accepted as the second most carcinogenic HPV type after HPV16 [1].Cancer of the cervix became the fourth most prevalent cancer in women globally in 2020 [2].HPV comprises various types, and among them, HPV type 16 and HPV type 18 are classified as highrisk oncogenic strains.These two types are particularly significant due to their association with certain cancers, especially cervical cancer, as well as other genital and anogenital cancers.Both HPV type 18 and HPV type 16 are transmitted through sexual contact and can lead to persistent infections, raising the risk of cancer development over time [3].The development of keratinocytes, the cells that make up the outer layer of the skin, is intricately linked to the life cycle of HPVs.HPV expresses its early genes after infecting undifferentiated basal cells, promoting the early phases of the viral life cycle.Late genes are expressed as a result of this process, which makes it easier to produce the structural proteins needed to build viral capsids [4].
In its genome, the HPV features a distinctive circular, double-stranded DNA molecule.The viral genome consists of three main segments: The early region (E Region), the late region (L Region), and the long control region (LCR).These segments play essential roles in the virus's life cycle and replication process.Conversely, the L Region contains viral late genes, notably L1 and L2.The viral capsid (Fig. 1), which encloses the viral DNA to produce new contagious viral particles, emerges by the gene expression toward the end of the viral life cycle.The HPV type 18 L1 (Late 1) gene is an essential part of the virus that codes for the important structural protein L1.Lukman et al. in 2022 [5] demonstrated the significance of positive interaction and its potential implications in the viral life cycle and good binding mode of the "major capsid protein L1 of HPV type 16" (PDB ID: 2R5H) with Echinacea purpurea components.By exploring this novel association, we aim to study the interaction of "major capsid protein L1 of HPV type 18" (PDB ID: 2R5H) with E. purpurea phytoligands (curcumin, echinacoside, and chicoric acid) [5].While different HPV types may have distinct genetic variations, the L1 protein is relatively conserved among HPV type 16 and 18 strains.Moreover, it is essential for the effective infection that L1 interacts with the heparan sulfate (HS) carbohydrates present on host cells during the first stage of papillomavirus infectious entrance [6].Blocking this interaction with soluble heparin or enzymatic removal of HS inhibits virion binding and infectious entry.A structural alteration brought on by the viral protein L1's binding to the cell surface makes protein L2 accessible.This promotes the virus's capacity to bind to a cell receptor and allows cellular furin protease to cleave L2.Lacking L1 and L2, virus particles are unable to release their DNA, essentially halting the progress of infection [6].

Previous research by
Purple coneflower, also known as E. purpurea, possesses antiviral, anti-inflammatory, and immunomodulatory activities that suggest it may be used as a chemotherapeutic [7].According to certain research, some chemicals in E. purpurea may slow the growth of cancer cells and trigger apoptosis.

METHODS
To find the best affinity of ligand molecules curcumin, echinacoside, and chicoric acid against the protein 2R5I, molecular docking was performed.2D structure analysis was performed to understand the structural configuration of 2R5I with the use of PDBsum (http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/).The Ramachandran plot was created with the use of PDBsum to determine the accuracy of the predicted protein structure.All the analysis was performed using 8.00 GB RAM and 11 th Gen Intel ® Core (TM) i3-1115G4 processor on Windows 11.Docking analysis was done in software AutoDock Vina and structures were visualized using DS Biovia Discovery Studio software.

Retrieval of ligands
Curcumin, echinacoside, and chicoric acid were selected to perform molecular docking against protein 2R5I.The ChEMBL database "https://www.ebi.ac.uk/chembl/" was used to retrieve molecular structure (Fig. 2), 3D SDF file, and SMILES data of ligands molecules.SMILES data were used further to perform SwissADME analysis and molecular structure prediction of ligands.PyMOL was used to visualize the ligand structure [8].

Retrieval of protein
The major capsid protein L1 of HPV type 18, represented by PDB ID 2R5I, was sourced using the RCSB PDB data bank (https://www.rcsb.org/) in PDB format [9].The protein had a 3.40 Å resolution and the X-ray diffraction method was used for protein retrieval.The visualization software Chimera 1.16 was used for visualization of protein 3D structure.

Protein and ligand preparation
The crystallographic structure of the protein is not supported by the free energy of water molecules so all water molecules of protein 2R5I were deleted with the use of software Chimera 1.16 [10].The proteinligand binding and docking results can be affected by the presence of water molecules.The protein structure was simplified by removing all additional chains.The X-ray diffraction method of protein retrieval does not contain charges and hydrogen atoms, so hydrogen atoms and Kollman charges were inserted in the protein structure for better optimization.All steps of protein purification were performed with the use of the software Chimera 1.16.The purified protein structure (Fig. 3) was used to analyze the Ramachandran plot and secondary structure prediction of protein with the use of PDBsum [10].Ligand preparation was done by selecting a molecule for Autodock4 in Autodock tool 1.5.7 for three ligands, this step was performed to add charges and merge non-polar hydrogens [11].

Grid preparation
The AutoDock tool 1.5.7 "https://ccsb.scripps.edu/mgltools/"was used for grid preparation.Dimensions of the grid were automatically predicted with the use of AutoDock tool 1.5.7 for docking because the binding site of the protein was not identified (Table 1).

Molecular docking
AutoDock Vina was used to perform docking of curcumin, echinacoside, and chicoric acid against protein 2R5I to determine their inhibitory properties (Eberhardt et al., 2021).Purified protein structure and ligands structure were converted into.Pdbqt format with the use of PyMOL before docking.The docking was performed for all three ligands against protein 2R5I with the dimensions predicted by AutoDock tool 1.5.7.AutoDock Vina predicted nine possible configurations of proteinligand binding according to the grid box for all three ligands against protein 2R5I.The lower binding affinity is considered the best degree of binding and best docking configuration.Echinacoside had the lowest binding affinity than curcumin and chicoric acid, and the best binding affinity of individual three ligands was visualized with the use of DS Biovia Discovery Studio [13].
Visualization DS Biovia Discovery Studio was used to visualize 3D and 2D structure of the best binding affinity of individual three ligands against protein 2R5I [13].3D and 2D structures of the best binding affinity of three ligands with protein were generated and downloaded to predict amino acids and bonds involved in protein-ligand binding.

Protein structure analysis
The Ramachandran plot was used to analyze the torsion angle distribution of a protein structure.The Ramachandran plot represented in Fig. 4 was performed using PDBsum, and the secondary structure of the protein (Fig. 5) was also created in PDBsum [10].Red areas on the graph represent stable peptide conformation, which is known as the sterically permissible region.The allowed region contains 80.2% or 4365 residues, whereas the disallowed region contains 0.3% or 15 residues.A total of 6345 residues are made up of 465 glycine residues, 375 proline residues, and 5445 non-glycine and non-proline residues.The secondary structure of 2R5I contains seven sheets, three helix-helix

SwissADME analysis
Screening of small molecules and their medication properties are determined using the Lipinski rule of five.All three ligands were accepted by Lipinski's rule of five according to ADMElab 2.0 [12].All minimum and maximum values of SwissADME characteristics, Lipinski rule of five, and ligand physicochemical properties can be studied from SwissADME [8].

Molecular docking and visualization
The ligand echinacoside had better binding affinity than curcumin and chicoric acid against HPV type 18. Possible nine binding affinities of individual three ligands derived from AutoDock Vina are listed in Table 5. RMSD i.b. represents intra-batch RMSD, where RMSD is calculated in a single docking batch.RMSD u.b. represents inter-batch RMSD, where RMSD is calculated in different docking batches.
The ligand curcumin, echinacoside, and chicoric acid had the best binding affinity scores −6.9, −7.9, and −6.3, respectively.All individual best binding affinity scores were analyzed with the use of DS Biovia Discovery Studio for 3D and 2D structure visualization of proteinligand binding (Figs. 6-8).Detailed information about the amino acids engaged in the interactions and the specific type of interactions established between 2R5I and phyto-ligands is presented in Table 6.

DISCUSSION
Two types of HPV strain (18 and 16) are responsible for 70% of HPV causing cancer of the cervix (World Health Organization, 2015) [2].Reduction of HPV L1 capsid antigen expression is observed in the progression of invasive cancer [14].HPV type 18 and type 16 are isolates of one HPV type according to their intratypic molecular variants, only <10% variation in the nucleotide sequence of L1 is observed [15].The L1 is a conserved region in HPV type 18 and HPV type 16 and heparin is observed in binding sites of both HPV type 18 and 16, which is involved in HPV infection [5,16].
HPV vaccination cost is beyond the limit for the average citizen, so E. purpurea as an affordable herbal therapy was reported in Nigeria for HPV infection.Immune system improvement and reduction in viral replication were reported with the use of Echinacea along with affordability and accessibility for the average citizen [5].At present, there is no scientific report to prove the efficiency of Echinacea, but the potentiality of the plant for research on E. purpurea for HPV infection was reported through molecular docking analysis.Molecular docking analysis of Echinacea ligands (curcumin, echinacoside, and chicoric acid) was done against the pentamer structure of 2R5H [5].L1 conserved region of protein 2R5H was targeted to check the binding affinity of Echinacea ligands and chicoric acid (−8.7 kcaL/moL) was reported as the best ligand according to docking results [5].
Depending upon the pentamer structure of HPV type 16 L1 (2R5H) docking analysis against Echinacea, we performed docking on the pentamer structure of HPV type 18 (2R5I) against Echinacea.In the present molecular docking analysis, the same ligands Echinacea were used against protein 2R5I because of their dissimilarity in the L1 region, the presence of heparin at the binding site of the protein, and the role of both proteins in cervical cancer.We checked the binding affinity of ligands Echinacea against protein 2R5I to compare their binding affinity with protein 2R5H for ideas of future research.The chicoric acid had the best binding affinity (−8.7 kcaL/moL) against protein 2R5H but chicoric acid was observed to have an overall low binding affinity against protein 2R5I.The echinacoside was observed to have the best docking affinity (−7.9 kcaL/moL) in protein 2R5I.
The best docking affinity score was −6.9 kcaL/moL, −7.9 kcaL/moL, and −6.3 kcaL/moL for curcumin, echinacoside, and chicoric acid, Many research works are ongoing for HPV-related diseases to provide promising results of therapeutic agents for the management of HPV infection.In silico analysis in bioinformatics and molecular modeling, advancement is a boon for validating such therapeutic agents.Although, research work has been done to check the inhibitory action of natural compounds against L1 protein (2R5I).L1 protein (2R5I) is a conserved region and contains heparin in the binding site, which is a risk factor for HPV infection.This docking analysis on protein 2R5I provides promising information on how HPV-related infections can be managed with the use of plant E. purpurea as an inhibitor when confirmed by in vivo techniques.

Fig. 1 :
Fig. 1: HPV has a non-enveloped, icosahedral capsid composed of L1 and L2 proteins, enclosing its circular double-stranded DNA genome with a regulatory LCR region and linked to proteins that resemble histones.This structure facilitates viral entry, replication, and pathogenesis in host cells, contributing to diseases such as genital warts and cancer

Table 5 : Ligands binding affinity result of docking against 2R5I Binding score of curcumin Binding score of echinacoside Binding score of chicoric acid
beta alpha beta unit, 21 strands, three beta hairpins, five beta bulges, 12 helices, 40 beta turns, and four gamma turns.