EXPLORATION OF ANTI-MELANOMA POTENTIAL OF PHYTOCHEMICALS FROM NYCTANTHES ARBORTRISTIS THROUGH COMPUTATIONAL STUDIES

Objective: The goal of the current research is to identify the dominant phytochemical from the plant Nyctanthesarbor-tristis Linn. and to investigate their binding affinities against the proteins BRaf Kinase mutant (3OG7) and Hsp90 Chaperone (2VCJ) that causes melanoma. Methods: In this work, Schrodinger software was utilized to investigate the anti-cancer potential of phytochemicals Nyctanthesarbor-tristis against specific target proteins, namely BRaf Kinase mutant (3OG7) and Hsp90 Chaperone (2VCJ) Inhibitors. Results: Based on the outcome of the docking investigation, phytochemicals that exhibited the highest binding affinity to the specified protein targets were subjected to induced fit docking and MM-GBSA computations using the Schrodinger Maestro version 2021.2 in prime module. According to the analysis, the compounds with the highest binding affinities for 2VCJ and 3OG7 are Arbortristoside D and Nicotiflorin, respectively. The compound that interacted with both proteins was Arbortristoside B. These phytochemicals appear to be more effective to the FDA-approved V600E-BRaf inhibitor Vemurafenib and Hsp90 Chaperone Inhibitor Diclonine. Conclusion: One of the most common, deadly, and dangerous malignant diseases with a high global prevalence rate is melanoma (skin cancer). The present study may prove more helpful in developing an ideal targeted drug delivery system of phytochemicals obtained from plant Nyctanthesarbor-tristis for treatment of melanoma. This suggests that these substances could be evolved into highly effective anti-melanoma drugs.


INTRODUCTION
Malignant melanoma is an aggressive form of skin cancer that accounts for approximately 75% of skin cancer-related deaths worldwide [1,2].According to American Cancer Society, estimate for melanoma in the United States for 2023 was about 97,610 new cases of invasive and 89,070 cases of in-situ melanoma will be diagnosed in United States, while 7,990 (approx) people are expected to die of melanoma Melanoma will be leading cause of death world wide.
Melanoma is reportedly affected by various environmental and genetic factors, such as ultraviolet exposure and carcinogenic BRAF mutations [3,4].Inspite of several available treatment modalities (surgery, radiation therapy, immunotherapy, and chemotherapy) [5,6] to cure melanoma, phytochemicals have been recognized as better anti-cancer therapies to prevent or inhibit carcinogenesis because of its fewer side effects and better biocompatibility [7].
Synthetic chemicals are widely used as medicines in the treatment of diseases, encompassing various side effects.Different plants were explored as a source of bioactive agents for the treatment of ailments like cancer.Plants pose immense biological properties due to the presence of different chemical substances which perform several important physiological functions.Among 4,22,000 flowering plants reported from the whole world, more than 50,000 plants are reported to have medicinal and pharmacological uses.A rich diversity of medicinal plants is found in India [8].
Nyctanthesarbor-tristis (NTA) Linn.(Oleaceae), commonly known as 'parijat' is a common wild shrub flourishing in the sub-Himalayan tract in the states of Uttar Pradesh, Assam, Bengal, Madhya Pradesh and in the south upto Godavari [9].NTA is a beautiful and fragrant plant.Its flowers bloom at night, drop off and fall early next morning for this reason, it is called as-sad tree‖.It is mainly characterized by the presence of phenylethanoid derivatives and iridoid derivatives.It is used in traditional medicine as stomachic, carminative, intestinal astringent, expectorant, in biliousness, piles, and various skin diseases and as hair tonic.It has also been reported to possess hepato-protective, anti-viral, antifungal and analgesic, anti-pyretic, ulcerogenic activities [10].
Juice of the leaf is used in chronic and bilious fever rheumatism as a laxative, diaphoretic and diuretic.The plant has been reported to be effective against leishmanial, viral and amoebic infections [11].It is also been used in the Ayurvedic system of medicine for the cure of snake bite, bites of wild animals, cancer, sores, ulcers, dysentery, menorrhagia and obstinate sciatica.Leaves are responsible for some Central Nervous System (CNS) activities such as hypnotic, tranquilizing, local anesthetic antiasthmatic activities [12].
Vemurafenib has potent inhibitory effect and is selective for BRAF kinase [13].It is used to treat diseases imposed on by V600E-BRAF.Despite these advances, a large number of patients experienced vemurafenib resistance, and reports have demonstrated keratoacanthoma and high rates of squamous cell carcinomas associated with identified inhibitors [14,15].HSP90 chaperones, including mutant B-raf, are essential for cell survival and advancement of malignancy.Thus, blocking HSP90 can effectively stop the signalling pathways that promote tumour growth and stop HSP90 from functioning in tumour cells [16].
Therefore, the current study focuses on targeting Hsp90 Chaperone protein 2VCJ and B-Raf Kinase mutant protein 3OG7.We investigated the anti-cancer properties of phytochemicals from NTA against these protein targets.Further, we performed structure-based screening of phytochemicals from NTA against skin cancer protein targets and combined molecular docking, quantum mechanical charge derivation (MMGBSA) in the binding site, followed by induced fit docking on the protein targets, which is responsible for producing melanoma cancer.

Protein preparation and receptor grid generation
The three-dimensional (3D) structures of melanoma target proteins were retrieved from RCSB with Protein Data Bank (PDB) ids2VCJ (Hsp90 Chaperone Inhibitors, 2.50 Å resolution) [17] and 3OG7 at (B-Raf Kinase mutant, 2.45 Å resolution) [18][19].The proteins were created using the Schrodingermodelling computations.The proteins' 3D structures have been imported using the "Protein preparation wizard" in accordance with the procedure described by Tutone et al. [20].Missing hydrogen atoms, Heavy atoms, water molecules, metal ions, co-crystallized ligands and incomplete and/or terminal amide groups could all be present in a protein structure and the wizard treated the metals, corrected the bond ordering, rectified the formal corrections, removed the extra water beyond the 3 Angstrom limit of the het atom, and added the missing protons.The most stable state for the protein's ligand's potential ionisation states was determined using Epik [21].The proteins were then optimised under regulated conditions employing the force field OPLS-2005 with a constrained RMSD tolerance of 0.3 [22].
The region wherein a receptor and ligand interacts is revealed through the establishment of receptor grids.The grid was built around the co-crystallized structure using the glide "receptor grid building" component of the Schrödinger suite [23].The centroid that develops across the co-crystallized structure serves as a sign of the protein's active site [24,25].

Ligands selection and preparation
The phytochemicals of NTA reported in literature [26,27] were retrieved from Pubchem and are given in table 1. Eliminating salt and unnecessary hydrogen atoms is necessary to achieve a proper and legal protein-ligand docked complex.In order to prepare the ligands, the "LigPrep" module of the Schrodinger Maestro suite was applied to each ligand in the form of its 3D structure.
Stereo-chemistries, desalting, and tautomer generation were chosen to obtain at least 32 conformations for each ligand and were optimized using Epikin pH between 7±2 and ring conformation [28].α-linolenic acid 36.

Molecular docking (Rigid and flexible)
The Maestro12.8versiontool was used in this investigation to perform rigid and flexible docking and to assess the binding affinities, ligand effectiveness, and inhibitory characteristic to the targets [29].The ligands were docked to the active binding site of the melanoma proteins 2VCJ and 3OG7 using Extra precision-Glide XP mode, which binds to determine the flexibility of the ligand.The phytochemicals would be able to reliably bind to the protein and ligand hydrophobically, avoiding the consequences and getting high docking scores.The electrostatic interaction of the hydrogen bonds involved both the hydrophobic contact and the salt bridge contacts [30].In order to predict precision of binding affinity of top-scoring NTA phytochemicals with anti-melanoma proteins, Induced fit docking (IFD) protocol was used.Using Glide and the Refinement module in Prime, IFD is an in silico method that accurately predicts ligand binding modes and related structural changes in the receptor [31].

Pose validation
In order to evaluate and validate the reproducibility of present computational method redocking of co-crystallized ligand into the proteins' active regions was done.The RMSD (root mean square deviation) of the bound ligands were found to be 1.418Å, and 1.302Å for 2VCJ and 3OG7, respectively and is illustrated in fig. 1.This demonstrates the reproducibility of the docking process.The ligand binding energy for each of the NTA phytochemicals and the recommended standard for inhibiting melanoma proteins was estimated using the MM-GBSA function in the Schrodinger Suite 2021 [32,33].This post-docking method is crucial because it exposes the proper ranking of ligands' binding and aids in accurately predicting the relative free binding energy [34].Here, the PRIME module built into Maestro was used to calculate the free binding energies of the docked complexes by utilizing the XP docking methods [35].

RESULTS AND DISCUSSION
Discovery of novel therapeutics significantly depends on in situ approaches.They predict the experimental binding mechanism and affinity of ligand in the binding site of target [36,37].Glide XP-Molecular docking, MM-GBSA and IFD was successfully performed between the selected ligands (phytochemicals from NTA) and two melanoma protein targets (PDB ID: 2VCJ and 3OG7) using Schrodinger software.Molecular docking results of top 5 ligands with target proteins is summarized in table 2. Arbortristoside D and Nicotiflorininteracted efficiently with 2VCJ and 3OG7, respectively, with highest binding energy.All top 5 phytochemicals have higher binding score than the reference compounds Dyclonine [38] (-3.445) and Vemurafenib (-4.440).The scores were higher than the cocrystallizedligand also.
The XP and IFD scores of investigated phytochemicals with melanoma proteins revealed that for protein target 2VCJ; Arbortristoside D generated highest binding affinity with glide XP docking score of-8.793kcalmol -1 which is significantly higher than the reference compound Dyclonine (-3.445 kcalmol -1 ) and cocrystallized ligand (-6.647).Arbortristoside D had the highest Glide emodel scores (-80.915kcal mol -1 ), followed by Arbortristoside B (-64.629 kcal mol -1 ), which were higher than the reference Dyclonine(-44.289 kcalmol -1 )while the cocrystallized ligand had higher glide emodel score (-109.343kcalmol -1 ).The MMGBSA binding score was higest for cocrystallized ligand ( -223.26)The present result is in agreement with previous studies that the selected plant has already been reported to have anti-cancer activity [39].
Forprotein target 3OG7, all the top five phytochemicals showed much higher binding score than the reference and cocrystallized ligand Vemurafenib with XP binding score of-4.440kcal mol -1 .Among the phytochemicals, the decreasing order of XP binding scores were Nicotiflorin>Arbortristoside-B>Arbortristoside E>Arborside D>Astraglin.The glide emodel score was highest for Arbortristoside E (-87.130 kcal mol -1 ), while the MMGBSA binding score was highest for reference Nicotiflorin (-99.63 kcalmol -1 ).The IFD binding scores for Nicotiflorinand Arbortristoside-B were 548.99 kcal mol -1 and-550.78kcal mol -1, respectively.Nicotiflorin showed 4 (LYS483, GLN530, CYS532, ASH594) while Arbortristoside-B and Vemurafenib (GLN530, CYS532, ASH594, TYR538, ASN580) showed 5 H-bond interactions with the same residues of protein target.The present study is in accordance with the finding that Arbortristoside A and B and iridoid glycosides are reported from seeds at 2.5 mg/kg in mice which possess anticancer activity against methylcholanthrene induced fibrosarcoma [40].Additionally Vemurafenib also formed two salt bridges (with LYS483 and ASH594).Table 2 shows the top five scoring phytochemicals' Glide XP docking and MMGBSA scores as well as the kind of ligand-protein interactions and the implicated residue.Fig. 2 shows the 2D and 3D interactions of the top-scoring bioactives from NTA with protein targets.Table 3 shows the IFD score and structure of top two phytochemicals.

CONCLUSION
Patients suffering from advanced BRAFV600-mutant melanoma can benefit substantially from BRAF inhibitors; however, developing resistance is still prevalent.Preclinical research showed that concomitant use of the HSP90 inhibitor could eliminate resistance.Our study's findings made it abundantly evident that the target proteins 2VCJ can bind and interact strongly with Arbortristosides B and Arbortristosides D, as demonstrated by their binding energies than those of the other ligands we investigated.Similarly, Nicotiflorin and Arbortristoside B showed the best interaction with target proteins 3OG7.Based on the findings of our investigation, it is likely stated that Arbortristosides B is a potent antimelanomaphytodrug with the ability to interfere with both the investigated melanoma targets BRAF mutant (2VCJ) and HSP90 inhibitor (3OG7).Hence, it is believed that Arbortristosides B will be devoid of the resistance associated with the BRAF inhibitors.On the basis of the predictions of our in silico investigations, additional in vitro and in vivo experiments are required to determine the anticancer potential of these phytochemicals of Nyctanthes Arbortristis for their anti-melanoma activity.