Ferroptosis Mediated Novel Drug Design Approach in the Treatment of Oral Squamous Cell Carcinoma

Background: Globally, Oral Squamous Cell Carcinoma (OSCC) is the highest prevalent type of oral cancer. Implementing a successful treatment plan for the aforementioned tumor has always been a primary concern. There are numerous targeted therapies of which Ferroptosis has been receiving increasing attention in the recent decade. A novel form of controlled cell death “Ferroptosis’ is caused by iron-dependent lipid peroxidation. A well-known mechanism for controlling ferroptosis is the Cysteine/GSH/GPX4 axis, in which System X͞c is crucial. System X͞c inhibitors have been proven earlier to improve chemotherapy sensitivity. Materials and methods: Five System X͞c inhibitors were selected from the literature. The structure of these molecules from Zinc15 and the protein sequence of the target from Protein Data Bank were obtained. Twenty new molecules were identified following pharmacophore modeling and were docked with the target protein using SwissDock. The binding energies of the new molecules with the target were compared with that of the reported molecules. Result: The molecular docking study showed that two new molecules (ZINC89362298 and ZINC1730544) resulted in the highest binding pattern (-8.64) than that of the reported molecules (-7.75). Conclusion: The present study concluded that ZINC89362298 and ZINC1730544 had better binding efficiencies than that of the reported System xc- inhibitors. Hence these two molecules could be used in targeted drug therapy and could be a promising lead in the management of oral cancer in the future.


Introduction
Oral squamous cell carcinoma (OSCC) remains a challenge for head and neck surgeons with a low 5-year survival rate despite improvements in diagnostic techniques and therapies (Bugshan and Farooq, 2020). Various factors including depth of invasion, tumor grade, and biomarkers predict the survival rate of cancer patients (Rivera et al., 2017). Numerous studies support identifying factors that affect the course of the treatment (Massano et al., 2006). Similar to how they aid with prognosis, medications also have their own role in a patient's survival and quality of life. The role of apoptosis and the genes involved have been sufficiently understood for decades to be used in diagnosis, prognosis, and treatment with targeted therapy being in trend (Dwivedi et al., 2020). Inducing apoptosis in oral malignant epithelial cells has shown encouraging outcomes to combat cancer (Hsu et al., 2004). Recently many other forms of regulated cell death have emerged with the motive to modify disease outcomes. A few other non-apoptotic cell death includes iron-dependent ferroptosis, vacuole-presenting methuosis, immune-reactive pyroptosis, and necroptosis (Yan et al., 2020). For instance, studies on pyroptosis were reported to influence tumor cell proliferation, metastasis, and invasion (Fang et al., 2020). Another form of cell death that researchers found exponential growth in the treatment of cancer in recent decades is Ferroptosis (Jiang et al., 2021). Ferroptosis, an oxidative stress-dependent regulated necrosis is a novel pathway for preventing cancer progression (Li et al., 2020). Ferroptosis has a complex and integrated pathway regulated precisely at multiple levels that includes transcriptional, epigenetic, posttranscriptional, and post-translational factors . The central role involved in ferroptosis is cysteine metabolism in which the glutamate-cystine antiporter, system X ͞ c is the prime target in cancer treatment (Xu et al., 2019). Tumors with overexpression of System X ͞ c are associated with increased cell proliferation and metastasis according to previous literature. Hence inhibition of System X ͞ c enhances chemotherapy sensitivity (Liu et al., 2021). System X ͞ c inhibitors reported in the literature are Artesunate, trigonelline, lanperisone, sulfasalazine, and sorafenib. Hence the study aims to identify existing drugs that can be potentially bound against System X ͞ c and to suggest potential substitutes as prospective leads for future stable drug designing. The objective of the study is to assess and compare the differences between the novel molecules' binding energies and those of the reported molecules with the target using computer-aided drug design in an in silico setting.

Materials and Methods
The study was carried out after obtaining approval from the scientific review board (SRB/SDC/UG-2102/22/ OPATH/013), in the Department of Oral Pathology. In this study, System X ͞ c was chosen as the target protein and its potential inhibitors were identified from the databases such as PubMed and Google Scholar. The five System X ͞ c _ inhibitors obtained from the search were Artesunate, Trigonelline, Lanperisone, Sulfasalazine and Sorafenib. These drugs have varied functions and clinical applications. Artesunate is an artemisinin derivative that has been used as an antimalarial agent, trigonelline is a plant alkaloid used as anti-diabetic drug (Roh et al., 2017), sulfasalazine is an anti-inflammatory drug used to treat ulcerative colitis (Patel et al., 2019), lanperisone is a muscle relaxant and sorafenib is a kinase inhibitor and has been known for its role in renal cancer (Liu et al., 2022). Hence this study attempts to assess the affinity of these drugs with the target System X ͞ c in the form of binding pattern with that of the new compounds derived from the molecular structure of these five molecules keeping them as a template. The steps to develop new pharmacophores and to assess the binding pattern of these molecules with the target are as follows with the abstract of the workflow presented in Figure 1.

Zinc 15 Database
Zinc15 is a free chemical database with purchasable compounds which are ready to dock (Irwin et al., 2005). The structure of the selected molecules (Artesunate, trigonelline, sulfasalazine, lanperisone, and sorafenib) was collected from the ZINC15 database. (Figure 2) The molecules' SDF (structure data file) format and SMILES (Simplified Molecular Input Line Entry Specification) format were also obtained for further assessment in SWISS ADME.

SWISS ADME (Absorption, Distribution, Metabolism and Excretion)
Swiss ADME is a free tool to assess the pharmacokinetic properties, drug-like nature, and medicinal chemistry friendliness of the selected molecules aiding in drug discovery (Daina et al., 2017). The SMILES format from Zinc15 of each molecule was used in Swiss ADME, where the properties were assessed such as Log P, drug likeliness rules such as Lipinski (Rule 1), Ghose (Rule 2), Veber (Rule 3), Egan (Rule 4) and Muegge (Rule 5) (Table 1) and topological polar surface area (TPSA).

RCSB PDB (Research Collaboratory for Structural Bioinformatics Protein Data Bank)
This an excellent tool that provides experimentally determined 3D structures of proteins, nucleic acids and other macromolecules along with its computed structure models (Rose et al., 2016). Protein sequence of the target System X ͞ c was collected from this Protein Data Bank (PDB) (Figure 3)

Pharmacophore Modeling
Pharmacophore modeling is done to develop pharmacophore models using the structural information about the molecules or compounds under study. To develop novel compounds from the existing System X ͞ c inhibitors, this study used ZINCPharmer, to identify the biologically relevant compounds related to the existing five System X ͞ c inhibitors and SwissDock, to determine the binding energies of the compounds with the target.

PharmaGist
PharmaGist is a free web server used to detect pharmacophores that enables a molecule to interact with a specific target (Duhovny et al., 2008). The sdf format of the molecules from the ZINC15 database was converted to mol2 (Molecule structure format) format using OpenBabel software which was then uploaded in PharmaGist. The results were obtained which were used for further analysis.

ZINCPharmer
ZINCPharmer, a free pharmacophore search software, identifies biologically relevant compounds automatically (Koes et al., 2012). The results from PharmaGist were loaded into ZINCPharmer where possible pharmacophores were obtained. Of these, the twenty best molecules with the highest score were selected for further analysis (Table 2).

Swiss dock
Swiss dock, a web service to predict the interaction between a target and a molecule was used in this study (Grosdidier et al., 2011). Each of the resulting 20 molecules from ZINCPharmer and the chosen 5 original molecules from the literature were docked individually with the target protein using SwissDock. The binding energies which were obtained from the results were then tabulated and compared.
This study where System X ͞ c is chosen as the target protein identified drugs/ molecules from the literature that bind with it. We searched various databases and articles related to System X ͞ c and its role in ferroptosis. The five system xc-inhibitors that resulted from the search were Artesunate, Trigonelline, Lanperizone, Sulfasalazine, Sorafenib. The drugs have varied functions and clinical applications. Artesunate is an artemisinin derivative that has been used as an antimalarial agent, trigonelline is a plant alkaloid used as anti diabetic drug, sulfasalazine is an anti inflammatory drug used to treat ulcerative colitis, lanperizone, a muscle relaxant and sorafenib is a kinase inhibitor has been known for its role in renal cancer. The structure of these molecules was collected from the ZINC15 database in order to assess its properties for which, Swiss ADME was used (Figures 1, 2). Protein sequence of the target system xc-was collected from Protein Data Bank (PDB) (Figure 3). Pharmacophore modeling was done using Pharmagist. Using Zinc Pharmer, which resulted in compounds with various Ferroptosis Mediated Novel Drug Design Approach and ZINC1730544) with the highest binding pattern (-8.64) than that of the existing System XC-inhibitors (-7.75) (Figure 4). The properties of all the molecules were tabulated for comparison in which these two novel compounds had better properties satisfying the rules required for a drug such as Lipinski (Rule 1), Ghose (Rule 2), Veber (Rule 3), Egan (Rule 4) and Muegge (Rule 5) ( Table 2). Other parameters such as bioavailability, LogP, and topological polar surface area (TPSA) were also compared ( Table 3).
The molecular docking study showed that two new molecules (ZINC89362298 and ZINC1730544) resulted with the highest binding pattern (-8.64) than that of the reported molecules (-7.75) (Figure 4). The properties of all the molecules were tabulated for comparison in which the two novel compounds had better properties satisfying all the rules (Table 1). Other parameters such as bioavailability, LogP and topological polar surface area (TPSA) were also compared.
combinations from the selected five drugs, the best 20 molecules were selected. Each of these resulted molecules and the chosen molecules were docked individually with the target protein for which Swissdock is used. The results showed binding energies which were then assessed and compared with the original molecules.

Results
The structure of the five selected System X ͞ c inhibitors from literature was obtained using ZINC15 (Figure 2). The properties of the molecules were assessed using SwissADME (Table 3). The structure of the protein System X ͞ c was obtained from protein data bank (Figure 3). The twenty new pharmacophore models with the highest score were filtered for the docking. The results of the molecular docking analysis using SwissDock showed two new molecules (ZINC89362298 and ZINC1730544) with the highest binding pattern (-8.64) than that of the existing System X ͞ c.
The results of the molecular docking analysis using SwissDock showed two new molecules (ZINC89362298

Discussion
The present study reported two molecules (ZINC89362298 and ZINC1730544) with better binding energies when compared to that of original molecules from the literature. Compounds binding with the concerned target is utmost important while developing a drug for which the ligands discovered from the present study demonstrated clearly that they bind strongly to the target protein.
A well-known, globally burden and the most prevalent oral malignancy, oral squamous cell carcinoma is derived from the epithelial lining of the oral mucosa. As easily accessible, the oral lesions should be detected more earlier than any other disorders. Light-based detection systems, fluorescence visualization, and brush cytology are a few emerging diagnostic techniques for oral cancer detection. (Lane et al., 2006).
A study by Yang et al investigated the importance of natural products in the treatment of cancer and infectious diseases in which they stressed on the fact that natural substitutes have their own place in drug discovery (Yang et al., 2012). Similarly this project included one natural compound trigonelline and it proved to be highly efficacious with the binding energy of -9.4 which is more than that of the reported compounds but with a disadvantage of violating few rules ( Table 2).
Establishment of oral sorafenib (400 mg bid) as the drug of choice for metastatic renal cell carcinoma following the phase I and phase II results (Wilhelm et al., 2006)     in the current study would be more appropriate to induce ferroptosis as it is already proven to resist tumorigenesis. It was studied that sulfasalazine and curcumin, by depleting glutathione and by increasing ROS induced ferroptosis binding with system xc-in breast cancer (Wang et al., 2021) which justifies the inclusion of sulfasalazine in this study. The side effects of these five molecules include metabolic and phenotypic alterations resulting in reduced anticancer activity (Dang et al., 2022). In accordance with these study findings the molecules such as Trigonelline, sulfasalazine and sorafenib violated one or two rules while the new identified molecules are in accordance with all 5 rules. Hence the reported molecules can serve as a potent anti cancer agent. Analyzing molecular dynamics of the drugs and the resulting molecules that were not performed in the present study is one of the major limitations of this study. Many more targets with respect to the ferroptosis pathway and their potential drugs/compounds need to be assessed which could be future recommendations.
In conclusion, the goal of this research is to identify ferroptosis-specific ligands that can be used to create commercial medications that are more effective and less harmful for an earlier diagnosis and potential treatment option. The two pharmacophore models (ZINC89362298 AND ZINC1730544) obtained from the reported System X ͞ c inhibitors could be a promising lead in developing commercial anticancer drugs. If proven successful in animal models, these molecules can be translated into novel therapeutic agent followed by human trials. Future in vitro studies and clinical trials are needed to be performed with these therapeutic combinations.

Author Contribution Statement
Conception and design of the study was done by Dr. Mukesh Doble and Dr. Dharini, data acquisition was carried out by Dr. Dharini. Analysis of data was carried out by Dr. Mukesh Doble and Dr. Dharini. Drafting of the article was done by Dr. Abilasha and Dr. Dharini. Final approval was done by Dr. Pratibha Ramani and Dr. Abilasha.