Sesquiterpene Lactones as Potential G1/S Phase Cell Cycle Inhibitors: A Molecular Docking

) complex and Eukaryotic Transcription Factor 2 protein (E2F-2). Methods: The inhibitory potential of SLs, namely ilicol, eucalyptone, and ascleposide E, was investigated using molecular docking analysis. The docking and visualization of ligand-protein complexes were performed using MGL Tools version 1.5.7, BIOVIA Discovery Studio version 21.1.0, and PyMol version 2.5.2. Additionally, drug likeness and pharmacokinetic properties of SLs were assessed via pkCSM and ADMET analysis. Results: Findings demonstrate that ilicol exhibit most favourable complex with CDK6 having binding energy of –7.8 kCal/mol and inhibition constant 1.81 μM. The visualization of ligand-receptor complexes reveals substantial hydrogen bonding interactions. Conclusions: Current study revealed that novel SLs show favourable drug likeness and promising ADMET pro�le along with strong inhibitory effect on G1/S regulatory proteins. The potency of SLs is in order of ilicol> ascleposide E>eucalyptone. To further validate the inhibitory effect of ilicol, implementation of comprehensive in vitro and in vivo investigations must be employed for progression of its development as a novel anti-cancer therapeutic.

Binding energy represents the strength of interaction between a ligand (drug candidate) and a receptor (target protein). It provides an estimate of the stability and a nity of the ligand-receptor complex. A lower binding energy indicates a stronger interaction, suggesting a higher likelihood of successful binding. Inhibition constants re ect the concentration of the ligand required to achieve a given level of inhibition. A lower inhibition constant indicates a more potent inhibitor, as it implies a lower ligand concentration is needed to achieve a desired level of inhibition. The investigation of SL compounds in this study consistently revealed low binding energies and inhibition constants, emphasizing their potential as potent anticancer agents (Table 1). checkpoint ensures that cells pass the G1 phase only when su cient growth signals, nutrient availability, and DNA integrity are present [6]. Inhibiting cyclin D, CDK4, CDK6, and E2F has become a promising approach for cancer treatment. By inhibiting the formation of cyclin D/CDK4-CDK6 complex, the phosphorylation of Rb is halted, leading to the restriction of E2F and subsequent inhibition of E2Fmediated gene transcription. As the expression of genes involved in DNA replication and cell cycle progression is reduced, it leads to cell cycle arrest and inhibition of cancer cell proliferation. This approach holds potential for developing targeted therapies that selectively suppress these proteins in cancer cells [5,7,8]. Plant compounds possess exhibit a lower risk of adverse effects and toxicity owed to their natural origin and evolutionary compatibility with human physiology. Secondly, the bioactive constituents in plant compounds can synergistically target multiple pathways and molecular targets involved in cancer progression, enhancing e cacy and minimizing the development of drug resistance [9][10][11]1]. Sesquiterpene lactones are a class of natural compounds characterized by their 15-carbon ring structure and diverse biological activities. SLs exhibit notable anti-cancer activity, attributed to their ability to interfere with various cellular processes involved in tumor growth and metastasis [12,13].
Molecular docking is a computational analysis extensively used in drug discovery to predict the binding a nity and mode of interaction between a ligand and receptor. It comprises of multiple steps, including retrieval of protein and ligand structures, optimization of their conformations, docking of the ligand within the receptor's binding site, and subsequent evaluation of the resulting docked complexes. Notably, this technique facilitates the identi cation of optimal ligand-receptor orientations and interactions, aiding in design and optimization of potential therapeutic agents [14,15]. The 3D crystal structures of G1/S cell cycle checkpoint proteins, including Cyclin D1 (PDB ID: 2W9Z), CDK4 (PDB ID: 1XO2), CDK6 (PDB ID: 2W96), and E2F-2 (PDB ID: 1N4M), were retrieved from the RCSB Protein Data Bank (PDB). These protein structures underwent processing in AutoDock (AD), which involved steps such as removal of water molecules, addition of polar hydrogen, assignment of Kollman charges, and conversion to PDBQT format. Bioactive SL molecules, namely eucalyptone (CID: 91885002), ascleposide E (CID: 10668005), and ilicol (CID: 44559657), were obtained from PubChem and optimized for docking in PDBQT format. Semi-exible docking was performed using AD, where the protein was kept rigid while the ligand was allowed to undergo conformational changes. Grid boxes, the docking search space centred around the protein's active site, were set up using AD with speci c dimensions, spacing (0.375), and size (40) for each protein. After generating multiple ligand conformations and ranking them based on binding energy, the conformation with the highest binding energy was selected as the most probable binding mode. Visualization of docked complexes and analysis of protein-ligand interactions were carried out using PyMol and BIOVIA Discovery Studio. To evaluate the pharmacokinetic properties of the investigated SLs, the online servers pkCSM and SwissADME were utilized. These tools provide an assessment of the Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) pro les for the potential lead compounds. Additionally, they evaluate drug likeness based on the Lipinski Rule of 5, which aids in predicting the absorption and permeability of small molecules and enables the identi cation of candidates with favorable pharmacokinetic properties. The SMILES notation of the selected ligands was obtained from PubChem and subjected to analysis using pkCSM [16,17]. Various types of protein-ligand interactions were observed by visualizing docked complexes. These interactions determine stability of ligand on protein's active site which can result in an effective protein inhibition. Binding energies and inhibition constants tend to decrease as a result of strong protein-ligand interactions. The most observed interactions are hydrogen bonds, Van der Waals interactions, hydrophobic and electrostatic interactions. Among the SLs ilicol showed the strongest binding a nities in complex with CDK6 and E2F-2 of -7.8 kcal/mol and lowest inhibition constant 1.81µM. The ilicol-CDK6 complex shows robust interactions including a hydrogen bond at residue VAL-150, bond distance of 2.44Å. Additionally, the complex includes alkyl hydrophobic interactions with protein residues LEU-185(5.39Å) VAL-77(5.32Å) ( Figure 1).     (Figure 4).   kcal/mol, respectively. Eucalyptone-Cyclin D 1 complex is characterized by four hydrogen bonds with active site residues ARG-26 (2.94Å and 2.80Å) and ARG-29 (2.68Å and 3.33Å). Hydrophobic pi-alkyl associations were observed at HIS-68 (5.26Å) and ILE-13 (5.42Å). Moreover, an electrostatic pi-anion interaction at residue ASP-129 (4.09Å) was also noticed in the complex, as depicted in The docking complex between eucalyptone and E2F-2 active site is characterized by ve hydrogen bonds at residues AL-23 (2.41Å), TYR-24 (1.85Å), ARG-60 (2.08Å) and ARG-144 (2.05Å) along with a hydrophobic association at ALA-23(3.15Å) (Figure 8).

M E T H O D S R E S U L T S
pharmacokinetic characteristics (Table 3). ADMET pro les are employed in drug design to assess and optimize the absorption, distribution, metabolism, excretion, and toxicity properties of potential drug candidates, ensuring their safety, e cacy, and overall The Lipinski Rule of 5 is employed in drug design to assess drug likeness based on speci c molecular properties. It comprises of four key parameters: molecular weight, lipophilicity (logP), number of hydrogen bond donors, and number of hydrogen bond acceptors (Table 2). The aim of this study was to explore the anti-cancer properties of newly discovered plant-based compounds as inhibitors of the cyclin D/CDK4-CDK6 and E2F-2, using insilico techniques. Our investigation demonstrated strong interactions between all three selected drug candidates and the residues of cyclin D1, CDK4, CDK6 and E2F-2, indicating their potential as inhibitors. Notably, ilicol exhibited the highest binding a nity of -7.8 kcal/mol. Numerous reports have highlighted the anti-cancer potential of various SLs through the inhibition of G1/S phase regulatory proteins in different cancer cell lines, thereby suppressing cell proliferation. Several studies regarding britannin, a pseudoguaianolide SL, have shown its potential as an anti-cancer agent by inhibiting G1/S phase progression via downregulation of its regulatory proteins, cyclin D and CDK4 in human breast cancer MCF7 and MDA-MB468 cell lines and colon cancer HCT116 cell line [18,19]. Recently, another SL alantolactone has gained attention as a promising anti-tumor drug due to its capacity to target multiple molecular pathways [20]. Alantolactone induces G1 phase cell cycle arrest in various cancer cell lines, such as squamous lung cancer SK-MES1 cells [21] and human myeloma cell lines RPMI-8226, MM1R, and NCI-H929 compound to have favourable drug-like characteristics, it should have a molecular weight below 500 Dalton, a logP value not exceeding 5, no more than 5 hydrogen bond donors, and no more than 10 hydrogen bond acceptors [24]. These criteria are derived from the observation that compounds adhering to these thresholds often demonstrate improved oral absorption, permeability, and bioavailability. However, it is important to note that the Lipinski Rule of 5 is a guideline rather than an absolute rule, and deviations from these parameters can still result in successful drug candidates [25]. The molecular pro le of the studied SLs aligns with all the guidelines (Table 2), except for the lipophilicity of eucalyptones, which was slightly higher (logP=5.18) than the threshold (logP < 5.00) de ned by Lipinski's rule. The ADMET pro le is of paramount importance in drug discovery as it provides essential insights into a drug properties, enabling optimization of potential therapeutic agents (Table 3). Absorbance is a critical parameter for evaluating the bioavailability and e cacy of a drug. Several parameters are considered to analyze absorbance, including water solubility (log S) in mol/L, measuring the drug's ability to dissolve in aqueous solutions. Higher water solubility generally enhances absorbance. All the investigated SLs demonstrate effective absorbance at 25˚C as they fall within the signi cant soluble log S range of -4 to -2 mol/L [26]. P-glycoprotein I/II is an e ux membrane transporter, upregulated in cancer cells and is responsible for impeding the absorption and bioavailability of chemotherapeutic drugs. P-glycoprotein I/II inhibitors have the potential to signi cantly enhance the uptake of anticancer drugs by several folds but they may also lead to adverse drug-drug interactions [27]. As demonstrated in Table 3, ilicol and ascleposide E are not P-glycoprotein I/II inhibitors however, eucalyptone being an inhibitor implicate higher d r u g a b s o r b a n c e a t t h e r i s k o f u n f a v o r a b l e pharmacokinetic interaction. All studied compounds show favorable intestinal absorption with absorbance >90% in case of eucalyptone and ilicol and 48% of ascleposide E, indicating e cient absorption compared to poorly absorbed molecules with values <30% [28]. The distribution parameter log VDss (volume of distribution at steady state) represents the extent of drug distribution throughout the body, ensuring a uniform concentration in the blood plasma [29]. The ideal VDss belongs within the range of -0.15 to 0.45 [30], as depicted in table 3, ascleposide E falls outside the range having low (log VDss= -0.21 L/Kg) distribution value, on the other hand, ilicol and eucalytone exhibit optimal VDss values. Cytochrome P450 (CYP450) is a family of enzymes found in the liver and other tissues that play a vital role in the metabolism of drugs and endogenous compounds. CYP450 isoforms, CYP3A4 and CYP2D6, are involved in the biotransformation of numerous drugs by oxidizing or modifying their chemical structures [30]. It is crucial to carefully evaluate the potential implications of CYP interactions, as they can lead to adverse effects or altered pharmacokinetics when combined with other medications [31]. Table 3 demonstrate that SLs show no signi cant interaction with CYP isoforms as either substrate or inhibitor, apart from eucalyptone having potential of being CYP3A4 substrate which can have both positive and negative implications. Being a substrate of CYP3A4 enzyme can facilitate the pronounced clearance and elimination of drug from body. However, it can also pose a challenge by potentially causing drug-drug interactions due to elevated enzyme activity and broad substrate speci city [32]. Renal OCT2 substrate relates to the drug's interaction with the organic cation transporter 2 (OCT2) in the kidney, which plays a signi cant role in the renal elimination of drugs and its metabolites from body. If a drug is identi ed as a Renal OCT2 substrate, it suggests that it undergoes active transport by OCT2 from the bloodstream into the urine, contributing to its excretion. All SLs under consideration are not renal OCT2 substrate which may prove advantageous as it can lead to reduced clearance consequently, increased drug concentrations and enhanced therapeutic effects but raise the risk of toxicity and drug interactions [31,33]. Total clearance represents the sum of all clearance mechanisms that remove the drug from the systemic circulation [33]. Ilicol and ascleposide E have high total clearance values, 1.129 and 1.142 log ml/min/kg meaning these compounds are eliminated rapidly from body. AMES toxicity analysis is a bacterial mutagenicity assay that assesses drug's potential to induce genetic mutations [31,34]. The early identi cation of mutagenic potential in compounds during drug discovery holds paramount signi cance, as it enables the prevention of the development of harmful drugs. All of the compounds studied exhibit a lack of mutagenic potential. Hepatotoxicity is the drug's potential to cause liver damage or dysfunction. All the SLs are nonhepatotoxic thus pose no danger to liver tissues. Maximum tolerable dose is the highest dose that can be administered to a patient without causing unacceptable or severe adverse effects [33,34]. Given that all three compounds possess a maximum tolerable dose below 0.477 log mg/kg/d [33], they can be deemed highly potent even at low concentrations, surpassing this limit may lead to potential toxicity.

C O N C L U S I O N S
Our study focused on investigating sesquiterpene lactones that speci cally target the Cyclin D1/CDK4-6 protein complex and E2F-2, with the objective of inducing cell cycle arrest at the G1/S phase. Through rigorous molecular docking analysis, we identi ed ilicol, ascleposide E and eucalyptone as compounds exhibiting substantial inhibition potential. These compounds demonstrated favourable hydrogen and hydrophobic interactions, which are critical for their inhibitory activity. To further validate and enhance our understanding of their inhibitory effects, we propose conducting comprehensive in vitro experiments. These experiments will provide crucial insights into the compounds' effectiveness as G1/S phase inhibitors, thereby advancing their potential as novel therapeutic agents. By assessing the compounds' activity against relevant biological targets and evaluating their impact on cell cycle regulation, we aim to obtain valuable information that will inform future research endeavors and optimize their therapeutic e cacy.

C o n  i c t s o f I n t e r e s t
The authors declare no con ict of interest.

S o u r c e o f F u n d i n g
The authors received no nancial support for the research, authorship and/or publication of this article.