Flavonoids as Strong Inhibitors of MAPK3: A Computational Drug Discovery Approach

Background Mitogen-activated protein kinase 3 (MAPK3) mediates the onset, progression, metastasis, drug resistance, and poor prognosis in various malignancies, including glioma, liver, ovarian, thyroid, lung, breast, gastric, and oral cancers. Negative regulation of MAPK3 expression using miRNAs has led to therapeutic effects in cancer. Objectives The present study performed molecular docking and dynamics simulation to identify potential MAPK3 inhibitors from natural flavonoids, possibly leading to drug development in cancer therapy. Methods A computational drug discovery approach was performed using the AutoDock tool to identify potential MAPK3 inhibitors from 46 plant-based flavonoids. A cross-validation study was executed using the Schrödinger Maestro docking tool. Molecular dynamics (MD) was executed to evaluate the stability of docked poses between the top-ranked compounds and the MAPK3 catalytic domain. Interactions among the most potent MAPK3 inhibitors and residues within the receptor's active site were studied using the BIOVIA Discovery Studio Visualizer before and after 100 ns MD simulations. Results Kaempferol 3-rutinoside-4′-glucoside, kaempferol 3-rutinoside-7-sophoroside, rutin, and vicenin-2 exhibited a magnificent binding affinity to the receptor's active site. In addition, the stability of the docked poses of these compounds seemed to be stable after ∼45 ns computer simulations. Conclusion The present study suggests that kaempferol 3-rutinoside-4′-glucoside, kaempferol 3-rutinoside-7-sophoroside, rutin, and vicenin-2 could strongly bind to the MAPK3 catalytic site and could be assigned as a potent inhibitor for MAPK3. These findings may be helpful in the treatment of various cancers. However, further validation experiments are needed.


Introduction
Mitogen-activated protein kinase 3 (MAPK3), called extracellular signal-regulated kinase 1 (ERK1), is a critical ERK/MAPK pathway cell signaling molecule. It mediates the transmission of signals from a cell's exterior to its interior. Te ERK/MAPK pathway regulates apoptosis, cell proliferation, and migration [1,2]. MAPK3 phosphorylates its downstream cytoplasmic protein, activating several nuclear transcription factors (e.g., c-Jun and c-fos) and participating in apoptosis and cell proliferation [3,4]. Te overexpression and/or hyperactivity of MAPK3 has been linked to the initiation, development, cancer cell migration, and drug resistance in various carcinomas, including liver, thyroid, lung, and gastric cancers [5]. Previously, Du et al. [6] reported that miR-143 upregulation reduced breast cancer cell proliferation by targeting MAPK3. Additionally, Cao et al. [5] demonstrated that the reduced miR-129 expression and MAPK3 overexpression were associated with cisplatin resistance in gastric cancer cells. MiR-129 overexpression also diminished the cell proliferation via downregulating MAPK3 leading to enhanced cell apoptosis. Tese results confrm the signifcant role of MAPK3 in tumorigenesis and drug resistance in diferent cancers.
Molecular docking analysis is a structural bioinformatics approach commonly used for drug discovery, identifying potential inhibitors for a given biological target. Molecular docking also unravels interaction modes between macromolecules and drug candidates [7,8]. Molecular docking analysis includes three steps: macromolecule structure, preparation of small molecule structure, and evaluation of binding afnity between ligand and receptor [9]. Postdocking studies such as molecular dynamics (MD) analysis are frequently used to understand better ligands' dynamic manner and stability within the receptors' binding site (e.g., proteins' active site). Moreover, MD enables assessing the fexibility of residues inside the catalytic site of proteins [10][11][12].
Flavonoids are secondary metabolites predominantly found in the plant kingdom, playing a signifcant role in plant development [13]. Te basic structure of favonoids has been shown in our previous study [14]. Generally, they comprise a C6-C3-C6 skeleton, in which the C6 rings are aromatic, and the C3 is a bridge linker [15]. Due to their high binding afnity to enzymes [16], favonoids have demonstrated a wide range of pharmacological behaviors in medicine, including anticancer, antioxidant, antiinfammatory, antiallergic, antibacterial, and antiviral properties [17,18]. Tere is growing evidence suggesting the curative potential of favonoids in diferent cancers such as breast [19], colorectal [20], oral [21], and lung cancers [22], as well as hepatocellular carcinoma [19].
Te present study suggested that favonoids may act as potential inhibitors of MAPK3 activity, leading to downregulating its downstream signaling pathways and reducing cell proliferation and migration. Terefore, we performed a molecular docking analysis to evaluate the binding afnity of several favonoids to the MAPK3 active site. According to estimated inhibition constant values (Ki) between studied ligands and the protein active site, top-ranked MAPK3 inhibitors were introduced, interaction modes among topranked favonoids and residues inside the MAPK3 catalytic site were analyzed, and the stability of the docked pose of the best MAPK3 inhibitor was studied by executing MD simulation. Te present results might be benefcial in cancer treatment.

Structural Preparation of MAPK3 and Flavonoids.
Te three-dimensional structure of MAPK3 was downloaded at 1.4Å X-ray resolution from the RCSB database [23], which is available at https://www.rcsb.org (PDB ID: 4QTB) [24]. Te 4QTB fle included two polypeptide chains: A and B. Te total number of residues in each chain was checked using the Notepad++ tool. Accordingly, chains A and B included 351 and 348 residues, respectively. Terefore, chain A was selected for further analysis. Critical amino acids within the active site were identifed by analyzing the two structure interactions among the 38Z (positive control inhibitor of the protein with a PDB ID of 24866313) and residues inside the active site of the protein using the BIOVIA Discovery Studio Visualizer version 19.1.0.18287, as well as reviewing the study by Chaikuad et al. [25]. Next, the 38Z molecule was eliminated from the PDB fle, and energy optimization was executed via the Swiss-pdbViewer version 4.1.0, available at https://spdbv.unil.ch [26].
Previous studies have reported about 6000 favonoids contributing to the colorful pigments of fruits, vegetables, and medicinal herbs [27]. Te present study selected 46 favonoids mainly found in commonly used fruits and vegetables, including onions, lettuce, kale, apples, tomatoes, berries, grapes, red grapes, red grapes, raspberries, strawberries, bilberries, merlot grapes, blueberries, and blackberries [27]. Terefore, 46 natural favonoids were considered for identifying possible MAPK3 inhibitors, and the binding afnity of a standard drug (name: Purvalanol; PubChem ID: 448991; DrugBank ID: DB02733) to the active site of MAPK3 was regarded as a positive control inhibitor in this study. Structural preparation and energy minimization of favonoids were explained in our previous study [28].

Cross-Validation Study.
Te most potent MAPK3 inhibitors achieved from the AutoDock tool were selected for cross-validation study. In this regard, the Schrödinger Maestro docking tool version 10.2 was used to calculate the docking scores [32,33]. Te lowest dock score (Glide score) was assigned as the best-docked model for each component. Furthermore, the prime MM-GBSA approach was utilized to indicate the relative binding energies [34].

Molecular
Dynamics. MD was executed in 100 ns (1,00,000 ps) simulations by Discovery Studio Client software version 16.1.0.15350 to evaluate the stability of the docked poses between the top-ranked favonoids, based on the AutoDock tool and Schrodinger Maestro docking software and MAPK3 active site. Advanced settings for MD simulation are mentioned in our previous report [28]. Furthermore, the root mean square fuctuation (RMSF) of MAPK3 residues and the time evolution of root mean square deviation (RMSD) of the receptor backbone atoms complexed with the top-ranked favonoids were analyzed. BIOVIA Discovery Studio Visualizer 19.1.0.18287 was used to unravel interactions between top-ranked favonoids and residues inside the MAPK3 active site and to illustrate twoand three-dimensional views of their docked poses.

Binding Afnity Assessment Using AutoDock.
According to the virtual screening analysis achieved by AutoDock 4.0, four and 32 compounds demonstrated Ki values at the micromolar (uM) and nanomolar (nM) scales, respectively. Besides, it was estimated that nine compounds, including orientin, kaempferol 3-rutinoside-7-sophoroside, rutin, isoquercitrin, vicenin-2, amentofavone, quercetin-3rhamnoside, nicotiforin, and sophorafavanone G, could potentially bind to the MAPK3 active site at the picomolar (pM) scale. Also, a salient binding afnity was observed between kaempferol 3-rutinoside-4′-glucoside (PubChem ID: 44258844) and MAPK3 catalytic site with the Ki and ΔG binding values of 731.68 femtomolar (fM) and −16.65 kcal/ mol, respectively. Terefore, the present study calculated the Ki value for ten compounds at either pM or fM concentrations. Tese favonoids were considered top-ranked MAPK3 inhibitors among the studied favonoids based on the AutoDock tool. Figure 1 demonstrates the chemical structures of these favonoids and purvalanol. Te ΔG binding value between purvalanol and MAPK3 active site was estimated as −8.53 kcal/mol. Accordingly, 39 favonoids demonstrated a higher binding afnity to the MAPK3 catalytic site than the positive control inhibitor. Figure 2 presents ΔG binding values between top-ranked favonoids achieved from the AutoDock tool, the standard drug, and MAPK3 active site. Te estimated binding energies and Ki values for 46 favonoids and the control inhibitor in this study are presented in Table 1. Furthermore, the details of energies between top-ranked inhibitors and MAPK3 catalytic domain are shown in Table 2.

Stability of the Docked Poses.
Regarding MD analysis, the docked poses between kaempferol 3-rutinoside-4′-glucoside, kaempferol 3-rutinoside-7-sophoroside, rutin, vicenin-2, and MAPK3 active site were stable after ∼45 ns computer simulations. Figure 3 demonstrates RMSF and RMSD for MAPK3 backbone atoms complexed with top-ranked favonoids in this study and the standard drug. Figure 4 illustrates the superimposed structures of top-ranked complexes before and after MD simulations.

ADMET
Assessment. SwissADME provides valuable information related to the pharmacokinetic features of compounds. Te following ADME was predicted for 46 favonoids studies in the present study: gastrointestinal (GI) and blood-brain barrier (BBB) permeability, P-gp (P-glycoprotein) substrate, cytochrome P-450 inhibition, and skin permeation coefcient (kp). Rutin and vicenin-2 revealed more appropriate ADME among top-ranked favonoids. Besides, none of the compounds demonstrated considerable toxicity. Table 5 lists the results of ADME and the toxicity of the compounds.

Discussion
MAPK3 is a serine/threonine kinase involved in the phosphorylation and translocation of several cytosolic proteins into the nucleus, leading to the dysregulation of several vital pathways and biological processes associated with apoptosis and cell proliferation [37]. Elevated expression and/or activity of MAPK3 is linked to the onset, development, drug resistance, and metastasis of various carcinomas such as ovarian cancer [38], glioma [39], lung cancer [40], and breast cancer [41]. Terefore, the present study executed a computational drug discovery approach to identify potential MAPK3 inhibitors from natural favonoids, which have widely exhibited anticanter efects [42].
Kaempferol 3-rutinoside-7-sophoroside formed fve hydrogen bonds with the Gly49, Ala52, Arg84, Asp166, and Asp184 located in the MAPK3 active site before MD simulation. Tis compound formed ten H bonds with the Tyr47, Glu50, Gly51, Arg84, Asp128, Lys131, Asp166, and Asp184, after 100 ns MD simulations. Besides, kaempferol showed   International Journal of Analytical Chemistry  International Journal of Analytical Chemistry a lower binding afnity to the MAPK3 catalytic site compared to that of its glycosylated forms with a ΔG binding value of −8.5 kcal/mol, indicating a positive correlation between binding sugar moieties to the rings A, B, and C and enhancing binding afnity between kaempferol and MAPK3 active site. According to a previous report [14], kaempferol    VAL24  GLN34  GLN44  GLY54  ARG64  SER74  ARG84  ARG94  ARG104  ALA114  LEU124  LYS134  CYS144  LEU154  HIS164  ILE174  ASP184  GLU194  TYR204  GLU214  LYS224  ILE234  ILE244  LEU254  PRO264  ASN274  LEU284  LYS294  LEU304  ASN314  ALA324  TYR334  GLU344  ASP354  ILE364  GLY374   14  Kaempferol is a yellow favonoid [46] mainly found in apples, tomatoes, grapes, pine, green tea, and angelica [47]. Antioxidant and anti-infammatory properties of kaempferol [46] may lead to therapeutic efects in various cancers by regulating cell cycle, apoptosis, metastasis, and angiogenesis [48]. Fouzder et al. [49] demonstrated that kaempferol diminished Nrf2 at mRNA and protein levels in nonsmall cell lung cancer cells, leading to the downregulation of Nrf2 downstream genes, including GST, AKR1C1, HO1, and NQO1, resulting in cancer cells sensitive to apoptosis. Pan et al. [50] executed an experimental study to examine the efects of kaempferol on rheumatoid arthritis (RA) in vitro and in vivo. Te authors examined the cell migration and invasion using scratch assays and the Boyden chamber approaches, respectively. Te cytoskeletal reorganization of RA fbroblast-like synoviocytes was tested using immunofuorescence staining. Real-time PCR and western blotting assays were used to examine the MMP expression levels. Pan et al. [50] reported that kaempferol diminished cell migration and invasion by downregulating the MAPK pathway, reduced the MMP expression, and inhibited the actin reorganization in RA FLSs. Huang et al. [51] demonstrated that kaempferol signifcantly inhibited the expression of several genes mediating infammatory response, including interleukin (IL)-1β, cyclooxygenase-2, and nitric oxide synthase. Furthermore, kaempferol diminished collagen II degradations by inhibiting MMP1, MMP3, and MMP13 expression. In addition, kaempferol downregulated the p38 MAPK pathway. Taken together, Huang et al. [51] introduced kaempferol as a compound with therapeutic efects in osteoarthritis.
Orientin was this study's second leading potential MAPK3 inhibitor with the ΔG binding and Ki values of −15.98 kcal/mol and 1.92 pM, respectively. Tis favonoid exhibited seven hydrogen and two hydrophobic interactions with the Gly49, Tyr53, Glu50, Val56, Asp128, Ser170, and    [54] and metastasis [55] is evident. Terefore, orientin could be assigned as a potential drug candidate for cancer treatment with inhibitory efects against proteins involved in cancer onset, development, and metastasis. Orientin is predominantly found in dayfower, millet, passion fruit, and pigeon pea leaves [56]. Several pharmacological features have been reported for orientin, such as antioxidant, anti-infammatory, antimicrobial, and radio-protective efects [57]. Kim et al. [58] demonstrated that orientin inhibited the invasive behavior of breast cancer cells via downregulating MMP9 and interleukin (IL-8) expression. Furthermore, Tian et al. [59] reported that orientin reduced cell proliferation and enhanced the apoptosis process in T24 human transitional cell bladder carcinoma cells in vitro by inhibiting nuclear factor-kappaB (NF-κB).
By analyzing the binding afnities between top-ranked MAPK3 inhibitors achieved from the AutoDock tool in this study and comparing the results with their corresponding structures, the following notes are suggested: (1) By comparing the results of kaempferol with its glycosylated forms, it might be suggested that binding a sugar moiety (or sugar moieties) to the basic structure of favonoids elevates the binding afnity of the compound to MAPK3 (2) By analyzing the ΔG binding values between kaempferol 3-rutinoside-4′-glucoside, kaempferol 3-rutinoside-7-sophoroside, rutin, vicenin-2, and MAPK3 catalytic site, it could be hypothesized that binding a disaccharide to the ring C or two monosaccharides to the ring A considerably elevates the binding affnity of the compound to the MAPK3

Data Availability
Te datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.