2.4.3. Molecular Docking of MAPK1 and MAPK3

To explore the binding relationships between the MAPK1 and MAPK3 proteins, compounds **10** and **11** were docked in comparison with Ravoxertinib, a known inhibitor of both MAPK1 and MAPK3 (see Supporting Information, Figure S16). From the binding energy calculations, **11** exhibited the lowest binding energies of −9.8 kcal/mol and −9.9 kcal/mol, respectively, for MAPK1 and MAPK3, which was better than those of **10** and Ravoxertinib (Figure 6A). Furthermore, docking studies were conducted for the protonated forms of compounds **10** and **11**, specifically protonated at the nitrogen at position 12, as shown in the Supporting Information (Figure S17). Both the neutral and protonated forms of compounds **10** and **11** exhibited similar binding energy values. Docking with MAPK-1 revealed that both the neutral and protonated forms of compounds **10** and **11** predominantly displayed comparable binding interactions with amino acid residues. Interestingly, the protonated forms of compounds **10** and **11** exhibited hydrogen bonding interactions rather than hydrophobic interactions. Docking with MAPK-3 demonstrated that the protonated forms of compounds **10** and **11** could engage in additional interactions at the enzyme site. However, these additional interactions primarily resulted from hydrophobic interactions, which are generally weaker compared to hydrogen bonding interactions. Upon considering the binding interactions of amino acid residues at the enzyme active site, the neutral forms of compounds **10** and **11** exhibited a closer match with the known ligand, Ravoxertinib, compared to the protonated forms. Specifically, for MAPK-1, the neutral forms of compounds **10** and **11** demonstrated interactions with amino acid residues, including ASP111, ILE31, ILE103, and LEU156. For MAPK-3, the neutral forms of compounds 10 and 11 displayed interactions with LEU173 and VAL56. The number of interacting bonds involving hydrogen, hydrophobic interactions, and electrostatic interactions demonstrated the stability of both **10** and **11** in the binding pocket of both MAPK1 and MAPK3. However, compound **11** interacted with both MAPK1 and MAPK3 with stronger hydrogen binding than that of **10** (Figure 6B,C). Regarding the amino acid residues of MAPK1 that interacted with **11**, the key binding site of MAPK1 comprised ALA52, ASN154, ASP111, CYS166, GLU71, and ILE103. The interactions between **11** and MAPK3 at the specific amino acid residues involved ASN171, GLN122, ILE48, LEU173, LYS71, and TYR130. Interestingly, compounds **10** and **11**, along with compound **9**, had similar binding sites, which confirmed that MAPK1 and MAPK3 are the biomolecular targets. The docking conformations of **11** with MAPK1 and MAPK3 demonstrated stable ligand–receptor binding and increased interaction, thereby affirming the necessity of validating the interaction stability through molecular dynamics simulation.

### 2.4.4. Molecular Dynamics Analysis of MAPK1 and MAPK3 with **11**

To gain an in-depth understanding of the protein–ligand interaction of **11**, a molecular dynamics study was conducted on the core proteins of MAPK1 and MAPK3, utilizing the protein–ligand complexes obtained from the results of the molecular docking simulations. When the trajectory exhibits steep changes, it indicates that the system has undergone a significant transition, whereas a smooth trajectory signifies that the system has reached equilibrium. One crucial parameter in molecular dynamics simulations is the root mean square deviation (RMSD), which serves as a measure of the stability of the trajectory. In this simulation, the conformation and movement of the ligand were analyzed using the RMSD value. The RMSD (Å) values of the ligand conformations for MAPK1 and MAPK3 were found to be 2.38 ± 0.15 and 1.79 ± 0.19, respectively, as depicted in Figure 7A. The ligand conformation analysis revealed that both MAPK1 and MAPK3 maintained consistent proximity throughout the entire 15 ns duration of the experiment. However, when comparing the RMSD values, the interaction between **11** and MAPK3 exhibited greater stability in the ligand conformation, and both protein targets did not show significant differences in terms of their interactions. Additionally, the RMSD (Å) of the ligand movement between **11** and these targets was assessed. The results indicated that the average RMSD values for MAPK1 and MAPK3 were 5.68 ± 0.52 and 5.63 ± 2.04, respectively (Figure 7B). A relatively small RMSD of the ligand movement indicates good proximity between the ligand and the targets. The stability of the interaction between compound **11** and MAPK3 showed a greater tendency to fluctuate at 5, 7.5, and 13.2 ns compared to the interaction with MAPK1, particularly at 5 ns intervals.

**Figure 6.** Molecular docking of **10** and **11** with both MAPK1 (ERK2) and MAPK3 (ERK1). (**A**) Binding energies indicate that the interactions of **10** and **11** with both MAPK1 and MAPK3, compared to Ravoxertinib, a known inhibitor. (**B**) Interactions between the amino acid residues of MAPK1 and both **10** and **11**. (**C**) Interactions between the amino acid residues of MAPK3 and both **10** and **11**.

**Figure 7.** Molecular dynamics study of the interactions between **11** and MAPK1 and MAPK3. Green line shows the interaction with MAPK1, whereas the red line shows the interaction with MAPK3. (**A**) The ligand conformation and (**B**) ligand movement interaction of **11** with MAPK1 and MAPK3.

Considering all these findings, we hypothesized that **11** interacts with multiple targets in the MAPK family. Among these targets, MAPK1 demonstrated the highest potential and consistently engaged in strong interactions with **11**, aligning with our previous investigation. According to previous studies investigating the structure-cytotoxicity relationship, which employed both in vitro and in silico approaches, the mechanistic pathways underlying the anti-lung cancer effects of marine alkaloids from the tetrahydroisoquinolinequinone family have been elucidated. Specifically, compounds such as 5-*O*-(*N*-Boc-L-alanine) renieramycin T, a renieramycin-ecteinascidin derivative [20], (1R,4R,5S)-10-(benzyloxy)-9 methoxy-8,11-dimethyl-3-(thiazol-5-ylmethyl)-1,2,3,4,5,6-hexahydro-1,5-epimi- nobenzo [d]azocine-4-carbonitrile (DH\_25), a right-half C–E ring analog of renieramycins [30], and 22-*O*-(4- -pyridinecarbonyl) jorunnamycin A, a 4- -pyridinecarbonyl substituted renieramycintype derivative [29], have demonstrated efficacy against non-small cell lung cancer (NSCLC) cells through modulation of the protein kinase B (Akt), myeloid cell leukemia-1 (MCL-1), and mitogen-activated protein kinase (MAPK) signaling pathways, leading to the induction of apoptosis. The proteins Akt, MCL-1, and MAPK are interconnected targets of cellular signaling networks involved in the regulation of apoptosis. While the MAPK pathway exerts pro-apoptotic effects, Akt signaling generally promotes cell survival. Furthermore, MCL-1, functioning as an anti-apoptotic protein, is influenced by both the MAPK and Akt pathways, thereby contributing to the delicate balance between cell survival and apoptosis in cancer cells [31,32]. Therefore, targeting these signaling pathways and their interactions holds promise for therapeutic interventions aimed at inducing apoptosis in cancer therapy.
