*2.4. In Silico Prediction of the Target Genes and Molecular Pathways*

The series of the 4- -pyridinecarbonyl ester of the renieramycin-type derivatives (**10** and **11**) exhibited significant cytotoxicity against NSCLC cells. The anticancer mechanisms of compounds **10** and **11** were not investigated because of their limited quantity. Regarding data mining from several databases, pharmaco-mapping, and computational strategies, network pharmacology has recently emerged as a comprehensive tool for elucidating the putative biomolecular targets, signaling pathway complexities, and protein– protein interactions (PPIs) of potential leads [26,27]. Therefore, the inhibition mechanism and molecular pathways of both **10** and **11** against NSCLC were investigated by molecular docking and molecular dynamics studies, enabling the identification of the primary target genes.

2.4.1. Investigation of the Potential Targets against NSCLC

The Swiss Target Prediction online tool was employed to compare the renieramycintype derivatives (**10** and **11**) to a massive internal pharmacophore model database to identify prospective drug targets using reverse pharmacophore alignment (Figure 4) [28]. One hundred and sixteen potential targets related to **10** and **11** were found. From the following four databases: GeneCards, Therapeutic Targets Database (TTD), Online Mendelian Inheritance in Man (OMIM), and DisGeNET, 6431 genes were found in association with NSCLC. Overlapping the corresponding genes using a Venn diagram, 94 prospective targets of **10** and **11** against NSCLC were generated (Figure 4C).

**Figure 4.** Structure and putative analysis of **10** and **11** against NSCLC. (**A**) Structure of **10**. (**B**) Structure of **11**. (**C**) Venn diagram analysis showing both **10** and **11** associated with 116 targets, whereas the NSCLC cells are associated with 6431 targets. The interception analysis showing 94 overlapping targets between **10**/**11** and NSCLC.

#### 2.4.2. Protein–Protein Interaction Network Construction and Core Target Identification

To envision the relationship between the 94 prospective targets matched with **10** and **11** against NSCLC, a PPI network was retrieved and visualized using Cytoscape v3.9.1 after the resulting putative targets were loaded into the STRING platform (Figure 5A). The PPI network illustrates the interactions between the targets, with highly linked proteins in the network having a comparatively high number of interactions. CytoHubba, a plugin for Cytoscape, was employed to perform a topological analysis, which revealed the top 10 proteins linked to the number of degrees (Figure 5B). The colors from yellow to red represent small to large degrees. The results revealed several proteins, including signal transducer and activator of transcription 3 (STAT3), mitogen-activated protein kinase 1 (MAPK1), mitogen-activated protein kinase 3 (MAPK3), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase 2 (CDK2), 90 kDa heat shock protein (HSP90AA1), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit

alpha (PIK3CA), cyclin D1 (CCND1), and cyclin E1 (CCNE1), as the 10 core hub proteins serving as potential therapeutic targets against NSCLC. Regarding the previous virtual study, gene ontology and KEGG pathway enrichment analyses depicted that MAPKs were potential targets of **9** toward the induction of NSCLC apoptosis [29]. Thus, we focused on MAPK1 and MAPK3 for further analysis due to the structural similarities between **9** and **11**, which are the 4- -pyridinecarbonyl esters of the tetrahydroisoquinolinequinone alkaloids.

**Figure 5.** Protein–protein interaction (PPI). (**A**) The main cluster of the PPI obtained from the 94 overlapping targets and visualized using Cytoscape 3.9.1. (**B**) Top 10 targets based on the number of degrees visualized by the CytoHubba plugin. More than 10 degrees are displayed for all the main targets. The intensity of the colors represents the degree values, where red, orange, and yellow correspond to large, medium, and small values, respectively. Red arrow indicates the MAPK family.
