3.1. Potential bioactive compounds from SB
A total of 35 compounds in GL were identified by the GC-MS analysis (Figure 2), and the name of compounds, retention time, and peak area (%) were enlisted in Table 1. All 35 compounds were checked and only 4,9a-Bis(acetyloxy)-3-[(acetyloxy)methyl]-4a, 7b-dihydroxy-1,1,6,8-tetramethyl-5-oxo-1a,1b,4,4a,5,7a,7b,8,9,9a-decahydro-1H cyclopropa[3,4]benzo[1,2-E]azulen-9-yl acetate # (PubChem ID: 538181) violated Lipinski’s rule (Molecular Weight ≤ 500g/mol; Moriguchi octanol-water partition coefficient ≤ 4.15; Number of Nitrogen or Oxygen ≤10; Number of NH or OH ≤5). Additionally, membrane permeability is typically restricted when topological polar surface area (TPSA) exceeds 140Ų [19]. The number of 33 compounds out of 35 did not surpass 140Ų. Both Formic acid, (3,5-dinitrophenyl)amino-, [3-(ethoxycarbonyl)prop-2-yl] ester (PubChem ID: 552329) and 4,9a-Bis(acetyloxy)-3-[(acetyloxy)methyl]-4a,7b-dihydroxy-1,1,6,8-tetramethyl-5-oxo 1a,1b,4,4a,5,7a,7b,8,9,9a-decahydro-1H-cyclopropa[3,4]benzo[1,2-E]azulen-9-yl acetate # (PubChem ID: 538181) were not accepted within the border. This information was exhibited in Table 2.
3.2. Overlapping genes between SEA and STP linked to 34 compounds
Based on the SMILES, a total of 423 genes from SEA and 551 genes from STP connected to 34 compounds were extracted (Supplementary Table S1). Venn diagram showed that 189 genes were overlapping between the two public databases (Figure 3).
3.3. Overlapping genes between 189 genes (SEA and STP overlapping genes) and AS-related genes
A total of 2,606 genes associated with AS were selected by using DisGeNET and OMIM human gene databases (Supplementary Table S2). The Venn diagram shows that 98 overlapping genes was identified between 2,606 genes related to AS and the 189 overlapping genes (Figure 4-A) (Supplementary Table S3). The gene-gene network of the 189 overlapping genes was constructed by STRING, which indicated 98 nodes and 134 edges (Figure 4-B).
3.4. Identification of a hub signaling pathway on a bubble plot
The KEGG analysis indicated that a total of overlapping 98 genes was enriched 27 signaling pathways (p-value < 0.05) (Figure 5) (Table 3). This analysis is for identifying the AS-related signaling pathways from the number of 98 genes. A hub signaling pathway (inactivation of MAPK signaling pathway) and connected to 7 key genes (HSPB1, PDGFRB, PRKCB, PRKCA, MAPK14, RELA, and PLA2G4A) against AS was identified on STRING analysis.
3.5 Acquisition of core genes related to signaling pathways
The number of 34 core genes obtained by KEGG pathway enrichment analysis and PRKCA manifested the highest degree (16), which was followed by PRKCB (15), MAPK14 (14), and RELA (14) (Figure 6), (Table 4). Accordingly, PRKCA was the uppermost gene of GL against AS.
3.6 MDT of 7 genes and 15 compounds related to MAPK signaling pathway
The number of 7 genes and 15 compounds associated with MAPK signaling pathway was identified by both SEA and STP databases (Figure-7A). Also, each 6 genes excluding PDGFRB gene was strongly related to MAPK signaling pathway (Figure-7b). The MDT was performed to evaluate the binding affinity energy of these 15 compounds against their related each gene, respectively. The MDT of A1-A2 on HSPB1 protein (PDB ID: 4MJH) was analyzed in the "Homo Sapiens" setting. According to the docking score, the priority of binding energy is given: A1>A2. The two binding energy of A1-HSPB1 and A2-HSPB1 revealed -8.8 and -6.6 kcal/mol, respectively. The Benzamide, 4-acetyl-N-(2,6-dimethylphenyl)- (PubChem ID: 147129) (A1) had the strongest affinity on HSPB1. The MDT between B1-B2 and PDGFRB protein (PDB ID: 3MJG) in the "Homo sapiens" setting shown binding affinity of each ligand, and the order of the priority of binding energy is as follows: B1>B2. The two-binding energy of B1-PDGFRB and B2-PDGFRB revealed -8.2 and -7.9 kcal/mol, respectively. The 3-[4-Methoxyphenyl] quinolin-4-ol (PubChem ID: 279953) (B1) had the strongest affinity on PDGFRB. The MDT between C1-C3 and PRKCB protein (PDB ID: 2I0E) in the "Homo sapiens" setting exposed binding affinity of each ligand, and the order of the priority of binding energy is as follows: C1>C3>C2. The two-binding energy of C1-PRKCB, C2-PRKCB and C3-PRKCB exhibited -8.4, -6.2, and -7.2 kcal/mol, respectively. The 1, 2, 3, 4-Tetrahydro-9-methyl-6-cyclohexyl-1-carbazolone (PubChem ID: 535444) (C1) had the strongest affinity on PRKCB. The MDT between D1-D11 and PRKCA protein (PDB ID: 3IW4) in the "Homo sapiens" setting shown binding affinity of each ligand, and the order of the priority of binding energy is as follows: D1>D2>D6>D4>D8>D10>D7>D11=D5>D9>D3. The eleven-binding energy of D1-PRKCA, D2-PRKCA, D3-PRKCA, D4-PRKCA, D5-PRKCA, D6-PRKCA, D7-PRKCA, D8-PRKCA, D9-PRKCA, D10-PRKCA, and D11-PRKCA revealed -8.0, -7.9, -4.4, -6.4, -5.2, -6.7, -5.6, -6.3, -5.0, -6.3, and -5.2 kcal/mol, respectively. The Benzamide, 4-acetyl-N-(2,6-dimethylphenyl)- (PubChem ID: 147129) (D1) had the strongest affinity on PRKCA (the uppermost gene) (Figure 8-A, B). KEGG pathway enrichment analysis showed that inhibition of PRKCA blocks phosphorylation of RafB associated with inflammation [27] (Figure 8-C, D). The MDT between E1-E5 and MAPK14 protein (PDB ID: 1WBW) in the "Homo sapiens" setting shown binding affinity of each ligand, and the order of the priority of binding energy is as follows: E1>E2>E5>E4>E3. The five-binding energy of E1-MAPK14, E2-MAPK14, E3-MAPK14, E4-MAPK14, and E5-MAPK14 revealed -7.4, -6.2, -3.6, -4.2 and -4.8 kcal/mol, respectively. The Benzamide, 4-acetyl-N-(2,6-dimethylphenyl)- (PubChem ID: 147129) (E1) had the strongest affinity on MAPK14. The MDT between F1-F2 and RELA protein (PDB ID: 2O61) in the "Homo sapiens" setting shown binding affinity of each ligand, and the order of the priority of binding energy is as follows: F1>F2. The two-binding energy of F1-RELA and F2-RELA revealed -8.2 and -4.0 kcal/mol, respectively. Interestingly, the MDT of 5 compounds (G1-G5) on PLA2G4A has inactive affinity scores (> -6.0 kcal*mol-1), thereby we did not consider them as promising molecule candidates against AS. The threshold of AutoDock Vina program is regarded as active molecules (binding affinity value < -6.0 kcal*mol-1) and inactive molecules (binding affinity value > -6.0 kcal*mol-1) [28]. The information is enlisted in Table 5.
3.7 Comparative MDT against positive controls on target proteins
The comparative MDT was performed to evaluate affinity between the highest docking score ligands and positive controls. Each positive control of target protein is as follows. The MDT of HSP27 inhibitor J2 (PubChem ID: 135384973) on HSPB1 protein (PDB ID: 4MJH) was -7.3 kcal/mol, while Benzamide, 4-acetyl-N-(2, 6-dimethylphenyl) - (PubChem ID: 147129) (A1) had -8.8 kcal/mol. The A1 was a stronger binding affinity than the positive control (HSP27 inhibitor J2). The MDT of Axitinib (PubChem ID: 6450551) on PDGFRB protein (PubChem ID: 3MJG) was -8.1 kcal/mol, whereas the 3-[4-Methoxyphenyl]quinolin-4-ol (PubChem ID: 279953) was -8.2 kcal/mol. The B1 exposed a stronger binding affinity than the positive control (Axitinib). The MDT of GO 6983 (PubChem ID: 5280335) on PRKCB (PubChem ID:2I0E) was -7.5 kcal/mol, where the 1,2,3,4-Tetrahydro-9-methyl-6-cyclohexyl-1-carbazolone (PubChem ID: 147129) (C3) was -8.4 kcal/mol. The C3 exhibited a stronger binding affinity than the positive control (GO 6983). The MDT of Sphingosine (PubChem ID: 3499) on PRKCA protein (PubChem ID: 3IW4) was -5.5 kcal/mol, while Benzamide, 4-acetyl-N-(2,6-dimethylphenyl)- (PubChem ID: 147129) (D11) had -8.0 kcal/mol. The D11 manifested stronger binding affinity than the positive control (Sphingosine). The MDT of JX-401 (PubChem ID: 1126109) on MAPK14 protein (PubChem ID: 1WBW) was -6.9 kcal/mol, whereas Benzamide, 4-acetyl-N-(2,6-dimethylphenyl)- (PubChem ID: 147129) (E1) was -7.4 kcal/mol. The E1 revealed a stronger binding affinity than the positive control (JX-401). The MDT of Curcumenol (PubChem ID: 167812) on RELA (PDB ID: 2O61) was -7.9 kcal/mol, while the 3-[4-Methoxyphenyl]quinolin-4-ol (PubChem ID: 279953) (F2) was -8.2 kcal/mol. The binding affinity of F2 was stronger than the positive control (Curcumenol). Conversely, the MDT of Berberine (PubChem ID: 2353) on PLA2G4A (PDB ID: 1BCI) was -6.6 kcal/mol, whereas the Erucic acid (PubChem ID: 5281116) (G3) was -4.6 kcal/mol. The Berberine (PubChem ID: 2353) had stronger affinity than Erucic acid in GL. The information is enlisted in Table 6.