2.1. Molecular docking simulations study
Molecular docking simulations were used to clarify the compounds' binding mode and obtain other information that could be utilized for further structural optimization 30,31. Our selected compounds and two reference fungicide compounds were docked against the four different target enzymes Scytalone dehydratase or SDH1 (1STD), Trihydroxynaphthalene reductase (1YBV), trehalose-6-phosphate synthase 1 or Tps1 (6JBI) and isocitrate lyase enzyme or ICL1 (5E9G). The docked compounds were ranked based on the maximum occupancy of binding pocket with minimum free energy, the strength of hydrogen bonding, and other potential non-covalent interactions. Out of 39 docked molecules, top-ranking docking poses were selected. Protein-ligand binding affinity is essential for biological processes, as these physical and chemical interactions determine biological recognition at the molecular level. In this way, it is possible to look for a ligand capable of inhibiting or activating a specific target protein through its interaction. Therefore, it is crucial to find a ligand that binds to a target protein with high affinity 32. The ranking criteria involved Lipinski’s rule of five, the number of hydrogen bond interactions and binding with the selected protein targets involving the binding pocket residues.
Compounds were docked with two enzymes of the melanin pathway, Scytalone dehydratase (1STD) and Trihydroxynaphthalene reductase (1YBV), to inhibit the melanin pathway is responsible for appressorium formation (Table S1). Compound Cryptocin, HDFO, Tanzawaic-acid-L and Camptothecin showed strong binding affinity − 10.1 kcal/mol, -9.3 kcal/mol, -9.2 kcal/mol and − 9.1 kcal/mol respectively against Scytalone dehydratase (1STD) (Table 1A).
Cryptocin was bound with Scytalone dehydratase (1STD) and formed a hydrogen bond with side chain A:TYR50, whereas hydrophobic interactions with residues A:LEU76, A:PRO149, A:ILE151, A:VAL70, A:VAL75, A:LEU54, A:MET69, A:TYR50, A:PHE53, A:HIS85, A:PHE158, A:PHE169 (Table 1A and Fig. 1A). Rest of the compounds were interactions with amino acid residues A:SER129, A:TYR50, A:VAL75, A:PRO149, A:VAL70, A:LEU54, A:ARG166, A:TYR30, A:PHE53, A:PHE158, A:PHE162, A:PHE169, A:HIS85, A:VAL108, A:TRP26, A:HIS110 (Table 1A and Fig. 1B). Bond distance and type of interactions were shown in Table 2 and Table S2.
All these compounds showed strong binding with Scytalone dehydratase (1STD) active site residues followed by Tanzawaic-acid-L, Camptothecin, and HDFO, whose binding affinity was lower than Cryptocin. On the other hand, compounds Camptothecin, GKK1032A2, Alternariol-monomethyl-ether, Arohynapene-A, and Tricyclazole exhibit the highest binding affinity − 9.5 kcal/mol, -9.5 kcal/mol − 8.9 kcal/mol, -8.7 kcal/mol and − 8.3 kcal/mol, respectively with Trihydroxynaphthalene reductase (1YBV) amongst all compounds (Table 1B). Camptothecin showed hydrogen bond with residues B:GLY210, B:TYR223, B:MET215, B:THR213, and B:SER164 hydrophobic non bonded interactions are formed with B:GLY40, B:ILE41, B:MET215 and B:ARG39 and other bonds are B:MET215 and B:MET162 (Table 1B and Fig. 2B). Other compounds showed interaction with B:TYR178, B:LYS182, B:THR213, A:ASN265, B:SER164, B:ILE41, B:ILE211, B:PRO208, B:MET162, A:ALA15, A:PRO17, A:LYS200, B:ARG248, B:LEU246, B:TYR223, B:CYS220, B:MET215, B:VAL219, B:ILE211, B:TYR216, B:TRP243, B:GLY209, B:GLY210, B:MET215 and B:MET283 as details shown in Table 1B and interaction of compound Alternariol-monomethyl-ether shown in Fig. 2A,B.
Bond distance and type of interactions were shown in Table 2 and Table S2. Camptothecin showed the highest binding affinity and more hydrogen bonds than other compounds, and all interactions are possessed in the active site residues of the protein. Likewise, in trehalose-6-phosphate synthase 1 or Tps1 (6JBI), the compound GKK1032A2, camptothecin, chaetoviridin-A and rocaglaol have been observed to bind through meaningful bonds having binding scores of -10.2 kcal/mol, -8.9 kcal/mol, -8.5 kcal/mol, and − 8 kcal/mol respectively (Table 1C). Hydrogen bonds favor the docking interactions of GKK1032A2 with A:MET390, A:HIS181, and A:LYS294, while non-bonded hydrophobic interactions are favored by A:LEU392, A:VAL393, and A:HIS181 (Table 1C and Fig. 3A). Other three compounds showed interactions with residues A:ASN21, A:ARG22, A:TYR99, A:HIS152, A:ARG327, B:LYS294, B:ASN391, B:VAL393, B:VAL287, A:THR46, A:HIS155, A:ASP153, A:TRP108, A:HIS112, A:HIS181, B:LEU371, B:LEU392, B:VAL324, A:HIS181, A:PRO24, A:LEU44, A:LEU48, as detailed in Table 1C and compound Camptothecin illustrated in Fig. 3B. Bond distance and type of interactions were shown in Table 2 and Table S2. GKK1032A2 showed strong binding with trehalose-6-phosphate synthase 1 or Tps1 (6JBI) active site residues and highest binding affinity compared to chaetoviridin-A, camptothecin and rocaglaol.
In case of the isocitrate lyase enzyme (5E9G), arohynapene-B and pannellin possess the highest binding affinity − 8.3 kcal/mol and − 8 kcal/mol, respectively amongst all the compounds (Table 1D). The binding conformations were analyzed, and we identified that arohynapene-B formed a hydrogen bond with A:ALA396, A:ALA399 and A:TYR38. In addition, several residues A:PRO397, A:PRO426, A:TYR38, A:TYR425 formed hydrophobic interactions (Table 1D and Fig. 4A). While pannellin formed hydrogen bond with A:LYS135, B:LYS135 and A:ASN134 and hydrophobic interactions with residues A:LYS135 and A:HIS138 whereas electrostatic interaction with A:ASP118 (Table 1D and Fig. 5B). Bond distance and type of interactions were shown in Table 2 and Table S2.
2.3. Bioactivity score assessment of selected potential natural products
The bioactivity score of the selected compound was predicted through the Molinspiration server. In this prediction, biological activity measured by the bioactivity score for enzyme inhibitor was evaluated enzyme (Table 4), which are classified into three different ranges: molecule having bioactivity score greater than 0.00 is most likely to illustrate meaningful biological activity, while scores extending from − 0.50 to 0.00 are expected to be moderately active, and if the score is less than − 0.50, it is presumed to be inactive. The bioactivity scores for the G protein-coupled receptor ligand (GPCR) are most active for all the selected compounds except alternariol-monomethyl-ether and GKK1032A2 are moderately active, whereas tricyclazole is biologically inactive. Meanwhile, the ion channel modulators' scores for the tanzawaic-acid-L, arohynapene-B, HDFO, azoxystrobin, and strobilurin are biologically active, and all other compounds are moderately active except tricyclazole is biologically inactive.
The result of kinase inhibitors scores for the compound camptothecin and azoxystrobin have biological active score values, and other compounds are moderately active, whereas the compound GKK1032A2 and tricyclazole are inactive. Moreover, the nuclear receptor score values, all the compounds are biologically active, whereas tricyclazole is biologically inactive according to the classification ranges of Linn et al. 33. Compounds alternariol-monomethyl-ether, tanzawaic-acid-L, arohynapene-A, camptothecin, pannellin, azoxystrobin, and strobilurin have moderately active score values; meanwhile, cryptocin, chaetoviridin-A, GKK1032A2, arohynapene-B, rocaglaol, and HDFO are biologically active, whereas compound Tricyclazole is inactive for Protease inhibitors. The structures of all compounds have score values for enzyme inhibitors greater than 0.00 considered biologically active. Meanwhile, the compound pannellin has moderately active score values, whereas the compound tricyclazole has inactive score values.