In silico molecular docking evaluation reveals high potencies of some natural antifungal metabolites on melanin biosynthesis and appressoria formation enzymes in Magnaporthe oryzae

In silico molecular docking evaluation reveals high potencies of some natural antifungal metabolites on melanin biosynthesis and appressoria formation enzymes in Magnaporthe oryzae Md. Arif Khan University of Development Alternative Md. Abdullah Al Mamun Khan Mawlana Bhashani Science and Technology University Jannatul Maowa Sanjana Mawlana Bhashani Science and Technology University Asif Ahsan Bangladesh Agricultural University Dipali Rani Gupta Bangabandhu Sheikh Mujibur Rahman Agricultural University M. Nazmul Hoque Bangabandhu Sheikh Mujibur Rahman Agricultural University Tofazzal Islam (  [email protected] ) Bangabandhu Sheikh Mujibur Rahman Agricultural University Article Keywords: Molecular docking, Antifungal metabolites, Melanin biosysthesis, Magnaporthe oryzae Posted Date: June 29th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1794320/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/32 Abstract Magnaporthe oryzae is one of the most notorious fungal pathogens that causes blast disease in cereals and results in enormous loss of grain production. Many chemical fungicides are being used to control the pathogen but none of them are effective against blast disease. Thus, there is a demand to discover potential and safe natural biofungicides to manage blast disease successfully. To nd out effective biofungicides, we performed in silico molecular docking analysis of some natural compounds targeting four enzymes namely, scytalone dehydratase, SDH1 (1STD), trihydroxynaphthalene reductase, 3HNR (YBV1), trehalose-6-phosphate synthase, Tps1 (6JBI) and isocitrate lyase, ICL1 (5E9G) of M. oryzae fungus that regulate melanin biosynthesis or appresorium formation. Thirty-nine natural compounds that previously reported to inhibit the growth of M. oryzae were subjected to rigid and exible molecular docking against aforementioned enzymes followed by molecular dynamics simulation and free energy analysis of protein-ligand complexes. The results of virtual screening showed that out of 39, 12 compounds showed good binding energy with any one of the target enzymes as compared to reference molecule azoxystrobin and strobilurin. Among the compounds, camptothecin GKK1032A2 and arohynapene-B bind more than one target enzymes of M. oryzae. All the compounds except tricyclazole showed good bioactivity score. Taken together, our results suggest that all of the 12 compounds have the potential to develop new fungicides but camptothecin, GKK1032A2 and arohynapene could act as multi-site mode of action fungicides against M. oryzae. 1. Introduction Blast disease caused by the Magnaporthe oryzae (anamorph: Pyricularia oryzae), is one of the most destructive diseases of cereals including rice, wheat, barley, nger millet etc. 1,2. Approximately 10–30% of the global rice production is lost by the infections caused by this recalcitrant pathogen 3. Although rice blast is prevalent in most rice-growing regions in the world wheat blast was con ned only to South American countries, including Brazil, Bolivia, Argentina, and Paraguay until 2015 4. The disease was rst time reported in an Asian country, Bangladesh, in February 2016 5. Some newspaper in India has also reported the existence of the disease in border districts of India that are close to wheat blast infected districts of Bangladesh 6. Very recently, this disease has also been reported in the Gambia, an African country, apart from South America and Asia 7. Air-borne inoculum is the mechanism of short-distance disease spread whereas, seed trading is thought to be the mechanism of disease spread over a long distance 4,8. Although the rate of devastation by the disease depends on several factors but can cause yield loss of up to 100% in a congenial environment 2,9. The genus of M. oryzae consists of several pathotypes and can cause blast disease on more than 50 Poaceae plants 10. Based on host speci city they are classi ed as Oryza pathotype (infecting rice), Triticum pathotype (infecting wheat), Setaria pathotype ((infecting foxtail millet), Lolium pathotype ((infecting ryegrass), and many other 11,12. Although the isolates from different hosts are genetically distinct, cross-infection does occur to some extent 4,13. As M. oryzae isolate of wheat can infect barley, maize triticale, durum and swam rice grass in laboratory conditions whereas M. oryzae isolates of rice can cause disease in the wheat plant 5,14,15. However, the virulence of these pathogens during cross-infection in eld conditions has not been tested yet. Page 2/32 The control of blast disease is di cult and mainly achieved through the use of chemical fungicides 16,17. However, extensive use of chemical fungicides has led to acquired resistance against fungicides 2,17. Moreover, traditional breeding strategies take a longer time to develop resistant variety and resistance often breakdown in eld conditions after certain years due to quick evolution of the pathogen 10,18. Therefore, development of new fungicides by searching for bioactive natural compounds is a novel approach to managing destructive diseases. Various research studies have been carried out to estimate the antifungal potential of natural compound against blast fungus in vitro however, led e cacy of these compounds are still unclear 19–21. Most of the natural compounds act directly on fungal cell whereas some compound act as speci c inhibitor of fungal cellular or metabolic process to inhibit the growth. Present researchers are focusing on identi cation of compounds that have speci c and multiple inhibitors for fungal cellular process or pathogenicity related factors. Proteins that are responsible for the cellular process or pathogenicity of speci c fungus would be the target for design of speci c inhibitors that block the growth of the fungus. Like many other fungal pathogens M. oryzae infect host by elaborating a specialized infection cell called an appressorium. Melanin deposition in appresoria has been reported to contribute to rapture the host cell wall and establish host-pathogen interaction. Therefore, enzymes in the melanin biosynthetic pathway are valuable target for development of fungicides. Scytalone dehydratase, SDH1 (1STD), an enzyme involved in the melanin biosynthesis in may phytopathogenic fungi including M. oryzae 22. SDH1 catalyzes the conversion of scytalone to 1, 3, 8trihydroxynaphthalene, and vermelone to 1, 8-dihydroxynaphthalene. Whereas, trihydroxynaphthalene reductase, 3HNR (1YBV), an essential enzyme for the biosynthesis of fungal melanin, catalyzes the conversion of trihydroxynaphthalene to vermelone 23. Subsequently, the 1, 8-dihydroxynaphthalene is polymerized into melanin. Moreover, during the morphogenesis of conidium to appresoriunm development, storage compounds like sugar and lipid present in the conidium are moved from conidium to appresorium as a source of energy required for generation of tugor pressure 24. One such sugar, trehalose, is present in conidia of M. oryzae and is mobilized during appressorium formation. The biosynthesis of trehalose is partly regulated by trehalose-6phosphate synthase, Tps1 (6JBI), and deletion of the Tps1 gene in M. oryzae abolishes its ability to cause disease in rice 25–27. Isocitrate lyase, ICL1 (5E9G), one of the principal enzymes of the glyoxylate cycle in the rice blast fungus M. oryzae helps cells to assimilate two-carbon compounds into the tricarboxylic acid cycle (TCA cycle) and channel these via gluconeogenesis to generate glucose. Icl1 mutant cells impaired in germ tube emergence, appressorium development and cuticle penetration and were less virulent compared to wild type strain 28. Recent study showed that bromophenols isolated from the red alga Odonthalia corymbifera exhibited potent ICL inhibitory activity and blocked appressoria formation by M. grisea as well as reduced disease severity was observed by the treatment of bromophenol in M grisea infected leaf 29. Molecular docking and other computational study of inhibitors on their target enzymes/proteins of a pathogenic fungus can contribute a great help to estimate the potential of the inhibitors that could prevent the activity of an enzyme. In silico analyses of molecular docking and protein – ligand interaction of antifungal metabolites on target enzymes or proteins are also important for understanding the mechanism of antifungal action and their potential as a novel candidate fungicide against M. oryzae. In this study, we aimed to screen some recently reported inhibitory natural products against blast fungus M. oryzae for understanding their mechanisms of action and promise as candidate fungicides using in silico molecular docking studies on some enzymes involved in pre-infectional development of the blast fungus. The speci c objectives of this study were Page 3/32 to (i) virtual screening and molecular docking simulation of 39 promising antifungal natural products on four enzymes viz. SDH1, 3HNR, TPS1 and ICL1 using PyRx 0.8, ii) assess fungicide-likeness, and iii) bioactivity of natural compounds using in silico analysis. 2. Results 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. Proteinligand binding a nity 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 speci c target protein through its interaction. Therefore, it is crucial to nd a ligand that binds to a target protein with high a nity 32. The ranking criteria involved Lipinski’s rule of ve, 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 a nity − 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 a nity was lower than Cryptocin. On the other hand, compounds Camptothecin, GKK1032A2, Alternariol-monomethyl-ether, Arohynapene-A, and Tricyclazole exhibit the highest binding a nity − 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 Page 4/32 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 a nity 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 a nity compared to chaetoviridin-A, camptothecin and rocaglaol. In case of the isocitrate lyase enzyme (5E9G), arohynapene-B and pannellin possess the highest binding a nity − 8.3 kcal/mol and − 8 kcal/mol, respectively amongst all the compounds (Table 1D). The binding conformations were analyzed, and we identi ed 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. 2.2. Fungicides likeness Physicochemical properties of the potential compounds were analyzed to evaluate their fungicide-likeness nature. We analyzed the fungicide-likeness of the natural compounds under the well-established fundamental rule of drug-likeness i.e. Lipinski rule of 5. Natural compounds physicochemical properties include molecular weight, number of rotatable bonds, number of hydrogen bond acceptors, and number of hydrogen bond donors, topological polar surface area, Fraction Csp3, Molar refractivity, and Synthetic accessibility were analyzed. The predicted results were listed in Table 3. Interestingly all selected natural compounds bear the Molecular Weight range from 189.24 to 434.48 (< 500) except GKK1032A2 and pannellin. The milogP values of the potential compounds were found to be below 5 (0.89to 5.08) except compound GKK1032A2. According to Lipinski’s rule, that most “drug-like” molecules have number of hydrogen bond acceptors ≤ 10, and number of hydrogen bond donors ≤ 5. Furthermore, the number of hydrogen bond donors was less than ve, and the number of hydrogen bond acceptors was less than 10. Besides, TPSA of all potential compounds was observed in the range 46.53 to 112.91 Ų. Page 5/32 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 classi ed 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 classi cation 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. 3. Discussion Blast caused by M. oryzae is a destructive disease of cereal crops that causes enormous economic losses by reducing the grain yield 18. Although the chemical fungicides provide a marginal protection against blast disease but increasing risk of health hazard and pathogen resistant is a matter of concern. Hence there is still a need to discover some alternative natural fungicides against M. oryzae infection that can be more effective as they will be environmentally suitable with less toxicity. To nd out potential fungicides against M. oryzae, we performed a literature-based survey and identi ed 12 compounds that differently inhibit the growth of M. oryzae fungus. Molecular docking study revealed that most of these compounds bind either 1STD or 1YBV or 6JBI and/or 5E9G enzymes responsible for either melanin biosynthesis or appresoria formation enzymes of M. oryzae. Scytalone dehydratase, SDH1 (1STD) and Trihydroxy naphthalene reductase, 3HNR (1YBV), two key enzymes of melanin biosynthesis pathway and found to be essential for virulence in plant pathogenic fungi 22,34. Various studies have reported that the SDH1 and 3HNR enzymes are promising molecular target for the identi cation of potential inhibitors 35,36. Up to date several synthetic fungicides namely, carpropamid, tricyclazole, pyroquilon and phthalide have been discovered that inhibit these two enzymes of melanin biosynthesis pathway. Although these compounds were effective against M. oryzae but extensive uses of these fungicides lead to development of resistance. For instance, carpropamid, ((1RS,3SR)-2,2-dichloro-NPage 6/32 [(R)-1- (4-chlorophenyl)ethyl]-1-ethyl-3-methylcyclopropanecarboxamide), a commercial fungicide, that targets the scytalone dehydratase enzyme and it has been widely used in Japan as the chemical agent for nursery-box treatment against leaf blast of rice 16. However, a single-point mutation in Sdh1 in M oryzae isolates causing substitution of one amino acid in the scytalone dehydratase gene showing decreased sensitivity to carpropamid 37. Two other enzymes namely trehalose-6-phosphate synthase, TPS1 (6JBI), Isocitrate lyase, ICL1 (5E9G), have also been shown are good targets to design fungicides. TPS1 is the key enzyme in trehalose biosynthetic pathways that catalyzes the transfer of glucose from uridinediphospho (UDP)-glucose to glucose 6-phosphate to generate trehalose 6-phosphate (T6P). TPS1 appeared to be dispensable for development and virulence M. oryzae, Fusarium verticillioides, Puccinia striiformis f. sp. tritici and Fusarium graminearum and Tps1 mutants showed reduced pathogenicity 38,39. Since fungal TPS1 shares minimal similarity to plant homologs therefore, inhibition of TPS1 may serve as a promising target for the development of new strategies to control fungal diseases 40. As for example, Validamycin A, a competitive inhibitor of Tps1 and have been used as potential fungicide 41. Isocitrate lyase (ICL), a key enzyme in carbon metabolism and is essential for the pathogenesis for both human and plant fungal pathogens 42. It has been shown that icl1 gene of Leptosphaeria maculans involved in successful host colonization of Brassica napus, whereas an M. grisea Icl1 regulates virulenceassociated functions such as germ tube emergence, appressorium development, and cuticle penetration. Δicl mutants exhibits less virulent than wild type and impaired virulence-associated function 43. Several natural compounds have been identi ed as inhibitor of Icl1. Halisulfate 1, a sesterterpene sulfate, isolated from tropical sponge Hippospongia spp., reduces both appresorium formation and infection of rice plants by the fungus M. grisea by potentially binding with Icl1 43. Bromophenols, another natural compound isolated from the red alga Odonthalia corymbifera exhibited potent ICL inhibitory activity and blocked appresoria formation of M. grisea in a concentration-dependent manner 29. Joshi et al. 44 used ICL as a molecular target to discover new antifungal compounds against F. graminarearum using molecular dynamic study. Four natural compounds namely, Melianoninol, Nimbinene, Vilasinin, and Fraxinellone from Melia azedarach identi ed as potent inhibitor of ICL1. Molecular dynamics simulation demonstrated that these four phytochemicals displayed considerable signi cant structural and pharmacological properties and could be probable antifungal drug candidates against F. graminarearum. Therefore, molecular docking and simulations studies could be utilized to predict the e ciency of binding of the ligand with biomolecules 45,46. In this study, a total of 39 compounds were subjected to molecular docking study and 12 of them showed good binding a nity with the aforementioned four enzymes of M. oryzae. Among the 12 compounds, four compounds viz. cryptocin, tanzawaic-acid-L, camptothecin, and HDFO strongly bind with 1STD whereas ve natural compounds namely, alternariol-monomethyl-ether, GKK1032A2, arohynapene-A, camptothecin, and tricyclazole strongly bind with trihydroxy naphthalene reductase (1YBV). Chaetoviridin-A, GKK1032A2, camptothecin, and rocaglaol showed strong binding a nity for 6JBI whereas, only two compounds viz. arohynapene-B and pannellin were bind with 5E9G with less energy requirement. Then, we have subjected the all the compound for analyzing fungicide-likeness by the Lipinski’s rule of 5. According to Lipinski’s rule of 5, a compound would be an active fungicide if it satis es all the properties of pharmacological and biological activity. Molecular weight of a chemical is an important criterion to determine its fungicides activity. The molecules that have low molecular weight (฀500) are readily transported, diffused and absorbed by the cell Page 7/32 membrane in comparison to large molecules 47. Molecular weight of the selected compounds was found more or less than 500 g/mol (189.24 g/mol to 506.5 g/mol). In addition, the positive LogP values indicate easier passage of compounds through bio-membranes and the acceptable limit is ฀5 48,49. Moreover, the lipophilic compounds easily permeable trough cell membrane by passive diffusion and bind with biomolecules to inhibit the vital metabolic enzymes in to the cell. Therefore, the membrane permeability depends on the lipophilic nature of a compound. The calculated log P value of natural compounds was ranging from 0.89 to 5.08 which is ideal for crossing the cell membrane. Recently, Steinberg et al. 50 reported that C18-SMe2+, a mono-alkyl lipophilic cations (MALCs) having LogP value 2.26 easily diffuse through plasma membrane. Although molecular weight and logP value of some compounds were exceed the expected limit mentioned in Lipinski’s rule 5 but it would be worth mentioning that this slight increase in molecular weight and milogP value will not have a signi cant impact on compound transportation and diffusion. It has been shown that the molecular mass of several FDA-approved drugs was more signi cant than 500 g/mol 51. Furthermore, the number of hydrogen bond donors was less than ve, and the number of hydrogen bond acceptors was less than 10 52. Besides, TPSA of all potential compounds was observed in the range 46.53 to 112.91 Ų which is also between the acceptable ranges (Lipinski 2004). In case of bioactivity score, all the compound poses a score value of − 0.50 to 0.00 which indicate the compounds are biologically active except tricyclazole. Among the compounds, camptothecin, a very well-known alkaloid isolated from plant origin showed strong binding a nity with 1STD, 1YBV and 6JBI. As evident in several reports camptothecin treatment inhibits the growth of M. oryzae, Rhizoctonia solani, Alternaria alternata, Colletotrichum gloeosporioides, Fusarium oxysporum, Botrytis cinerea, Sphaerotheca fuliginea and Pseudoperonospora cubensis 1,53. Among the reported fungi, camptothecin was found to be most effective against M. oryzae, at a concentration 1.53 µg/mL (EC50 value) whereas, it was effective against mycelial growth of A. alternate and F. oxysporum with EC50 value 250 µg/ mL and for C. gloeosporioides, it was 500µg/mL. The molecular simulation result showed that CPT could binds to the interface of DNA-topoisomerase I complex of M. oryzae and affecting the translation and carbohydrate metabolism/energy metabolism leading to cell death 53. Four other natural compounds namely, GKK1032A2 tanzawaic-acid-L, arohynapene-A and arohynapene-B were identi ed from fungus Penicillium sp. showed conidial germination inhibition in M. oryzae 54. While the compound GKK1032A2 was effective at a concentration 3 µg/mL, the rest of the compounds were effective at 25–50 µg/mL. However, GKK1032A2 has been found to be ineffective against other phytopathogenic fungi including F. graminearum, B. cinerea and P. infestans but the antifungal activity of tanzawaic-acid-L arohynapene-A and arohynapene-B on other phytopathogenic fungus have not been tested yet. Interesting, HDFO, (3aS,4aR,8aS,9aR)-3a-hydroxy8a-methyl-3,5-dimethylenedecahydronaphto [2,3-b]furan-2(3H)-one, a newly identi ed compound from Biscogniauxia sp. O821 completely inhibited condial germination of M oryzae at a concentration of less than 5 ppm whereas blast lesion formation was signi cantly reduced in the presence of 5 and 10 ppm HDFO. In addition, this compound had antifungal activity against A. alternata, Cochliobolus miyabeanus, Colletotrichum orbiculare, Corynespora cassiicola, Fusarium oxysporum f.sp. conglutinans and Fusarium oxysporum f.sp. spinaciae at > 50 ppm 55. Like as HDFO, cryptocin also inhibit the growth of a wide range of fungi including M. oryzae with Minimum Inhibitory Concentration (MIC) value less than 1.0 µg/mL 20. Engelmeier et al. 56 reported rocaglaol and pannellin inhibit germ tube formation at 1.6 and 3 µg/mL respectively. Therefore, all the 12 Page 8/32 compounds could be used as lead compound or biofungicide for the inhibition of aforementioned enzymes in M. oryzae. Although there are a number of fungicides are available for the control of many diseases, the blast disease caused by the different pathotypes of M. oryzae remain to be managed effectively. Recent studies suggest that most of the important plant pathogenic fungi acquired resistance against chemical fungicides 57,58. Currently used fungicides generally target a single enzyme which can be overcome by single point mutation 17,37. For instance, extensive use of strobilurin (QoI) fungicides in Brazil has led to a widespread distribution of cyt b mutations conferring resistance in strains isolated from wheat and other grasses 17. Therefore, an alternative approach such as multi-site mode of action fungicides are needed to be discovered for the management of devastative blast disease. It is hypothesized that fungicides with multi-site mode of action would not be easily overcome by the emergence of resistance 58. In the current study we identi ed three natural products viz. camptothecin, GKK1032A2 and arohynapene-A have multiple enzymes target for inhibition of blast fungus. Therefore, these compounds merit further study in vivo evaluation for considering them as potential fungicides or lead compound to control blast disease. 4. Conclusions The control of blast diseases in major cereal crops viz. rice, wheat, maize etc. using natural compounds is an advanced and risk-free method for disease management in agriculture. The results of present study clearly revealed that the camptothecin, GKK1032A2 and arohynapene-A could act as potential lead compounds for the development of effective fungicides against the most notorious blast fungus. All these compounds showed greatest binding a nity more than one target proteins along with a good number of H-bond and bioactivity score compared to the currently available fungicide for blast disease management. These compounds hold ideal logP values and low molecular weights. Therefore, these compounds could cross the cell membranes and are able to inhibit the target enzymes in M. oryzae that involved in pathogenesis related factors in blast fungus. Our results convincingly suggest that at least three antifungal natural compounds viz. camptothecin, GKK1032A2 and arohynapene-A target multiple enzymes involved in melanin biosynthesis and appressoria formation in the blast fungus M. oryzae. Both these processes are essential for successful infection of host plants by the M. oryzae. Further in vivo molecular and eld study are required for con rming the ndings of this in silico study before recommending camptothecin, GKK1032A2 and arohynapene-A as fungicides or lead compounds against M. oryzae. 5. Materials And Methods 5.1. Protein preparation The crystal structure of Magnaporthe oryzae Scytalone dehydratase (PDB ID: 1STD at 2.90 Å resolution), Trihydroxynaphthalene reductase (PDB ID: 1YBV at 2.80 Å resolution), trehalose-6-phosphate synthase 1 (Tps1) (PDB ID: 6JBI at 2.50 Å resolution) and Isocitrate lyase (PDB ID: 5E9G at 2.10 Å resolution), was retrieved from Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) 59 and considered as a template for all molecular docking simulation. For protein preparation, we used Discovery Studio 2019 molecular visualization software 4.5 60 and PyMOL 2.3.3 software 61. First, the proteins were Page 9/32 uploaded to the software and found unnecessary objects such as default given ligands, ions, and water molecules were removed from the PDB le. Finally, the les were saved in PDB le format for further analysis. List of the enzymes/proteins and their biological functions are given in spplementary Table 1. 5.2. Ligand dataset preparation The canonical smiles of 39 compounds were retrieved from the PubChem database 62, and their 3D structures were generated using Online SMILES Translator and Structure File Generator 63. Afterward, each of the compounds was ready as ligands for molecular docking study with the target proteins. The 2D chemical structure of best-docked compounds is illustrated in Fig. 5. 5.3. Molecular docking simulation In structural biology, molecular docking is a well-known and reliable technique, especially in computer-aided drug design (CADD) processes 64. The technique ensures the best prediction of binding mode between a small molecule and a speci c macromolecule 65. The active site of the proteins was identi ed before going to the molecular docking simulation study through metaPocket (https://projects.biotec.tu-dresden.de/metapocket/) and cross-checked by another server named CASTp (http://sts.bioe.uic.edu/castp/index.html?2r7g). Molecular docking simulation was carried out using PyRx 0.8 virtual screening software 66. For simulating the best interaction, the docking was performed setting the center in axis x- 32.0745, axis y- 36.2698 and axis z17.8046 with the dimension was in axis x- 38.0525 Å, axis y- 33.7716 Å and axis z- 51.7508 Å for Scytalone dehydratase (PDB ID: 1STD); center in axis x- 71.3457, axis y- 13.7945 and axis z- 31.7174 with the dimension was in axis x- 70.3723 Å, axis y- 60.4891 Å and axis z- 66.3320 Å for Trihydroxynaphthalene reductase (PDB ID: 1YBV); center in axis x- 23.3577, axis y- (-0.9662) and axis z- 25.0398 with the dimension was in axis x61.2168 Å, axis y- 98.7383 Å and axis z- 95.6880 Å for Tps1 (PDB ID: 6JBI); center in axis x- (-21.315), axis y34.7671 and axis z- 30.1677 with the dimension was in axis x- 147.9292 Å, axis y- 83.7344 Å and axis z82.9492 Å for Isocitrate lyase enzyme (PDB ID: 5E9G).. After docking simulation, the protein data bank partial charge & atom type (pdbqt) le format, given by PyRx as output, was saved for further protein-ligand interaction analysis. 5.4. Protein-ligand interaction analysis For a clear view of protein-ligand interaction of the best-docked complexes, 2D plots of protein-ligand interactions were analyzed through Discovery Studio 4.5. It generates a 2D graph of hydrogen bonds, electrostatic interactions, and hydrophobic interactions, contributing to the a nity of the drug-like molecules within the active site of M. oryzae proteins. 5.5. Fungicides likeness The physicochemical parameters of the most promising compounds were predicted using the web tool SwissADME (http://www.swissadme.ch/index.php). The predicted parameters included the number of rotatable bonds, number of hydrogen bond acceptors, number of hydrogen bond donors, partition coe cient log p (miLog P), molecular weight, synthetic accessibility, and topological polar surface area (TPSA). 5.6. Bioactivity score prediction Page 10/32 The online Molinspiration Cheminformatics server (http://www.molinspiration.com) was utilized to evaluate the biological activity of selected compounds. The prediction was based on the enzyme inhibition score such as G-protein-coupled receptor (GPCR), Ion channel modulator, Kinase inhibitor, Nuclear receptor ligand, Protease inhibitor, and Enzyme inhibitor. The results are calculated according to previously published recommendations 33. Therefore, it is recommended that if the value is equal to or greater than 0.00, the more active it will be, while if the values are between -0.50 and 0.00, it is moderately active, and, if the score is less than -0.50, it will be considered inactive 67,68. Tables e 1: Summary of top-ranked compounds screened against Scytalone dehydratase (1S ydroxynaphthalene reductase (1YBV), trehalose-6-phosphate synthase 1 or Tps1 (6JBI) and isocit e enzyme (5E9G) with their respective binding energy and interacting amino acid residues. Page 11/32 ounds STD Binding Energy (kcal/mol) Residues involved in Hydrogen bond Interaction ocin -10.1 waicL -9.2 A:SER129 -9.1 A:TYR50 tothecin O ence ystrobin) ence bilurin) YBV -9.3 -7 -6.9 nariolmethyl- -8.9 032A2 -9.5 tothecin -9.5 lazole -8.3 napene- ence -8.7 -8.9 A:TYR50 A:TYR50 A:GLN84 A:LYS148 B:TYR178, B:LYS182, B:GLY210, B:THR213, B:PRO208, A:ASN265 B:THR213, B:TYR223 B:GLY210, B:TYR223, B:MET215, B:THR213, B:SER164 B:SER164, B:TYR178, B:TYR223 B:ARG39, Residues involved in Hydrophobic interaction A:LEU76, A:PRO149, A:ILE151, A:VAL70, A:VAL75, A:LEU54, A:MET69, A:TYR50, A:PHE53, A:HIS85, A:PHE158, A:PHE169 A:VAL75, A:PRO149, A:VAL70, A:LEU54, A:ARG166, A:TYR30, A:TYR50, A:PHE53, A:PHE158, A:PHE162, A:PHE169 A:HIS85, A:PRO149, A:LEU54, A:TYR50, A:PHE53 A:PRO149, A:VAL108, A:TRP26, A:TYR30, A:HIS85, A:HIS110, A:PHE158 A:TYR24, A:MET20, A:TYR16, A:TRP92, A:ALA27, A:ILE87, A:VAL23 A:LEU132, A:ALA130, A:ILE101, A:TYR103, A:TRP134 B:MET215, 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:GLY40, B:ILE41, B:MET215, B:ARG39 B:TYR223, B:GLY209, B:GLY210, B:MET215, B:VAL219 B:ILE41, B:MET215, Page 12/32 Electrostatic interaction Other A:MET20 A:ASP150 B:MET215 B:MET283 B:MET21, B:MET162 B:CYS220, B:MET283 xystrobin) ence bilurin) BI oviridin- 032A2 tothecin -7.4 -8.5 -10.2 -8.9 glaol -8 ence -7.8 ence -7.4 xystrobin) bilurin) E9G napene- ellin ence xystrobin) ence bilurin) -8.3 -8 -7 -6.7 B:ALA61, B:ASN62, B:SER63, B:MET215, B:GLY36, B:ILE41 B:VAL274 B:ALA61 A:ASN21, A:ARG22, A:TYR99, A:HIS152, A:ARG327 A:MET390, A:HIS181, A:LYS294 B:LYS294, B:ASN391, B:LEU392, B:VAL393, B:VAL287 A:THR46, A:HIS155, A:ASP153, A:TYR99 A:TRP108, A:HIS112, A:HIS152, A:HIS181 A:ARG289, A:LYS294, A:ASP288, A:ASP153 B:ASN100, B:TYR154, B:ARG327, B:ARG22, B:TYR99 A:ALA396, A:ALA399, A:TYR38 A:LYS135, B:LYS135, A:ASN134 A:LYS214 A:LYS214, A:ARG380, B:ASP521 *Active site amino acids are bolded B:ARG190, B:ALA207, B:LYS273, B:ILE165, B:VAL274 A:ASP153 A:LEU392, A:VAL393, A:HIS181 B:LEU371, B:LEU392, B:VAL324 A:HIS152, A:HIS155, A:HIS181, A:PRO24, A:LEU44, A:LEU48, A:ARG22, A:TYR99, A:TRP108 A:HIS181, A:TRP108, A:TYR154, A:HIS155, A:VAL393 A:ASP153 B:TYR99, B:ALA95, B:ARG327, B:TRP62 A:PRO397, A:PRO426, A:TYR38, A:TYR425 A:LYS135, A:HIS138, A:LEU124, B:ALA517, B:VAL520, B:LYS525 A:LEU260, A:LEU124, A:LYS214, A:MET218 Page 13/32 A:ASP118 B:ASP521 Table 2: Type of interactions, interacting residues and bond distance of Scytalone dehydratase (1STD), Trihydroxynaphthalene reductase (1YBV), trehalose-6-phosphate synthase 1 or Tps1 (6JBI) and isocitrate lyase enzyme (5E9G) with their best binding energy compound. Page 14/32 Compounds 1YBV vs. Camptothecin Interacting amino acid residues B:ARG39 B:GLY40 B:ILE41 B:MET162 B:SER164 B:GLY210 Bond distance (Å) 5.35551 3.84091 3.34078 5.34854 3.32811 3.07126 Interaction category Hy Bond Hy Bond Hy Bond Other H Bond H Bond B:MET215 2.23423 H Bond B:THR213 5E9G vs. Arohynapene-B 6JBI vs. GKK1032A2 H Bond B:MET215 B:MET215 B:TYR223 5.16755 3.14003 3.14331 Hy Bond Other H Bond A:TYR50 A:PHE53 A:LEU54 A:VAL70 A:VAL75 A:LEU76 A:MET69 A:HIS85 A:PRO149 A:ILE151 A:PHE158 A:PHE169 A:TYR38 A:TYR38 A:ALA396 5.36041 4.93096 4.45416 4.64779 4.05694 5.39137 4.3566 4.83013 4.08713 4.81867 4.80565 4.43414 3.35874 5.23978 2.1551 Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond H Bond Hy Bond H Bond A:TYR425 A:PRO426 A:HIS181 5.1667 4.49266 1.9225 Hy Bond Hy Bond H Bond A:TYR50 1STD vs. Cryptocin 2.77025 A:PRO397 A:ALA399 A:HIS181 A:LYS294 A:MET390 A:LEU392 A:VAL393 H= Hydrogen, Hy= Hydrophobic 2.95919 4.70296 2.88171 4.23591 3.41725 2.66371 4.53888 5.01831 Page 15/32 H Bond Hy Bond H Bond Hy Bond H Bond H Bond Hy Bond Hy Bond Type of Interaction Pi-Alkyl Pi-Sigma Pi-Sigma Pi-Sulfur Carbon H Bond Conventional H Bond Conventional H Bond Conventional H Bond Alkyl Sulfur-X Conventional H Bond Conventional H Bond Pi-Alkyl Pi-Alkyl Alkyl Alkyl Alkyl Alkyl Alkyl Pi-Alkyl Alkyl Alkyl Pi-Alkyl Pi-Alkyl Carbon H Bond Pi-Alkyl Conventional H Bond Alkyl Conventional H Bond Pi-Alkyl Alkyl Conventional HBond Pi-Alkyl Pi-Cation Conventional H Bond Alkyl Alkyl Table 3: Physicochemical properties of selected potential compound and reference fungicide compound. Compound Alternariol-monomethylether Cryptocin Chaetoviridin-A GKK1032A2 Tanzawaic-acid-L Arohynapene-A Arohynapene-B Camptothecin Rocaglaol Pannellin Tricyclazole HDFO Azoxystrobin Strobilurin MW (g/mol) 272.25 361.48 432.89 503.67 288.38 286.37 286.37 348.35 434.48 506.5 189.24 248.32 403.39 442.5 RB HBA HBD miLogP Lipinski 3 6 1 3 3 4 1 5 6 0 0 8 6 4 6 2 3 3 3 5 6 9 2 3 8 7 1 1 4 2 2 2 1 2 2 0 1 0 1 0.89 3.22 5.08 3.19 3.12 3 2.03 4.48 4.28 2.4 1.51 3.38 4.92 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 1 5 2 2.74 Yes TPSA (Å฀) 79.9 74.68 89.9 75.63 57.53 57.53 57.53 81.2 77.38 112.91 58.43 46.53 103.56 83.45 Synthetic accessibility 2.83 4.76 5.68 7.28 5.16 4.12 4.01 3.84 4.85 5.35 2.44 4.02 3.42 5.6 MW- Molecular weight, RB- Rotatable Bond, HBA- Hydrogen bond acceptor, HBD- Hydrogen bond donor, TPSA- Topological surface area. Table 4: Prediction of bioactivity of the selected compounds and reference fungicide compounds. Compounds Alternariolmonomethyl-ether Cryptocin Chaetoviridin-A GKK1032A2 Tanzawaic-acid-L Arohynapene-A Arohynapene-B Camptothecin Rocaglaol Pannellin Tricyclazole HDFO Azoxystrobin Strobilurin GPCR ligand Ion channel modulator Kinase inhibitor 0.08 0 -0.16 0.17 0.36 0.29 0.46 0.18 0.08 -1.23 0.14 0.25 0.27 -0.25 -0.35 -0.17 0.03 -0.01 0.12 -0.15 -0.01 -0.15 -0.74 0.13 0.03 0 -0.48 -0.44 -0.76 -0.47 -0.17 -0.22 0.27 -0.1 -0.29 -0.73 -0.25 0.09 -0.17 -0.41 -0.44 -0.25 Supplementary Tables Nuclear receptor ligand 0.23 0.2 0.09 0.13 0.61 0.41 0.3 0.07 0.4 0.35 -2.08 0.87 0.25 0.51 Protease inhibitor Enzyme inhibitor 0.24 0.02 0.07 -0.11 -0.05 0.08 -0.1 0.01 -0.04 -1.89 0.03 -0.12 -0.03 0.18 0.28 0.05 0.41 0.29 0.35 1.11 0.1 -0.01 -0.63 0.69 0.19 0.17 -0.48 0.03 Table S1: Type of interactions, interacting residues and bond distance of Scytalone dehydratase (1STD), Trihydroxynaphthalene reductase (1YBV), trehalose-6-phosphate synthase one or Tps1 (6JBI), isocitrate lyase enzyme (5E9G), with the selected fungicide compound. Page 16/32 Compounds 1STD vs. Azoxystrobin 1STD vs. Strobilurin 1YBV vs. Azoxystrobin Interacting amino acid residues A:TYR16 A:MET20 A:MET20 A:VAL23 A:TYR24 A:ALA27 A:GLN84 Bond distance (Å) 5.1848 4.64773 5.62552 5.4992 4.70402 4.6699 3.23819 Interaction category Hy Bond Hy Bond Other Hy Bond Hy Bond Hy Bond H Bond A:ASP150 B:GLY36 4.91626 2.69794 Other H Bond A:ILE87 A:TRP92 A:ILE101 A:TYR103 A:ALA130 A:LEU132 A:TRP134 A:LYS148 B:ARG39 B:ILE41 B:ILE41 B:ALA61 6JBI vs. Azoxystrobin 6JBI vs. 3.00579 2.99912 H Bond B:MET215 3.01993 A:LYS294 2.97898 A:VAL393 B:ARG22 H Bond H Bond Hy Bond H Bond 4.71583 3.15534 B:MET215 B:VAL274 B:ARG190 B:ALA207 B:LYS273 B:ILE165 B:VAL274 A:TRP108 A:ASP153 A:TYR154 A:HIS155 A:HIS181 A:ASP288 A:ARG289 Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond H Bond 4.12944 4.66549 3.12197 B:ALA61 B:ASN62 B:SER63 1YBV vs. Strobilurin 5.4548 4.92997 5.27854 4.86174 3.67326 3.84036 4.93422 2.80766 Hy Bond H Bond H Bond 5.04026 4.10961 5.32698 3.68404 4.84762 4.87589 4.85917 4.70808 3.54525 5.12316 4.74001 3.94673 3.10102 3.13091 Hy Bond H Bond Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond H Bond Hy Bond Hy Bond Hy Bond H Bond H Bond 4.64628 4.17472 Hy Bond H Bond Page 17/32 H Bond Type of Interaction Pi-Alkyl Alkyl Pi-Sulfur Pi-Alkyl Pi-Pi Stacked Pi-Alkyl Conventional H Bond Pi-Alkyl Pi-Alkyl Alkyl Pi-Alkyl Alkyl Pi-Sigma Pi-Alkyl Conventional H Bond Pi-Anion Conventional H Bond Conventional H Bond Pi-Donor H Bond Pi-Alkyl Conventional H Bond Pi-Alkyl Conventional H Bond Conventional H Bond Conventional H Bond Pi-Alkyl Pi-Donor H Bond Alkyl Alkyl Alkyl Alkyl Pi-Alkyl Pi-Pi T-shaped Carbon H Bond Pi-Alkyl Pi-Alkyl Pi-Sigma Carbon H Bond Conventional H Bond Conventional H Bond Pi-Alkyl Pi-Donor H Bond Strobilurin B:TRP62 B:ALA95 B:TYR99 B:TYR99 B:ASN100 4.86579 4.10978 3.94277 5.74107 2.90799 Hy Bond Hy Bond H Bond Hy Bond H Bond B:ARG327 3.02748 H Bond B:TYR154 B:ARG327 5E9G vs. Azoxystrobin 5E9G vs. Strobilurin 2.89275 H Bond 4.56151 Hy Bond A:LEU124 A:LYS214 3.54711 3.1052 Hy Bond H Bond A:LYS214 A:MET218 A:LEU260 A:ARG380 B:ASP521 4.45867 4.54471 4.93381 3.49642 3.61028 Hy Bond Hy Bond Hy Bond H Bond H Bond B:ALA517 B:VAL520 B:ASP521 B:LYS525 A:LEU124 A:LYS214 5.12416 5.25347 3.50407 5.29024 4.76458 3.19943 H= Hydrogen, Hy= Hydrophobic Page 18/32 Hy Bond Hy Bond Other Hy Bond Hy Bond H Bond Pi-Alkyl Alkyl Pi-Donor H Bond Pi-Pi T-shaped Conventional H Bond Conventional H Bond Conventional H Bond Alkyl Pi-Sigma Conventional H Bond Pi-Alkyl Pi-Alkyl Pi-Anion Pi-Alkyl Alkyl Conventional H Bond Alkyl Alkyl Alkyl Carbon H Bond Carbon H Bond Table S2: Type of interactions, interacting residues and bond distance of Scytalone dehydratase (1STD), Trihydroxynaphthalene reductase (1YBV), trehalose-6-phosphate synthase 1 or Tps1 (6JBI), isocitrate lyase enzyme (5E9G), with the other selected compounds. Page 19/32 Compounds 1STD vs. Tanzawaic-acid-L 1STD vs. Camptothecin 1STD vs. HDFO 1YBV vs. Alternariolmonomethyl-ether Interacting amino acid residues A:TYR30 A:TYR50 A:PHE53 A:LEU54 A:VAL70 A:VAL75 A:SER129 Bond distance (Å) 4.57912 4.74722 5.39016 4.22005 4.86485 4.66304 2.46023 Interaction category Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond H Bond A:TYR50 A:PHE53 A:LEU54 A:HIS85 A:PRO149 A:TRP26 A:TYR30 A:TYR50 4.99408 4.0495 4.17104 5.86733 4.97318 5.07574 5.18325 2.91435 Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond H Bond B:LYS182 3.13789 H Bond A:PRO149 A:PHE158 A:PHE162 A:ARG166 A:PHE169 A:TYR50 A:HIS85 A:VAL108 A:HIS110 A:PRO149 A:PHE158 B:ILE41 B:MET162 B:TYR178 B:PRO208 B:PRO208 B:GLY210 B:ILE211 B:THR213 1YBV vs. GKK1032A2 B:MET215 B:MET215 A:ALA15 A:PRO17 A:LYS200 B:LEU246 B:ARG248 A:ASN265 Page 20/32 5.47299 4.66232 3.49342 4.51563 4.9856 3.63955 4.85989 4.00524 4.39831 4.84767 5.31703 4.74505 5.26285 3.16042 2.24883 4.76055 3.32215 3.9343 2.95007 3.68835 4.10695 5.06932 5.2637 5.3372 4.53376 3.92783 2.36537 Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond H Bond Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond H Bond H Bond Hy Bond H Bond Hy Bond H Bond Hy Bond Other Hy Bond Hy Bond Hy Bond Hy Bond Hy Bond H Bond Type of Interaction Pi-Alkyl Pi-Alkyl Pi-Alkyl Alkyl Alkyl Alkyl Conventional H Bond Alkyl Pi-Alkyl Pi-Alkyl Alkyl Pi-Alkyl Pi-Donor H Bond Pi-Alkyl Pi-Alkyl Alkyl Pi-Pi T-shaped Alkyl Pi-Alkyl Pi-Alkyl Conventional H Bond Pi-Alkyl Alkyl Pi-Alkyl Alkyl Pi-Alkyl Alkyl Pi-Alkyl Conventional H Bond Conventional H Bond Conventional H Bond Pi-Alkyl Conventional H Bond Alkyl Conventional H Bond Pi-Sigma Pi-Sulfur Alkyl Alkyl Alkyl Alkyl Alkyl Conventional H Bond 1YBV vs. Arohynapene-A 1YBV vs. Tricyclazole B:ILE211 B:THR213 5.15363 2.7626 Hy Bond H Bond B:TYR223 B:TRP243 B:MET283 B:SER164 3.32618 4.58665 5.79345 1.80365 Hy Bond Hy Bond Other H Bond B:GLY209 4.39859 Hy Bond B:MET215 B:VAL219 B:CYS220 B:TYR223 4.18564 5.30239 4.89439 3.57611 Hy Bond Hy Bond Other H Bond B:MET215 B:TYR216 B:VAL219 B:CYS220 B:TYR223 B:TYR178 B:GLY210 6JBI vs. Chaetoviridin-A 2.11557 4.39859 B:TYR223 B:MET283 A:ASN21 4.03173 5.37817 2.97488 A:TYR99 3.01488 A:HIS152 A:ASP153 A:HIS181 A:ARG327 4.13512 3.2513 5.38791 3.15163 A:ARG22 A:TRP108 A:HIS112 A:HIS152 6JBI vs. Camptothecin 4.21542 4.45903 5.34854 5.09712 4.00308 3.06606 Hy Bond Hy Bond Hy Bond Hy Bond H Bond H Bond Hy Bond Hy Bond Other H Bond H Bond H Bond 4.61561 4.79727 3.11406 Hy Bond Hy Bond HBond B:VAL287 3.57577 H Bond B:VAL324 B:LEU371 B:ASN391 5.34311 4.74267 3.19834 Hy Bond Hy Bond H Bond B:LEU392 B:VAL393 4.8772 3.36434 Hy Bond H Bond B:LYS294 B:LEU392 Page 21/32 2.91239 3.16196 Hy Bond Other Hy Bond H Bond H Bond H Bond Alkyl Conventional H Bond Alkyl Pi-Alkyl Alkyl Alkyl Pi-Donor H Bond Pi-Sigma Pi-Alkyl Pi-Sulfur Conventional H Bond Conventional H Bond Amide-Pi Stacked Amide-Pi Stacked Alkyl Alkyl Pi-Sulfur Pi-Donor H Bond Pi-Pi Stacked Pi-Sulfur Conventional H Bond Conventional H Bond Conventional H Bond Pi-Alkyl Pi-Alkyl Conventional H Bond Pi-Alkyl Pi-Anion Pi-Alkyl Conventional H Bond Carbon H Bond Conventional H Bond Pi-Alkyl Alkyl Conventional H Bond Conventional H Bond Alkyl Conventional 6JBI vs. Rocaglaol A:ARG22 A:PRO24 A:LEU44 A:THR46 4.67738 4.62125 4.28721 2.99097 Hy Bond Hy Bond Hy Bond H Bond A:TYR99 A:TRP108 A:HIS152 A:ASP153 A:ASP153 A:HIS155 4.92019 4.90005 3.75287 3.68267 4.60817 3.31986 Hy Bond Hy Bond Hy Bond H Bond Other H Bond A:LEU48 A:TYR99 A:HIS155 A:HIS181 5E9G vs. Pannellin H= Hydrogen, Hy= Hydrophobic 4.95312 3.6587 5.01862 5.12842 A:ASP118 A:ASN134 4.73743 3.66467 B:LYS135 2.87842 A:LYS135 A:LYS135 A:HIS138 3.09262 4.5186 4.53618 Hy Bond H Bond Hy Bond Hy Bond Other H Bond H Bond H Bond Hy Bond Hy Bond H Bond Alkyl Alkyl Alkyl Conventional H Bond Alkyl Carbon H Bond Pi-Alkyl Pi-Alkyl Pi-Pi Stacked Carbon HBond Pi-Anion Conventional H Bond Pi-Pi Stacked Pi-Pi Stacked Pi-Anion Carbon H Bond Conventional H Bond Conventional H Bond Alkyl Pi-Alkyl Declarations Ethical approval Not applicable. Funding This study received no nancial support. Con icts of interests The authors declare no competing interests. Data availability The sequence data reported in this article are available in the FungiDB database (https://fungidb.org/fungidb/app/record/dataset/DS) and Protein Data Bank (https://www.rcsb.org/). Page 22/32 Acknowledgements The authors would like to thank those who provided us the samples. Supplementary information Supplementary information supporting the results of the study are available in this article as Table S1 and S2. Authors’ contributions M.A.K. and T. I. conceived and designed the study. M.A.K., M.A.A.M.K., J.M.S. and A.A. executed the bioinformatics analysis, interpreted the results and drafted the manuscript. D.R.G., M.N.H. and T.I. contributed intellectually to the interpretation and presentation of the results. Finally, all authors have approved the manuscript for submission. References 1. 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(B) Interaction between compound HDFO and Scytalone dehydratase illustrated. 2D interaction analysis shown at left and 3D interaction analysis shown at right side. Page 28/32 Figure 2 (A) Interaction between Trihydroxynaphthalene reductase and compound Alternariol-monomethyl-ether illustrated. (B) Camptothecin docked in complex with Trihydroxynaphthalene reductase (PDB ID: 1YBV); 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. 2D interaction analysis shown at left and 3D interaction analysis shown at right side. Page 29/32 Figure 3 (A) GKK1032A2 Docked in complex with trehalose-6-phosphate synthase 1 or Tps1 (PDB ID: 6JBI); Hydrogen bonds favor the docking interactions of GKK1032A2 with A:MET390, A:HIS181, and A:LYS294, while nonbonded hydrophobic interactions are favored by A:LEU392, A:VAL393, and A:HIS181. (B) Interaction between trehalose-6-phosphate synthase 1 or Tps1 and compound Camptothecin illustrated. 2D interaction analysis shown at left and 3D interaction analysis shown at right side. Page 30/32 Figure 4 (A) Arohynapene-B docked in complex with isocitrate lyase enzyme (PDB ID: 5E9G); The binding conformations were analyzed, and we identi ed 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. (B) 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. 2D interaction analysis shown at left and 3D interaction analysis shown at right side. Page 31/32 Figure 5 Two-dimentional (2D) chemical structure of the 12 top ranked selected compounds. Page 32/32