*6. In silico* **molecular mechanism of action of fungicidal compounds**

Computational biology has now become an indispensable part in research to understand the biological process in a better way at a short period. *In silico* molecular docking is an application area in bioinformatics, which studies the interaction between the molecules by fitting the molecules in a 3D space. The interactions between protein-protein, protein – DNA, protein – small molecule (drug) and even within carbohydrates and lipid molecules can be studied using docking tools.

Docking tools are automated, available both in online and offline, most of the offline tools are commercial tools. Each tool has its own advantage. Some of the online tools are patch dock (bioinfo3d.cs.tau.ac.il/PatchDock/), Zdock server (zdock.bu.edu/), Dock blaster (blaster.docking.org/). Few non commercial docking tools are Hex, Autodock, Dock, MSdock and few commercial tools are Flexidock, GOLD, HADDOCK.

Molecular docking studies can also be performed for fungicides. As fungicides are the chemical compounds used to kill or inhibit fungi or fungal spores. The fungicides act primarily by inhibiting any of the process: electron transport chain, nucleic acid synthesis, mitosis and cell division, protein synthesis, lipid and membrane synthesis, sterol biosynthesis. The enzyme involved in any of the above mentioned process can be considered as a target receptor and the fungicide as ligand.

In case of a fungicide that targets sterol biosynthesis (DM inhibitors), CYP 51 enzyme (14 –α demethylase) involved in the ergosterol biosynthesis can be chosen as a target (Yang, *et al.,* 2009). The docking studies require the 3D structure of the target enzyme. If the 3D structure of the target enzyme is not already available, then homology modeling can be performed.

The docking tools calculate the binding energy between the fungicide and the enzyme target. The binding energy is denoted as E value. The E value for the docked complex should be more negative. The more negative the E value, more stable the docking complex formed.

action and is useful in treating cases of Candidiasis, extracutaneous sporotrichosis,

Igarashi *et al*. (2003) screened for novel antifungal compound, Yatakemycin from the *Streptomyces* species TP – AO 356. Yatakemycin were obtained by solvent extraction of the fermentation broth and preparative HPLC. NMR elucidated the structure of Yatakemycin and CID – MS/MS experiments as a novel antibiotic belonging to a family of CC – 1065 and duocarmycins known to be DNA alkylating agents. Yatakemycin inhibited the growth of pathogenic fungi such as *Aspergillus fumigatus* and *Candida albicans* with the MIC values of 0.01 – 0.03 μg/ml more potent than amphotericin B (MIC 0.1 – 0.5 μg/ml). It also showed

Datta *et al*. (2001) studied the Ju-2 a novel phosphorous-containing antifungal antibiotic from *Streptomyces kanamyceticus* M8. Ellaiah *et al.* (2005) studied the chracteristics of oligosaccharide antibiotic by 1H NMR and 13C NMR spectra and elucidated the structural formula as C14H86O17. Separation, purification and structural elucidation of Irumamycin and 17-hydroxy-venturicidin were established by IR, ESI-MS, 1H and 13C NMR data (Fourati *et al.,* 2005). The numerous fungicidal compounds from actinobacterial genera are summarized

Computational biology has now become an indispensable part in research to understand the biological process in a better way at a short period. *In silico* molecular docking is an application area in bioinformatics, which studies the interaction between the molecules by fitting the molecules in a 3D space. The interactions between protein-protein, protein – DNA, protein – small molecule (drug) and even within carbohydrates and lipid molecules

Docking tools are automated, available both in online and offline, most of the offline tools are commercial tools. Each tool has its own advantage. Some of the online tools are patch dock (bioinfo3d.cs.tau.ac.il/PatchDock/), Zdock server (zdock.bu.edu/), Dock blaster (blaster.docking.org/). Few non commercial docking tools are Hex, Autodock, Dock, MS-

Molecular docking studies can also be performed for fungicides. As fungicides are the chemical compounds used to kill or inhibit fungi or fungal spores. The fungicides act primarily by inhibiting any of the process: electron transport chain, nucleic acid synthesis, mitosis and cell division, protein synthesis, lipid and membrane synthesis, sterol biosynthesis. The enzyme involved in any of the above mentioned process can be

In case of a fungicide that targets sterol biosynthesis (DM inhibitors), CYP 51 enzyme (14 –α demethylase) involved in the ergosterol biosynthesis can be chosen as a target (Yang, *et al.,* 2009). The docking studies require the 3D structure of the target enzyme. If the 3D structure of the target enzyme is not already available, then homology modeling can be performed.

The docking tools calculate the binding energy between the fungicide and the enzyme target. The binding energy is denoted as E value. The E value for the docked complex should be more negative. The more negative the E value, more stable the docking complex

Mucormycosis and some cases of hyalohyphomycosis and Phaeohyphomycosis.

potent cytotoxicity against cancer cell lines with the IC50 of 0.01 – 0.3 μg/ml.

*6. In silico* **molecular mechanism of action of fungicidal compounds** 

dock and few commercial tools are Flexidock, GOLD, HADDOCK.

considered as a target receptor and the fungicide as ligand.

in the Table. 3

formed.

can be studied using docking tools.


Table 3. Fungicidal secondary metabolites produced by actinobacteria

Applications of Actinobacterial Fungicides in Agriculture and Medicine 45

*In silico* tools are fruitful to gain insight into the mode of action of fungicides and help the

research to move a step ahead leading to the rational drug discovery.

Fig. 4. Cytochrome 51 docking complex with a fungicidal compound(4P1NPA)

Fig. 5. H bond formation between cytochrome 51 and fungicidal compound(4P1NPA)

Computational advancement also provides way to modify the functional group of the fungicide, leading to the creation of analogues of the fungicide. These docking approaches provide insight into the structure based drug designing. Structure based drug designing assist in the creation of novel fungicides that can be used to treat already existing drug resistant fungal pathogens.

The interaction between the fungicide and the enzyme target will always by hydrogen bond formation between electropositive and electronegative atom. For an enzyme target to get disrupted, the H bond formation of the fungicide should be within the active site of the target.

The knowledge about the active site of the target enzyme can be obtained using online active site predicting tools. Few such tools include: CASTp – Computed Atlas of Surface Topography of proteins (http://sts.bioengr.uic.edu/castp/calculation.php), Q-site finder (http://www.modelling.leeds.ac.uk/qsitefinder/), and Pocket finder (http://www.modelling.leeds.ac.uk/pocketfinder/).

The antifungal compound isolated from the marine *Streptomyces* sp. DPTB16 was characterized as 4-Phenyl-1-Napthyl Phenyl Acetamide and its 1D structure was also elucidated via spectral analysis (Fig.2). The structure of the compound was submitted to Pubchem compound database with accession number CID: 49786168 (Fig.3,4,5).

Fig. 2. The elemental representation of 4 - Phenyl 1-Napthyl Phenyl Acetamide

Fig. 3. The 3D structure of 4P1NPA in Pubchem compound database (CID: 49786168)

Computational advancement also provides way to modify the functional group of the fungicide, leading to the creation of analogues of the fungicide. These docking approaches provide insight into the structure based drug designing. Structure based drug designing assist in the creation of novel fungicides that can be used to treat already existing drug

The interaction between the fungicide and the enzyme target will always by hydrogen bond formation between electropositive and electronegative atom. For an enzyme target to get disrupted, the H bond formation of the fungicide should be within the active site of the

The knowledge about the active site of the target enzyme can be obtained using online active site predicting tools. Few such tools include: CASTp – Computed Atlas of Surface Topography of proteins (http://sts.bioengr.uic.edu/castp/calculation.php), Q-site finder (http://www.modelling.leeds.ac.uk/qsitefinder/), and Pocket finder

The antifungal compound isolated from the marine *Streptomyces* sp. DPTB16 was characterized as 4-Phenyl-1-Napthyl Phenyl Acetamide and its 1D structure was also elucidated via spectral analysis (Fig.2). The structure of the compound was submitted to

Pubchem compound database with accession number CID: 49786168 (Fig.3,4,5).

Fig. 2. The elemental representation of 4 - Phenyl 1-Napthyl Phenyl Acetamide

Fig. 3. The 3D structure of 4P1NPA in Pubchem compound database (CID: 49786168)

resistant fungal pathogens.

(http://www.modelling.leeds.ac.uk/pocketfinder/).

target.

*In silico* tools are fruitful to gain insight into the mode of action of fungicides and help the research to move a step ahead leading to the rational drug discovery.

Fig. 4. Cytochrome 51 docking complex with a fungicidal compound(4P1NPA)

Fig. 5. H bond formation between cytochrome 51 and fungicidal compound(4P1NPA)

Applications of Actinobacterial Fungicides in Agriculture and Medicine 47

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#### **7. Conclusion**

Actinobacteria isolated from marine habitat have potential for novel fungicidal compounds. The significance of finding actinobacteria in marine soil sample lies in the intrinsic economic importance in biotechnological perspectives. The chapters reinforce the view that the unexplored actinobacteria for bioprospecting novel fungicidal compound in the development of Biocontrol agents and formulations of drugs. Further investigation should address the relationship between structure relationship activity of fungicidal compounds, rapid methods for large scale production, purification and application in managing fungal infection in agriculture crops, human and animals.

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**3** 

*USA* 

**Naturally Occurring Antifungal** 

**Agents and Their Modes of Action** 

Yeast fermentations are involved in the manufacturing of foods such as bread, beer, wines, vinegar, and surface ripened cheese. Most yeasts of industrial importance are of the genus *Saccharomyces* and mostly of the species *S. cerevisiae*. These ascospore-forming yeasts are readily bred for desired characteristics. However, yeasts are undesirable when they cause spoilage to sauerkraut, fruit juices, syrups, molasses, honey, jellies, meats, wine, beer, and other foods (Frazier and Westhoff, 1988). Finishing process of the fermentation is usually either through filtration or pasteurization. However, the use of the latter is limited to certain foods since it is a heat treatment and hence denaturalizes proteins, and the former is also limited to clear liquids. Neither process can be applicable to some foods such as sauerkraut and "miso" (soy bean pastes). *Zygosaccharomyces bailii*, is a food spoilage yeast species. It is known for its capacity to survive in stress environments and, in particular, in acid media with ethanol, such as in wine. In addition, spoilage of mayonnaise and salad dressing by this osmophilic yeast is

In our continuing search for naturally occurring antimicrobial agents, a bicyclic sesquiterpene dialdehyde, polygodial (**1**) (see Figure 1 for structures), was isolated from various plants (Kubo, 1995). This sesquiterpene dialdehyde exhibited potent antifungal activity particularly against yeasts such as *Saccharomyces cerevisiae* and *Candida albicans*  (Taniguchi et al., 1988), although it possessed little activity against bacteria (Kubo et al., 2005). Because of the potent antifungal activity, polygodial can be used as a leading compound to search for new antifungal drugs. This involves the study of their structure and antifungal activity relationships (SAR). However, the study of SAR required the synthesis of a series of analogues differing in the hydrophobic bicyclic portion, and because of this,

Subsequently, 2*E*-alkenals and alkanals were characterized from various edible plants such as the coriander *Coriander sativum* L. (Umbelliferae) (Kubo et al., 2004), the olive *Olea europaea* L. (Oleaceae) (Kubo et al., 1995a; Bisignano et al., 2001) and the cashew *Anacardium occidentale* (Anacardiaceae) (Muroi et al., 1993), and these aldehyde compounds exhibited broad antimicrobial activity (Table 1) (Kubo et al., 1995b). The maximum antimicrobial activity of 2*E*alkenals is dependent on the balance of the hydrophobic alkyl (tail) chain length from the hydrophilic aldehyde group (head) (Kubo et al., 1995b and 2003a). The hydrophobicity of

well described. Therefore, safe and effective antifungal agents are still needed.

polygodial may not be practical to use as a leading compound.

**1. Introduction** 

Isao Kubo, Kuniyoshi Shimizu and Ken-ichi Fujita *Department of Environmental Science, Policy and Management,* 

*University of California, Berkeley, California* 

