In silico screening of phytogenic compounds against Rhizoctonia solani trehalase enzyme

The trehalase enzyme of Rhizoctonia solani is the target site for validamycin, a common fungicide used for controlling the sheath blight disease of rice. However, rampant use of validamycin has resulted in emergence of fungicide resistance necessitating the search for newer fungicide molecules. Thus, molecular docking analysis was carried out to screen phytogenic compounds with high trehalase inhibitory effect. The 3-dimensional structure of the protein was generated by Swiss-model using the sequence information available in UniProt database (entry no. L8WUM1). Eighteen compounds from plants previously reported to have antagonistic effect against R. solani were selected for the study. Molecular docking carried out by Autodock 4.2 showed that the compounds, Cycloartenol (− 8.64), β-Sitosterol (− 8.58), Nimbiol (− 8.29), Nimbandiol (− 7.32), Menthone (− 7.31), Nimbin (− 7.22) had high binding energies whereas validamycin had a binding energy of − 4.08. Among these, Nimbiol, Nimbandiol, Menthone, Nimbin were obeying all the Tice rule criteria and appeared to be good fungicide candidates against R. solani.


Introduction
Rice is the staple food for about one-fifth of world population (FAOSTAT 2017). The ever increasing population requires increased rice production. However, the rice diseases are among the major stumbling blocks for increased production. Among the diseases, the sheath blight disease caused by Rhizoctonia solani has been causing considerable yield loss which may go upto 50% (Yugander et al. 2015;DRR 2017).
The disease is mostly controlled by application of fungicides like validamycin, propiconazole, carbendazim, etc. (Molla et al. 2020). However, because of rampant use of these chemicals, chances of development of resistance of pathogens towards them have increased. In addition, there are environmental concerns associated with the use of synthetic pesticides. Therefore, efforts are being made to search biogenic compounds that could be used for managing plant diseases and pests. Molecular docking has emerged as a useful tool for screening compounds with drug/pesticidal potential. This tool considerably minimizes the time required for searching drug/pesticide candidates (Kurbanova et al. 2022). In this context, molecular docking analysis was carried out to screen phytogenic compounds against the trehalase enzyme of Rhizoctonia solani. The trehalase enzyme in fungi is responsible for the degradation of trehalose, which in turn acts as a reactive oxygen species (ROS) scavenger (Wang et al. 2018). It is also the target molecule for antifungal agents like validamycin and thus was selected as the target molecule in the present study.

Protein structure generation and validation
The protein structure for the trehalase enzyme of Rhizoctonia solani is not available in the protein data bank. Therefore, the amino acid sequence for the protein was retrieved from the SwissProt database in FASTA format. The 3-dimensional structure of the protein was generated using Swiss-model online server (https:// swiss model. expasy. org/). "Automodel" option was selected for the model generation which used neutral trehalase of Saccharomyces cerevisiae as template (sequence identity of 52.89% and a Global Model Quality Estimation (GMQE) value of 0.43). The PRO-CHECK Ramachandran plot was used for model validation.

Physico-chemical properties analysis, binding site prediction
The protein model generated by Swiss-model server was saved in.pdb format and it was used for physico-chemical property analysis using Protparam online (https:// web. expasy. org/ cgi-bin/ protp aram/ protp aram). The CastP server (http:// sts. bioe. uic. edu/ castp/ index. html? 1bxw) was used for determining the binding pockets for the modeled protein.
The largest positive patch in the protein was determined using PatchFinderPlus server (http:// pfp. techn ion. ac. il/).

Compound selection
Extracts from plants like Withania somnifera, Ziziphus jujuba, Azadirachta indica, Mentha piperita, Ocimum basilicum, Eucalyptus, Juniperus polycarpus, Juniperus sabina have been found to have antifungal activities against Rhizoctonia solani (EL-Hefny et al. 2020;Kagale et al. 2011;Ali et al. 2017; Abd-El-Khair and El-Gamal Nadia 2011; Khani et al. 2017). It was hypothesized that some of the compounds present in these plant extracts could be responsible for the antifungal activity of the extracts. Thus, the principal constituents of the plant extracts were considered for the study. Compounds like Rutin, Kaempferol are constituents of W. somnifera; β-Sitosterol, Stigmasterol, d-5-avenasterol, Squalene, Cycloartenol are constituents of Z. jujuba; Nimbiol, Nimbin, Gedunin, Nimbandiol, Nimbolide are constituents of A. indica; Menthol, Menthone, Limonene are found in Mentha piperita; Linalool, Eugenol are present in Ocimum basilicum, and a-Pinene is among the principal constituents of Eucalyptus, J. polycarpus, and J. sabina. Along with these compounds, Validamycin which is a known inhibitor of trehalase was included in the study. Propiconazole, another fungicide, recommended for controlling the disease was also included in the study. Three dimensional structures of these compounds in.sdf format were downloaded from PubChem database (https:// pubch em. ncbi. nlm. nih. gov/) and were converted to pdbqt format using Openbabel software for docking.

Molecular docking
Molecular docking was performed using Autodock 4.2 software following methodology published earlier (Mahanty et al. 2021;Sidhu et al. 2020;Kumar et al. 2021). Briefly, Auto-grid program was used to generate the grid maps. The grid dimensions were 90 A° with points separated by 0.375 A°. The grid was centered at − 32.584, − 0.973, − 5.096 to encompass entire active site.

Pesticide likeliness test
The pesticide likeliness was computed only for the compounds which had high binding energy using the online tool available at http:// www. molin spira tion. com (Mahanty et al. 2021;Yadav et al. 2014).

Protein structure generation and validation
The sequence retrieved from the SwissProt database was found to be having 774 amino acids. The 3-dimensional structure of the protein was generated using the Swiss-model tool (Fig. 1a) and it was validated using Ramachandran plot. It showed that 86.5% of the amino acid lied in the most favored region, additionally 11.5% was in the allowed region suggesting the validity of the model. The Ramachandran plot for the modeled protein is presented in Fig. 2.

Physico-chemical properties analysis, binding site prediction
ProtParam online server was used for determining the physico-chemical properties of the protein. The molecular weight of the protein was calculated to be 88,550.10 Da and the theoretical pI was calculated to be 5.73. The estimated half-lives of the protein in different organisms are as follows: 30 h (mammalian reticulocytes, in vitro), > 20 h (yeast, in vivo), > 10 h (Escherichia coli, in vivo) which indicate the stability of the protein. The CastP program predicted the presence of five pockets with area > 100. However, the largest pocket was found to be having an area of 3586.440 and volume 3323.21. This binding pocket which fell in between the amino acids ILE-134 and TRP-655 was used for the grid preparation (Fig. 1b).

Molecular docking
Molecular docking analysis showed the binding energies of the compounds. Table 1 presents the binding energies of different compounds along with the number of hydrogen bonds formed and the amino acids involved in it. Among the phytogenic compounds, Cycloartenol (− 8.64), β-Sitosterol (− 8.58), Nimbiol (− 8.29), Nimbandiol (− 7.32), Menthone (− 7.31), Nimbin (− 7.22), Nimbolide (− 7.16) were found to be having high binding affinity. Validamycin which is a known trehalase inhibitor had a binding energy of − 4.08. Similarly, Propiconazole which is recommended as a fungicide to control the disease was found to be having a binding energy of − 5.44. This shows that the afore-mentioned compounds have higher affinity for the trehalase enzyme compared to validamycin.

Pesticide potency
The pesticide potency of the compounds with high binding energy was evaluated by the online server described in materials and methods section. The server works on the principle of Tice rule according to which, the chemicals to be used as pesticides should have the following properties; molecular weight ≤ 500 g/mol, no. of hydrogen-bond donors (OH + NH) ≤ 3, no. of hydrogen-bond acceptors ≤ 12 (O + N), logP (logarithm of octanol/water partition coefficient) ≤ 5 and the number of rotatable bonds ≤ 12 (Tice 2001). The compounds Cycloartenol and β-Sitosterol which were having the highest binding energies were found to be having one violation to the Tice rule (logP > 5). The other compounds with high binding energies like Nimbiol, Menthone, Nimbin, and Nimbolide were meeting all the criteria set by the Tice rule (Table 2) (Tice 2001).

Potential pesticide candidate
Trehalose is a disaccharide found in fungi and insects which is synthesized under stress condition. When the cells return to normal state, trehalose gets degraded by the trehalase enzyme into two glucose molecules. Thus, the dynamics of trehalose and trehalase play an important role in the physiology of the fungus and trehalose inhibitors like Validamycin are used for controlling the fungal diseases. Validamycin works as a competitive or non-competitive inhibitor of trehalase (García and Argüelles 2021;Wang et al. 2022). Although, the exact binding site of trehalase to which Validamycin attaches has not been structurally elucidated, the present study showed that the amino acids GLU-269, LEU-238,  are involved in the H-bonding. In the present study, a number of compounds like Cycloartenol, Beta-sitosterol, Nimbiol, Nimbandiol, Menthone, and Nimbin had higher binding energies than Validamycin indicating that like Validamycin, these could also inhibit the activity of trehalase. Out of these compounds, Nimbiol, Menthone, Nimbin, and Nimbolide also met all the criteria set by Tice rule and thus appear to be better candidates to control the sheath blight disease of rice. The compounds Nimbiol, Nimbin, and Nimbolide are active compounds present in extracts of neem plant (Azadirachta indica) whereas menthone is found in Mentha piperita. These plants are readily available in the tropic to subtropical countries and could be used for extraction of the compounds which could lead to production of economical and environment friendly fungicides for controlling the sheath blight disease of rice.
This preliminary investigation identified the compounds which have the potential to inhibit the growth of R. solani the causal agent of sheath blight disease. However, in vitro antifungal assays such as the "poisoned food method" needs to be employed for validating the findings of the present study (Adak et al. 2020;Thankaraj et al. 2022). Further, in vivo studies in pot cultures and field condition need to be carried out to see if these compounds can really control the disease. Moreover, the mechanism through which these compounds could be inhibiting the activity of trehalase enzyme needs to be investigated.