Fusarium head blight (FHB) is a destructive disease of wheat and other cereals. FHB occurs in Europe, North America and around the world causing significant losses in production and endangers human and animal health. In this article, we provide the strategic steps for the specific target selection for the phytopathogen system wheat-Fusarium graminearum. The economic impact of FHB leads to the need for innovation. Currently used fungicides have been shown to be effective over the years, but recently cereal infecting Fusaria have developed resistance. Our work presents a new perspective on target selection to allow the development of new fungicides. We developed an innovative approach combining both genomic analysis and molecular modeling to increase the discovery for new chemical compounds with both safety and low environmental impact. Our protein targets selection revealed 13 candidates with high specificity, essentiality and potentially assayable with a favorable accessibility to drug activity. Among them, three proteins: trichodiene synthase, endoglucanase-5 and ERG6 were selected for deeper structural analyses to identify new putative fungicides. Overall, the bioinformatics filtering for novel protein targets applied for agricultural purposes is a response to the demand for chemical crop protection. The availability of the genome, secretome and PHI-base allowed the enrichment of the search that combined experimental data in planta. The homology modeling and molecular dynamics simulations allowed the acquisition of three robust and stable conformers. From this step, approximately ten thousand compounds have been virtually screened against three candidates. Forty-five top-ranked compounds were selected from docking results as presenting better interactions and energy at the binding pockets and no toxicity. These compounds may act as inhibitors and lead to the development of new fungicides.
Fusarium Head Blight disease (FHB), also known as scab, leads to significant losses in crop yield, low grain weights, low seed germination and contamination of grain with mycotoxins. Several species of Fusarium can cause FHB. Among these, F. graminearum (teleomorph Gibberella zeae (Schwein.) Petch), infects different parts of the plant. Fusarium penetrates through the stomata to the palea and lemma and destroys these tissues. The disease has a symptomless phase where the hyphae advance between the plant cells prior to host cell death [
Synthetic fungicides are globally used to control plant diseases. According to Cools and Hammond-Kosack (2013) 95.5% of the wheat-growing area in UK is receiving fungicide sprays [
Searching for novel targets to control wheat head blight disease remains a major challenge [
At the early phase of infection, fungal growth is symptomless representing the majority of mycelium colonizing the host tissues. Secondly the hyphal colonization is observed where the plant cell collapses. During the infection process the fungus maintains living host cells, probably with the expression of various secreted proteins, that might increase during the progress of the infection [
Data set | Target | FGSG locus number | Putative protein function | Phenotype in PHI-base | Cell localization | #copies in Fusarium genome | Protein size (aa) |
---|---|---|---|---|---|---|---|
EST analysis | 1 | FGSG_03537 | trichodiene synthase | Mixed outcome | cytoplasm* | 1 | 375 |
2 | FGSG_05906 | triacylglycerol lipase precursor | Reduced virulence | cytoplasm | 2* | 349 | |
PHI-base | 3 | FGSG_08731 FGSG_16980 | casein kinase | Lethal | cytoplasm | 2* | 371 |
Refined secretome | 4 | FGSG_03795 | endoglucanase 3 | Not in PHI-base | unknown | 1 | 382 |
5 | FGSG_00989 | rhamnogalacturonase B | Not in PHI -base | unknown | 1 | 540 | |
6 | FGSG_08196 | aspergillopepsin-2 | Not in PHI-base | cell periphery! | 1 | 260 | |
7 | FGSG_11048 | arabinogalactan | Not in PHI-base | extracellular | 1 | 350 | |
8 | FGSG_03813 | arabinofuranosidase | Not in PHI-base | extracellular | 1 | 499 | |
9 | FGSG_03628 | exoglucanase-6A | Not in PHI-base | extracellular | 1 | 458 | |
10 | FGSG_02658 | endoglucanase-5 | Not in PHI-base | extracellular! | 1 | 380 | |
11 | FGSG_11190 | guanyl-specific ribonuclease | Not in PHI-base | extracellular! | 1 | 132 | |
Known targets | 12 | FGSG_02783 FGSG_16532 | D-(24)-sterol C-methyltransferase | Not in PHI-base | cytoplasm | 2* | 381 |
13 | FGSG_09530 FGSG_06611 | beta-tubulin | Not in PHI-base | cytoplasm | 2 | 446 447 |
*Number of copies showed below, !validated by proteome experiments; (PHI-base reference number 1 PHI: 44; 2 PHI:432; 3 PHI:1235).
and of the 5,094 from Fusarium graminearum (named subgroup EST FG) are described in
The selection strategy is summarized in
From 19,892 sequences only 2835 candidates were retained after considering protein annotation, phenotype description and expression in planta. Followed by the filtering for redundancy, cell localization and accessibility (cytoplasmic proteins), only 92 satisfied the criteria. Among them, 30 had a low number of copies in the genome and were considered small proteins. The final filter produced a set of 13 candidates, which had no orthologues within non host genomes. Among them, only three were finally cho- sen for the 3D modeling phase, namely trichodiene synthase, endoglucanase-5 and the ERG6 sterol C-methyltransferase. These proteins were considered as potential targets
Locus number | Function | Genbank accession | Fungi | Ortholog | Reference | ||
---|---|---|---|---|---|---|---|
Plants | Insects | human | |||||
FGSG_02658 | Endoglucanase-5 | XP_382834.1 | F. pseudograminearum F. fujikuroi F. oxysporum F. verticillioides Magnaporthe oryzae Melanocarpus albomyces | None | None | None | [ |
FGSG_03537 | Trichodiene synthase-TRI5 | ESU09673.1 | F. culmorum F. mesoamericanum F. austroamericanum F. boothii F. asiaticum F. pseudograminearum F. cerealis F. meridionale F. lunulosporum F. sambucinum F. sporotrichioides | None | None | none | [ |
FGSG_02783 | Sterol C-methyltransferase-ERG6 | XP_382959.1 | F. pseudograminearum F. avenaceum F. fujikuroi F. oxysporum F. verticillioides | None | None | None | [ |
representing a group of essential genes, for life or virulence on wheat, with a broad diversity of functions and cellular processes. The three potential targets are significant, unique and have a single function for the pathogen; all are expressed only by the pathogen and are assayable. Therefore, these candidates were selected as potentially and readily inhibited by low molecular weight compounds.
The gene FGSG 02658 is located on chromosome 1 and is predicted to encode an endoglucanase type K protein (E.C. number 3.2.1.4.), belonging to the cellulase class that catalyze the hydrolysis of the β-(1,4) glycosidic bonds of cellulose that primarily hydrolyses less well-ordered regions of cellulose by cutting at internal glycosidic bonds. Cellulases play an important role during infection, enabling the degradation of plant cell walls, pathogen penetration and growth through plant tissue [
Protein sequence alignments between Fusarium endoglucanase FGSG 02658 and orthologs show high similarity with a well-defined conserved domain glycosyl hydrolase family 61 (GH61). To obtain a 3D homology model corresponding to the protein from Fusarium FGSG 02658 sequence (residue 1 to 380) was necessary to use complementary protocols for molecular modeling. Complete models were proposed by the HHPRED (one model), PHYRE2 (one model), TASSER (5 models) and ROBETTA (one model) server (
Method | Number of proposed models | Length of the model sequence | PDB templates used |
---|---|---|---|
MODWEB | 4 | 22 - 229 | 1L8F |
21 - 227 | 2ENG | ||
344 - 378 | 1AZJ | ||
346 - 378 | 4BMF | ||
HHPRED | 1 | 1 - 380 | 1AZ6 |
M4T | 1 | 20 - 230 | 10AZ 1HD5 |
PHYRES2 | 1 | 1 - 380 | 1L8F 2ENG 4BMF 1CBH |
SwissProt | 1 | 20 - 227 | 1L8F |
TASSER | 5 | 1 - 380 1 - 380 1 - 380 1 - 380 1 - 380 | 3ENG 3CM9 1W0S 1W0R 2OCN |
ROBETTA | 1 | 1 - 380 | 3ENG 3IOX 4BMF |
quence (residues 20 - 220) is very similar in all predictions (and even with the predictions not retained for the incomplete sequence), an enormous diversity of proposals is given. These models were all submitted to a short 10 ns of molecular dynamics (MD) simulation to check their conformational changes compared to the starting homology structures. According to our selection procedure, the model given by the Robetta server appeared to be the most suitable and was the most stable from short 10 ns of MD simulations. Consequently, this last model (
The trichodiene synthase gene (TRI5) is located on chromosome 2, locus FG03537.1
described as TRI5 GIBZE Trichodiene synthase (sesquiterpene cyclase) (TS). It contains two exons and encodes for a protein with 375 amino acids (XP 383713). Recently, the TRI5 gene was studied in terms of phylogenetic relationships among Fusarium chemotypes and reported as having a highly conserved organization and a common expression pattern [
The homology modeling procedure used was similar to the one described above for FGSG 02858. Nevertheless, the homology modeling phase was straightforward as all homology servers gave similar results and due to the crystal structure PDB templates existing for an ortholog for trichodiene synthase (1 PDB structure for an apo and 5 for complexes forms), such as the 1JFA structure of trichodiene synthase from Fusarium sporotrichioides [
The ERG6 gene encodes for the enzyme D-(24)-sterol C-methyltransferase that catalysis the attachment of a methyl group acting in a bifurcation point of the ergosterol biosynthesis pathway, locus FGSG 02783. The protein is located in the endoplasmic reticulum with a transmembrane portion and an active site positioned toward the cytoplasm [
All the 10,240 compounds selected from our virtual screening campaign were docked within the binding sites of the targets, as described above, for the major conformers’ population. These molecules were next ranked according to their docking scores. After analysis of the whole scores distributions, the best 15 scored compounds obtained for each target were selected. These compounds strongly interact with several target amino acid residues through polar, aromatic and hydrophobic interactions to form the more stable protein/ligand complexes. These 45 ligands thus selected from their docking scores were consequently retained as valuable candidates for further experimental validation. Detailed information for each target is presented below:
VS on Endoglucanase-5 (FGSG 02658)
The top 15 candidate compounds retained for the endoglucanase-5 target are shown in
VS on Trichodiene synthase (FGSG 03537)
The trichodiene synthase docking results are shown in
VS on ERG6-D-(24)-sterol C-methyltransferase (FGSG 02783)
Rank | Name | IUPAC name | GOLD Score |
---|---|---|---|
1 | F0922-0875 | N-(2-methoxyethyl)-4-[1-[(3-nitrophenyl)methyl]-2,4-dioxoquinazolin-3-yl]butanamide | 86.56 |
2 | F0529-0702 | benzyl 2-[[5-[(2-benzylsulfanylacetyl)amino]-1,3,4-thiadiazol-2-yl]sulfanyl]acetate | 86.33 |
3 | F2507-0625 | benzyl 2-[(6-pyridin-2-yl-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)sulfanyl]acetate | 86.07 |
4 | F0590-0218 | 1-(2,3-dihydroindol-1-yl)-2-[1-[(3-fluorophenyl)methyl]indol-3-yl]sulfonylethanone | 84.07 |
5 | F1126-0777 | 1-carbazol-9-yl-3-[2-(3,4-dimethoxyphenyl)ethylamino]propan-2-ol | 83.24 |
6 | F3382-0749 | 5-(1-benzyl-2,4-dioxoquinazolin-3-yl)-N-(thiophen-2-ylmethyl)pentanamide | 82.98 |
7 | F2563-0302 | N-[(4-methoxyphenyl)methyl]-2-[(5-phenyl-4-pyrrol-1-yl-1,2,4-triazol-3-yl)sulfanyl]acetamide | 82.98 |
8 | F5037-0106 | 3-phenylsulfanyl-N-[2-[(3-phenyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)oxy]ethyl]propanamide | 82.8 |
9 | F5037-1259 | N-[2-[(3-phenyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)oxy]ethyl]-1-thiophen-2-ylcyclopentane-1-carboxamide | 82.51 |
10 | F3398-1046 | N-(2-fluorobenzyl)-3-(4-(piperidin-1-yl)-[1,2,4]triazolo[4,3-a]quinoxalin-1-yl)propanamide | 82.5 |
11 | F5016-2418 | N1-((1-(benzofuran-2-carbonyl)piperidin-4-yl)methyl)-N2-(2-(cyclohex-1-en-1-yl)ethyl)oxalamide | 82.5 |
12 | F3010-0040 | 3-phenyl-N-(2-phenylethyl)-3-[(5-phenyl-1H-1,2,4-triazol-3-yl)sulfanyl]propanamide | 82.45 |
13 | F3406-4638 | 2-((5-(3-(benzo[d][1,3]dioxol-5-yl)-1,2,4-oxadiazol-5-yl)- 2-oxopyridin-1(2H)-yl)methyl)-4H-pyrido[1,2-a]pyrimidin-4-one | 82.4 |
14 | F2650-0437 | 2-[[5-[(4-chlorophenyl)methyl]-4-pyrrol-1-yl-1,2,4-triazol-3-yl]sulfanyl]-N-(2-fluorophenyl)acetamide | 82.15 |
15 | F5037-1244 | 2-phenyl-N-(2-((3-phenyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)oxy)ethyl)-2H-1,2,3-triazole-4-carboxamide | 82.08 |
Rank | Name | IUPAC name | GOLD Score | Conformation |
---|---|---|---|---|
1 | F5258-0045 | N-(2-((6-(pyridin-3-ylamino)pyridazin-3-yl)amino)ethyl)-3-(trifluoromethyl)benzenesulfonamide | 80.46 | 3 |
2 | F5147-0899 | N-(3-(6-oxo-3-phenylpyridazin-1(6H)-yl)propyl)-3-(phenylsulfonyl)propanamide | 79.3 | 3 |
3 | F5485-0615 | N-(2-(1H-indol-3-yl)ethyl)-2-(2-((2-fluorobenzyl)thio)-6-oxo-1,6-dihydropyrimidin-4-yl)acetamide | 78.86 | 2 |
4 | F0554-0301 | 4-fluoro-N-[2-[3-(2-morpholin-4-yl-2-oxoethyl)sulfanylindol-1-yl]ethyl]benzamide | 78.73 | 3 |
5 | F1604-0352 | 2-[3-(benzenesulfonyl)-6-methyl-4-oxoquinolin-1-yl]-N-(2-methylphenyl)acetamide | 78.16 | 3 |
6 | F5864-0089 | N-(2-((6-(1H-pyrazol-1-yl)pyrimidin-4-yl)amino)ethyl)-3-((6-methylpyridazin-3-yl)oxy)benzamide | 77.89 | 3 |
7 | F5913-0359 | N-(2-(1H-indol-3-yl)ethyl)-2-(6-oxo-4-(thiophen-2-yl)pyrimidin-1(6H)-yl)acetamide | 77.86 | 3 |
8 | F2902-1688 | 2-(2-fluorophenoxy)-N-(2-(4-(phenethylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)acetamide | 77.71 | 3 |
9 | F5630-0003 | 2-(benzo[d]isoxazol-3-yl)-N-(2-((4-methyl-6-(piperidin-1-yl)pyrimidin-2-yl)amino)ethyl)acetamide | 76.46 | 2 |
10 | F0554-0055 | N-[2-[3-[2-(2-fluoroanilino)-2-oxoethyl]sulfanylindol-1-yl]ethyl]benzamide | 76.28 | 3 |
11 | F2563-0139 | 2-[(4-pyrrol-1-yl-1,2,4-triazol-3-yl)sulfanyl]-N-[4-(trifluoromethoxy)phenyl]acetamide | 76.23 | 3 |
12 | F5097-2588 | N-[4-[2-oxo-2-(2,2,2-trifluoroethylamino)ethyl]phenyl]-3,3-diphenylpropanamide | 76.15 | 3 |
13 | F2902-1364 | 2-(1,2-benzoxazol-3-yl)-N-[2-[4-[(4-fluorophenyl)methylamino]pyrazolo [3,4-d]pyrimidin-1-yl]ethyl]acetamide | 76.1 | 1 |
14 | F3407-3740 | 4-[(2,4-dioxo-1H-quinazolin-3-yl)methyl]-N-[2-(1H-indol-3-yl)ethyl]benzamide | 75.99 | 2 |
15 | F0617-0211 | N-[2-[3-[2-oxo-2-(oxolan-2-ylmethylamino)ethyl]sulfanylindol-1-yl]ethyl]thiophene-2-carboxamide | 75.96 | 3 |
The docking results for this target are presented in
Overall, the searching approach for novel protein targets applied for agricultural purposes is a response to the demand for chemical crop protection. The search for new fungicides or antifungal compounds should be specific with no effect on the host or other organisms. The first part of this analysis was to select specific targets to start the virtual screening selection of chemical compounds. The availability of the genome, secretome, and PHI-base allowed and enriched the search, when used in combination with the experimental data provided from various in planta experiments (ESTs).
Several steps of selection enabled the choice of three major targets: an endoglucanase-5, a trichodiene synthase, and a 24-sterol C-methyltransferase. These three proteins are likely to represent a promising target group: the endoglucanase may be re-
Rank | Name | IUPAC name | Score | Conformation |
---|---|---|---|---|
1 | F0554-0055 | N-[2-[3-[2-(2-fluoroanilino)-2-oxoethyl]sulfanylindol-1-yl]ethyl]benzamide | 98.41 | 1 |
2 | F3407-4949 | 1-(4-(4-chlorophenyl)piperazin-1-yl)-2-(5-((2-methoxyphenyl)amino)-1,3,4-thiadiazol-2-yl)ethanone | 98.26 | 2 |
3 | F3382-3282 | N-(2,6-dimethylphenyl)-2-(3-(4-fluorobenzyl)-2,4-dioxo-3,4-dihydropteridin-1(2H)-yl)acetamide | 97.95 | 2 |
4 | F3382-6099 | 2-(5-amino-3-(ethylamino)-4-(3-(o-tolyl)-1,2,4-oxadiazol-5-yl)-1H-pyrazol-1-yl)-N-benzylacetamide | 97.37 | 2 |
5 | F3382-1309 | N-cyclohexyl-2-(3-(4-methoxybenzyl)-4-oxo-3H-pyrimido[5,4-b]indol-5(4H)-yl)acetamide | 95.69 | 2 |
6 | F3382-0379 | 5-((2-(2-ethoxyphenyl)-5-methyloxazol-4-yl)methyl)-2-(p-tolyl)pyrazolo[1,5-a]pyrazin-4(5H)-one | 95.59 | 1 |
7 | F3382-7502 | N-(2-(1H-indol-3-yl)ethyl)-3-(7-methyl-4-oxo-3,4,5,6,7,8- hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)propanamide | 95.58 | 2 |
8 | F6089-1853 | N-(2-(2-(3-fluorophenyl)thiazolo[3,2-b][1,2,4]triazol-6-yl)ethyl)- 1-methyl-3-phenyl-1H-pyrazole-5-carboxamide | 95.36 | 1 |
9 | F5253-0280 | N-(3-((4-(4-fluorophenyl)piperazin-1-yl)sulfonyl)propyl)-1H-indazole-3-carboxamide | 95.03 | 2 |
10 | F5485-0615 | N-(2-(1H-indol-3-yl)ethyl)-2-(2-((2-fluorobenzyl)thio)-6-oxo-1,6-dihydropyrimidin-4-yl)acetamide | 94.46 | 1 |
11 | F2471-0720 | N-(2-chlorobenzyl)-2-(5-((2-fluorobenzyl)oxy)-2-methyl-4-oxopyridin-1(4H)-yl)acetamide | 94 | 2 |
12 | F6089-8342 | Phenyl 4-((3-benzhydrylureido)methyl)piperidine-1-carboxylate | 93.63 | 1 |
13 | F2605-0233 | 2-(4-ethoxyphenyl)-N-[2-[[5-(4-fluorophenyl)-1H-imidazol-2-yl]sulfanyl]ethyl]acetamide | 93.62 | 2 |
14 | F3406-0517 | N-(2-(cyclohex-1-en-1-yl)ethyl)-2-(3-oxo-6-(p-tolylthio)- [1,2,4]triazolo[4,3-b]pyridazin-2(3H)-yl)acetamide | 93.42 | 1 |
15 | F2902-1688 | 2-(2-fluorophenoxy)-N-(2-(4-(phenethylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)acetamide | 93.41 | 1 |
quired for cell wall degradation and, therefore, the possibility to stop the disease before the development of symptoms; the trichodiene synthase, known to be required for mycotoxin synthesis, once inhibited could reduce the amount of toxins produced during infection; the 24-sterol C-methyltransferase is a well-known target for cellular growth inhibition, especially due to its specificity and absence in mammals.
The structure modeling was an important aspect when searching for new targets. Homology modeling and molecular dynamics proposed convincing 3D models at the atomic level, enhancing the future perspectives for the development of novel, efficient and specific fungicides to control plant diseases that do not impact on the environment. The experimental validation of the present virtual screening campaigns using the three targets models is under development.
To rank and select proteins from the Fusarium graminearum genome according to their potential as fungicides targets, we screened 13,321 proteins from the F. graminearum PH-1 genome [
Our systematic criteria for target filtering were defined in terms of priority, as our primary objective was to identify genes that could be used as targets for the development of new control options: 1) protein annotation, phenotype description and transcriptomic evidence coming from EST analysis as unique genes expressed in early days of infection; 2) we discarded redundant sequences, proteins with low structure similarity within the Protein Databank (PDB), cell localization prediction in nuclei, and candidates with low accessibility to chemical compounds; 3) the number of gene copies in the genome, and protein molecular size; 4) the possible target selection also considered the absence of orthologues in another organism such as insects, plants, humans and evolutionary conservation among other fungi. Therefore, our selection process consisted of several elimination rounds. The first step of this funnel consisted in using protein annotation, phenotype characterization at PHI-base and gene expression in the early infection phase from EST data collections. The next stage of our selection was: 1) the elimination of redundancy and choice of candidates expressed during the early days of infection; followed by 2) a B2GO categorization and cell localization within the cytoplasm and accessibility to chemicals; and finally 3) a BLAST search of the retained candidates against the PDB [
In the absence of experimentally solved 3D structures, computational methods were used to predict 3D protein models and provide information regarding protein functions and structures. Homology modeling has been shown to be efficient in methods to reach reasonable theoretical 3D models as soon as a suitable sequence alignment exists between the sequences of the template and the query [
Virtual screening uses computer-based methods to discover new ligands on the basis of biological structures. This technique reduces the molecular database to a few hit compounds for a protein target based on structural features such as size and toxicity of the synthesizable chemical structures. The chemical library construction was performed according to the steps described at Beautrait et al. [
The authors gratefully acknowledge CNPq (funding grant 400432/2012-9) EMBRAPA Labex-Europe, University of Brasilia and CAPES for postdoc fellowship (#51/2013). MU and KHK receive support from BBSRC ISP Grant 20:20 Wheat (BB/J/00426X/1). The authors would also thank the funding resources of PHI-base, the UK Biotechnology and Biological Sciences Research Council (BBSRC) (BB/I/001077/1, BB/K020056/1) and receives additional support from the BBSRC as a National Capability (BB/J/ 004383/1).
Martins, N.F., Bresso, E., Togawa, R.C., Urban, M., Antoniw, J., Maigret, B. and Hammond-Kosack, K. (2016) Searching for Novel Targets to Control Wheat Head Blight Disease― I-Protein Identification, 3D Modeling and Virtual Screening. Advances in Microbiology, 6, 811-830. http://dx.doi.org/10.4236/aim.2016.611079