Back
 AiM  Vol.6 No.11 , September 2016
Searching for Novel Targets to Control Wheat Head Blight Disease—I-Protein Identification, 3D Modeling and Virtual Screening
Abstract: 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.
Cite this paper: Martins, N. , Bresso, E. , Togawa, R. , 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. doi: 10.4236/aim.2016.611079.
References

[1]   Goswami, R.S. and Kistler, H.C. (2004) Heading for Disaster: Fusarium graminearum on Cereal Crops. Molecular Plant Pathology, 5, 515-525.
http://dx.doi.org/10.1111/j.1364-3703.2004.00252.x

[2]   Ravensdale, M., Rocheleau, H., Wang, L., Nasmith, C., Ouellet, T. and Subramaniam, R. (2014) Components of Priming-Induced Resistance to Fusarium Head Blight in Wheat Revealed by Two Distinct Mutants of Fusarium graminearum. Molecular Plant Pathology, 15, 948-956.

[3]   Cools, H.J. and Hammond-Kosack, K.E. (2013) Exploitation of Genomics in Fungicide Research: Current Status and Future Perspectives. Molecular Plant Pathology, 14, 197-210.
http://dx.doi.org/10.1111/mpp.12001

[4]   Yonn M.Y., Cha, B. and Kim J.C. (2013) Recent Trends in Studies on Botanical Fungicides in Agriculture. Plant Pathology Journal, 29, 1-9.
http://dx.doi.org/10.5423/PPJ.RW.05.2012.0072

[5]   Gardiner, D.M., Stiller, J. and Kazan, K. (2014) Genome Sequence of Fusarium graminearum Isolate CS3005. Genome Announcements, 2, e00108-14.
http://dx.doi.org/10.1128/genomeA.00227-14

[6]   Ma, L.-J., van der Does, H.C., Borkovich, K.A., Coleman, J.J., Daboussi, M.-J., Di Pietro, A., et al. (2010) Comparative Genomics Reveals Mobile Pathogenicity Chromosomes in Fusarium. Nature, 464, 367-373.
http://dx.doi.org/10.1038/nature08850

[7]   Wong, P., Walter, M., Lee, W., Mannhaupt, G., Münsterkotter, M., Mewes, H.-W., et al. (2011) FGDB: Revisiting the Genome Annotation of the Plant Pathogen Fusarium graminearum. Nucleic Acids Research, 39, D637-D639.
http://dx.doi.org/10.1093/nar/gkq1016

[8]   Kazan, K., Gardiner, D.M. and Manners, J.M. (2012) On the Trail of a Cereal Killer: Recent Advances in Fusarium graminearum Pathogenomics and Host Resistance. Molecular Plant Pathology, 13, 399-413.
http://dx.doi.org/10.1111/j.1364-3703.2011.00762.x

[9]   Agüero, F., Al-Lazikani, B., Aslett, M., Berriman, M., Buckner, F.S., Campbell, R.K., et al. (2008) Genomic-Scale Prioritization of Drug Targets: The TDR Targets Database. Nature Reviews Drug Discovery, 7, 900-907.
http://dx.doi.org/10.1038/nrd2684

[10]   Beautrait, A., Leroux, V., Chavent, M., Ghemtio, L., Devignes, M.-D.D., Smail-Tabbone, M., et al. (2008) Multiple-Step Virtual Screening Using VSM-G: Overview and Validation of Fast Geometrical Matching Enrichment. Journal of Molecular Modeling, 14, 135-148.
http://dx.doi.org/10.1007/s00894-007-0257-9

[11]   Abadio, A.K.R., Kioshima, E.S., Teixeira, M.M., Martins, N.F., Maigret, B. and Felipe, M.S.S. (2011) Comparative Genomics Allowed the Identification of Drug Targets against Human Fungal Pathogens. BMC Genomics, 12, 75-85.
http://dx.doi.org/10.1186/1471-2164-12-75

[12]   Brown, N.A., Antoniw, A., Hammond-Kosack, K.E. (2012) The Predicted Secretome of the Plant Pathogenic Fungus Fusarium graminearum: A Refined Comparative Analysis. PLoS ONE, 7, e33731.
http://dx.doi.org/10.1371/journal.pone.0033731

[13]   Hamilton, J.P., Neeno-Eckwall, E.C., Adhikari, B.N., Perna, N.T., Tisserat, N., Leach, J.E., et al. (2011) The Comprehensive Phytopathogen Genomics Resource: A Web-Based Resource for Data-Mining Plant Pathogen Genomes. Database, 2011, bar053.
http://dx.doi.org/10.1093/database/bar053

[14]   Wise, R.P., Caldo, R.A., Hong, L., Shen, L., Cannon, E. and Dickerson, J.A. (2007) BarleyBase/PLEXdb. Methods in Molecular Biology, 406, 347-363.

[15]   Kikot, G.E., Hours, R.A. and Alconada, T.M. (2009) Contribution of Cell Wall Degrading Enzymes to Pathogenesis of Fusarium graminearum: A Review. Journal of Basic Microbiology, 49, 231-241.
http://dx.doi.org/10.1002/jobm.200800231

[16]   Jenczmionka, N.J. and Schafer, W. (2005) The Gpmk1 MAP Kinase of Fusarium graminearum Regulates the Induction of Specific Secreted Enzymes. Current Genetics, 47, 29-36.
http://dx.doi.org/10.1007/s00294-004-0547-z

[17]   King, B.C., Waxman, K.D., Nenni, N.V., Walker, L.P., Bergstrom, G.C. and Gibson, D.M. (2011) Arsenal of Plant Cell Wall Degrading Enzymes Reflects Host Preference among Plant Pathogenic Fungi. Biotechnology for Biofuels, 4, 4.
http://dx.doi.org/10.1186/1754-6834-4-4

[18]   Stepień, L. (2014) The Use of Fusarium Secondary Metabolite Biosynthetic Genes in Chemotypic and Phylogenetic Studies. Critical Reviews in Microbiology, 40, 176-185.
http://dx.doi.org/10.3109/1040841X.2013.770387

[19]   Foroud, N.A., Ouellet, T., Laroche, A., Oosterveen, B., Jordan, M.C., Ellis, B.E., et al. (2011) Differential Transcriptome Analyses of Three Wheat Genotypes Reveal Different Host Response Pathways Associated with Fusarium Head Blight and Trichothecene Resistance. Plant Pathology, 61, 296-314.
http://dx.doi.org/10.1111/j.1365-3059.2011.02512.x

[20]   Zook, M., Johnson, K., Hohn, T. and Hammerschmidt, R. (1996) Structural Characterization of 15-Hydroxytrichodiene, a Sesquiterpenoid Produced by Transformed Tobacco Cell Suspension Cultures Expressing a Trichodiene Synthase Gene from Fusarium sporotrichioides. Phytochemistry, 43, 1235-1237.
http://dx.doi.org/10.1016/S0031-9422(96)00382-2

[21]   McCormick, S.P., Stanley, A.M., Stover, N. and Alexander, N.J. (2011) Trichothecenes: from Simple to Complex Mycotoxins. Toxins, 3, 802-814.
http://dx.doi.org/10.3390/toxins3070802

[22]   Rynkiewicz, M.J., Cane, D.E. and Christianson, D.W. (2001) Structure of Trichodiene Synthase from Fusarium sporotrichioides Provides Mechanistic Inferences on the Terpene Cyclization Cascade. Proceedings of the National Academy of Sciences of the United States of America, 98, 13543-13548.
http://dx.doi.org/10.1073/pnas.231313098

[23]   Cuzick, A., Urban, M. and Hammond-Kosack, K.E. (2008) Fusarium graminearum Gene Deletion Mutants Map1 and Tri5 Reveal Similarities and Differences in the Pathogenicity Requirements to Cause Disease on Arabidopsis and Wheat Floral Tissue. New Phytologist, 177, 990-1000.
http://dx.doi.org/10.1111/j.1469-8137.2007.02333.x

[24]   Cane, D.E., Shim, J.H., Xue, Q., Fitzsimons, B.C. and Hohn, T.M. (1995) Trichodiene Synthase. Identification of Active Site Residues by Site-Directed Mutagenesis. Biochemistry, 34, 2480-2488.
http://dx.doi.org/10.1021/bi00008a011

[25]   Abadio, A.K.R., Kioshima, E.S., Leroux, V., Martins, N.F., Maigret, B. and Felipe, M.S.S. (2015) Identification of New Antifungal Compounds Targeting Thioredoxin Reductase of Paracoccidioides Genus. PLoS ONE, 10, e0142926.
http://dx.doi.org/10.1371/journal.pone.0142926

[26]   Gaber, R.F., Copple, D.M., Kennedy, B.K., Vidal, M. and Bard, M. (1989) The Yeast Gene ERG6 Is Required for Normal Membrane Function but Is Not Essential for Biosynthesis of the Cell-Cycle-Sparking Sterol. Molecular and Cell Biology, 9, 3447-3456.
http://dx.doi.org/10.1128/MCB.9.8.3447

[27]   David, W., Jayasimha, P., Zhou, W., Kanagasabai, R., Jin, C., Jaradat, T.T., et al. (2004) Sterol Methyltransferase: Functional Analysis of Highly Conserved Residues by Site Directed Mutagenesis. Biochemistry, 43, 569-576.
http://dx.doi.org/10.1021/bi035257z

[28]   Vandeputte, P., Tronchin, G., Larcher, G., Ernoult, E., Bergès, T., Chabasse, D., et al. (2008) A Nonsense Mutation in the ERG6 Gene Leads to Reduced Susceptibility to Polyenes in a Clinical Isolate of Candida glabrata. Antimicrobial Agents and Chemotherapy, 52, 3701-3709.
http://dx.doi.org/10.1128/AAC.00423-08

[29]   Azam, S.S., Abro, A., Raza, S. and Saroosh, A. (2014) Structure and Dynamics Studies of Sterol 24-C-Methyltransferase with Mechanism Based Inactivators for the Disruption of Ergosterol Biosynthesis. Molecular Biology Reports, 41, 4279-4293.
http://dx.doi.org/10.1007/s11033-014-3299-y

[30]   Nes, W.D., Marshall, J., Jia, Z., Jaradat, T.T., Song, Z. and Jayasimha, P. (2002) Active Site Mapping and Substrate Channeling in the Sterol Methyltransferase Pathway. Journal of Biological Chemistry, 277, 42549-42556.
http://dx.doi.org/10.1074/jbc.M204223200

[31]   Jensen-Pergakes, K. (1998) Sequencing, Disruption, and Characterization of the Sterol Methyltransferase (ERG6) Gene: Drug Susceptibility Studies in Erg6 Mutants. Antimicrobial Agents Chemother, 42, 1160-1167.

[32]   Felipe, M.S.S., Andrade, R.V., Arraes, F.B.M., Nicola, A.M., Maranhao, A.Q., Torres, F.A.G., et al. (2005) Transcriptional Profiles of the Human Pathogenic Fungus Paracoccidioides brasiliensis in Mycelium and Yeast Cells. The Journal of Biological Chemistry, 280, 24706-24714.
http://dx.doi.org/10.1074/jbc.M500625200

[33]   Cuomo, C.A., Güldener, U., Xu, J.R., Trail, F., Turgeon, B.G., Di Pietro, A., et al. (2007) The Fusarium graminearum Genome Reveals a Link between Localized Polymorphism and Pathogen Specialisation. Science, 317, 1400-1402.
http://dx.doi.org/10.1126/science.1143708

[34]   Winnenburg, R., Baldwin, T.K., Urban, M., Rawlings, C., Kohler, J. and Hammond-Kosack, K.E. (2006) PHI-Base: A New Database for Pathogen Host Interactions. Nucleic Acids Research, 34, D459-D464.
http://dx.doi.org/10.1093/nar/gkj047

[35]   Mayer, K.F.X., Rogers, J., Dole el, J., Pozniak, C., Eversole, K., Feuillet, C., et al. (2014) A Chromosome-Based Draft Sequence of the Hexaploid Bread Wheat (Triticum aestivum) Genome. Science, 345, 1251788.
http://dx.doi.org/10.1126/science.1251788

[36]   Ling, H.-Q., Zhao, S., Liu, D., Wang, J.J., Sun, H., Zhang, C., et al. (2013) Draft Genome of the Wheat A-Genome Progenitor Triticum urartu. Nature, 496, 87-90.
http://dx.doi.org/10.1038/nature11997

[37]   Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, H., et al. (2000) The Protein Data Bank. Nucleic Acids Research, 28, 235-242.
http://dx.doi.org/10.1093/nar/28.1.235

[38]   Cavasotto, C.N. and Phatak, S.S. (2009) Homology Modeling in Drug Discovery: Current Trends and Applications. Drug Discovery Today, 14, 676-683.
http://dx.doi.org/10.1016/j.drudis.2009.04.006

[39]   Berman, H.M., Bhat, T.N., Bourne, P.E., Feng, Z., Gilliland, G., Weissig, H., et al. (2000) The Protein Data Bank and the Challenge of Structural Genomics. Nature Structural Biology, 7, 957-959.
http://dx.doi.org/10.1038/80734

[40]   Zhang, Y. (2008) Protein Structure Prediction: When Is It Useful? Current Opinion in Structural Biology, 19, 145-155.
http://dx.doi.org/10.1016/j.sbi.2009.02.005

[41]   Zhang, Y. (2009) I-TASSER: Fully Automated Protein Structure Prediction in CASP8. Proteins, 77, 100-113.
http://dx.doi.org/10.1002/prot.22588

[42]   Kopp, J. and Schwede, T. (2006) The SWISS-MODEL Repository: New Features and Functionalities. Nucleic Acids Research, 34, D315-D318.
http://dx.doi.org/10.1093/nar/gkj056

[43]   Kelley, L.A. and Sternberg, M.J.E. (2009) Protein Structure Prediction on the Web: A Case Study Using the Phyre Server. Nature Protocols, 4, 363-371.
http://dx.doi.org/10.1038/nprot.2009.2

[44]   Fernandez-Fuentes, N., Madrid-Aliste, C.J., Rai, B.K., Fajardo, J.E. and Fiser, A. (2007) M4T: A Comparative Protein Structure Modeling Server. Nucleic Acids Research, 35, W363-W368.
http://dx.doi.org/10.1093/nar/gkm341

[45]   Soding, J., Biegert, A. and Lupas, A.N. (2005) The HHpred Interactive Server for Protein Homology Detection and Structure Prediction. Nucleic Acids Research, 33, W244-W248.
http://dx.doi.org/10.1093/nar/gki408

[46]   Pieper, U., Webb, B.M., Dong, G.Q., Schneidman-Duhovny, D., Fan, H., Kim, S.J., et al. (2014) ModBase, a Database of Annotated Comparative Protein Structure Models and Associated Resources. Nucleic Acids Research, 42, D336-D346.
http://dx.doi.org/10.1093/nar/gkt1144

[47]   Kim, D.E., Chivian, D. and Baker, D. (2004) Protein structure prediction and analysis using the Robetta Server. Nucleic Acids Research, 32. W526-W523.
http://dx.doi.org/10.1093/nar/gkh468

[48]   Bresso, E., Leroux, V., Urban, M., Hammond-Kosack, K.E., Maigret, B. and Martins N.F. (2016) Structure-Based Virtual Screening of Hypothetical Inhibitors of the Enzyme Longiborneol Synthase—A Potential Target to Reduce Fusarium head blight disease. Journal of Molecular Modeling, 22, 163-176.
http://dx.doi.org/10.1007/s00894-016-3021-1

[49]   Phillips, J.C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., et al. (2005) Scalable molecular dynamics with NAMD. Journal of Computational Chemistry, 26, 1781-1802.
http://dx.doi.org/10.1002/jcc.20289

[50]   Humphrey, W., Dalke, A. and Schulten, K. (1996) VMD: Visual molecular dynamics. Journal of Molecular Graphics, 14, 33-38.
http://dx.doi.org/10.1016/0263-7855(96)00018-5

[51]   Yang, J., Ursu,O., Bologa, C., Waller, A., Sklar, L. and Oprea, T. (2013) The BADAPPLE promiscuity plugin for BARD Evidence-based promiscuity scores. Journal of Chemionformatics, 8, 29-43.
http://dx.doi.org/10.1186/s13321-016-0137-3 https://bard.nih.gov/BARD/static/documentation/BARD_ACS_3_Sep2013.pdf

[52]   Baell, J.B. and Holloway, G.A. (2010) New Substructure Filters for Removal of Pan Assay Interference Compounds (PAINS) from screening libraries and for their exclusion in bioassays. Journal of Medicinal Chemistry, 53, 2719-2740.
http://dx.doi.org/10.1021/jm901137j

[53]   Drwal, M.N., Banerjee, P., Dunkel, M., Wettig, M.R. and Preissner, R. (2014) ProTox: A Web Server for the in Silico Prediction of Rodent Oral Toxicity. Nucleic Acids Research, 42. W53-W58.
http://dx.doi.org/10.1093/nar/gku401

[54]   Akella L.B. and DeCaprio, D. (2010) Cheminformatics Approaches to Analyze Diversity in Compound Screening Libraries Current Opinnion in Chemical Biology, 14, 325-330.

[55]   Huang, B. and Schroeder, M. (2006) LIGSITEcsc: predicting ligand binding sites using the Connolly surface and degree of conservation. BMC Structural Biology, 6, 19.
http://dx.doi.org/10.1186/1472-6807-6-19

[56]   Verdonk, M.L., Cole, J.C., Hartshorn, M.J., Murray, C.W. and Taylor, R.D. (2003) MPROVED Protein-Ligand Docking Using GOLD. Proteins: Structure, Function and Genetics, 52, 609-623.
http://dx.doi.org/10.1002/prot.10465

[57]   Liebeschuetz, J.W., Cole, J.C. and Korb, O. (2012) Pose Prediction and Virtual Screening Performance of GOLD Scoring Functions in a Standardized Test. Journal of Computer-Aided Molecular Design, 26, 737-748.
http://dx.doi.org/10.1007/s10822-012-9551-4

[58]   Korb, O., Olsson, T.S.G., Bowden, S.J., Hall, R.J., Verdonk, M.L., Liebeschuetz, J.W., et al. (2012) Potential and Limitations of Ensemble Docking. Journal of Chemical Information and Modeling, 52, 1262-1274.
http://dx.doi.org/10.1021/ci2005934

[59]   Wang, Y., Yang, Xu, L., , H., , Q., Ma, Z. and Chu, C. (2005) Differential Proteomic Analysis of Proteins in Wheat Li Spikes Induced by Fusarium graminearum. Proteomics, 5, 4496-4503.
http://dx.doi.org/10.1002/pmic.200401317

[60]   Guo, L., Han, L., Yang, L., Zeng, H., Fan, D., Zhu, Y., et al. (2014) Genome and Transcriptome Analysis of the Fungal Pathogen Fusarium oxysporum f. sp. cubense Causing Banana Vascular Wilt Disease. PLoS ONE, 9, e95543.
http://dx.doi.org/10.1371/journal.pone.0095543

[61]   Yang, F., Jacobsen, S., Jorgensen, H.J.L., Collinge, D.B., Svensson, B. and Finnie, C. (2013) Fusarium graminearum and Its Interactions with Cereal Heads: Studies in the Proteomics era. Frontiers in Plant Science, 4, 37.
http://dx.doi.org/10.3389/fpls.2013.00037

[62]   Valente, M.T., Infantino, A. and Aragona, M. (2011) Molecular and Functional Characterization of an Endoglucanase in the Phytopathogenic Fungus Pyrenochaeta lycopersici. Current Genetics, 57, 241-251.
http://dx.doi.org/10.1007/s00294-011-0343-5

 
 
Top