Structural Investigation and Molecular Modeling Studies of Strobilurin-Based Fungicides Active Against the Rice Blast Pathogen Pyricularia Oryzae
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International Journal of Molecular Sciences Article Structural Investigation and Molecular Modeling Studies of Strobilurin-Based Fungicides Active against the Rice Blast Pathogen Pyricularia oryzae Andrea Kunova 1 , Luca Palazzolo 2, Fabio Forlani 1 , Giorgia Catinella 1 , Loana Musso 1 , Paolo Cortesi 1, Ivano Eberini 2,3,*, Andrea Pinto 1,* and Sabrina Dallavalle 1 1 Department of Food, Environmental and Nutritional Science (DeFENS), University of Milan, 20133 Milan, Italy; [email protected] (A.K.); [email protected] (F.F.); [email protected] (G.C.); [email protected] (L.M.); [email protected] (P.C.); [email protected] (S.D.) 2 Department of Pharmacological and Biomolecular Sciences (DiSFeB), University of Milan, 20133 Milan, Italy; [email protected] 3 Data Science Research Center (DSRC), University of Milan, 20133 Milan, Italy * Correspondence: [email protected] (I.E.); [email protected] (A.P.) Abstract: The increasing emergence of fungicide-resistant pathogens requires urgent solutions for crop disease management. Here, we describe a structural investigation of new fungicides obtained by combining strobilurin and succinate dehydrogenase inhibitor pharmacophores. We identified compounds endowed with very good activity against wild-type Pyricularia oryzae, combined in some cases with promising activity against strobilurin-resistant strains. The first three-dimensional Citation: Kunova, A.; Palazzolo, L.; model of P. oryzae cytochrome bc1 complex containing azoxystrobin as a ligand was developed. Forlani, F.; Catinella, G.; Musso, L.; The model was validated with a set of commercially available strobilurins, and it well explains Cortesi, P.; Eberini, I.; Pinto, A.; Dallavalle, S. Structural Investigation both the resistance mechanism to strobilurins mediated by the mutation G143A and the activity and Molecular Modeling Studies of of metyltetraprole against strobilurin-resistant strains. The obtained results shed light on the key Strobilurin-Based Fungicides Active recognition determinants of strobilurin-like derivatives in the cytochrome bc1 active site and will against the Rice Blast Pathogen guide the further rational design of new fungicides able to overcome resistance caused by G143A Pyricularia oryzae. Int. J. Mol. Sci. 2021, mutation in the rice blast pathogen. 22, 3731. https://doi.org/10.3390/ ijms22073731 Keywords: rice blast; crop protection; cytochrome bc1 complex; antifungal compounds; homology modeling; molecular docking Academic Editor: Isabelle Callebaut Received: 22 February 2021 Accepted: 1 April 2021 1. Introduction Published: 2 April 2021 The mitochondrial respiratory chain is one of the most important targets for the Publisher’s Note: MDPI stays neutral development of agricultural fungicides [1]. Different fungicide classes act on enzymes with regard to jurisdictional claims in within the respiratory chain, the most important being succinate dehydrogenase inhibitors published maps and institutional affil- (SDHI, or complex II) and strobilurins (quinone outside inhibitors, QoI, or complex III) [2–6]. iations. Strobilurins interact with the cytochrome bc1 complex (complex III) involved in electron transfer, oxidation of hydroquinone, and reduction of cytochrome c in the mitochondrial respiratory chain. Strobilurin fungicides have been a milestone in the fungicide market, and they are still among the best-selling agrochemicals worldwide. Succinate dehydrogenase (SDH), on the other side, is a particular enzyme on the interface between the tricarboxylic Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. acid cycle (TCA) and the mitochondrial respiratory chain. It couples the oxidation of This article is an open access article succinate to fumarate in TCA with the reduction of ubiquinone to ubiquinol in the electron distributed under the terms and transfer chain. SDHI are the fastest growing class of fungicides in terms of newly discovered conditions of the Creative Commons products [7]. Attribution (CC BY) license (https:// Notwithstanding their enormous potential, both fungicide classes have a single-site creativecommons.org/licenses/by/ mode of action, which makes them prone to resistance development in fungal pathogen 4.0/). populations. The resistance to strobilurins is most often determined by a single amino Int. J. Mol. Sci. 2021, 22, 3731. https://doi.org/10.3390/ijms22073731 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 18 Int. J. Mol. Sci. 2021, 22, 3731 2 of 16 populations. The resistance to strobilurins is most often determined by a single amino acid acidsubstitution substitution from from glycine glycine to alanine to alanine (G143A) (G143A) and and until until now now has has been been detected detected in in approximatelyapproximately 5050 differentdifferent pathogens, while diverse mutations in the B, C C,, and and D D subunits subunits ofof thethe SDH SDH enzyme enzyme are are known known in ca. in 20 ca. fungal 20 fungal species species [8–10]. Strict[8–10]. anti-resistance Strict anti-resistance measures aremeasures being appliedare being particularly applied particularly to strobilurin to use strobilurin to ensure use their to long-lasting ensure their activity long-lasting and to delayactivity the and spread to delay of fungicide the spread resistance, of fungicide e.g., resistance, limited number e.g., limited of treatments, number preferablyof treatments, in a mixturepreferably or in rotation a mixture with or fungicides rotation with having fungicides a different having mode a different of action mode [11]. of action [11]. Rice blast, caused by the fungus Pyricularia oryzae ,, is is one one of of the the most most serious serious fungal fungal diseasesdiseases ofof cultivated cultivated rice worldwide,rice worldwide, causing causing each year each 10–30% year yield 10 losses–30% correspondingyield losses tocorre ca.sponding 70 billion to USD ca. 70 of economicbillion USD loss of [economic12–14]. It loss is managed [12–14]. It predominantly is managed predominantly by fungicides, especiallyby fungicides, in regions especially where in regions traditional where rice traditional blast-susceptible rice blast varieties-susceptible are varieties grown, andare breedinggrown, and of resistantbreeding varietiesof resistant has varieties been neglected. has been neglected. During the During years, the diverse years fungicide, diverse classesfungicide have classes been have used been against usedP. oryzaeagainst, among P. oryzae which, amongb-tubulin which inhibitorsβ-tubulin inhibitors (e.g., benomyl), (e.g., sterolbenomyl), biosynthesis sterol biosynthesis inhibitors (e.g., inhibitors propiconazole), (e.g., propiconazole), melanin biosynthesis melanin inhibitors biosynthesis (e.g., tricyclazole),inhibitors (e.g. antibiotics, tricyclazole), (e.g., kasugamycin), antibiotics (e.g. or, QoI/strobilurins kasugamycin), (e.g.,or QoI/strobilurins azoxystrobin) [ 15(e.g.,16,]. Inazoxystrobin) Europe and particularly[15,16]. In inEurope Italy, onlyand strobilurinsparticularly and in demethylationItaly, only strobilurins inhibitors(DMI) and aredemethylation currently approved inhibitors [17 (DMI)]. Therefore, are currently to augment approved the portfolio [17]. Therefore, of fungicides to augment for rice blast the managementportfolio of fungicides and to delay/overcome for rice blast themanagement resistance developmentand to delay/o againstvercome at-risk the fungicides,resistance newdevelopment solutions against are urgently at-risk needed. fungicides, new solutions are urgently needed. InIn the search search for for innovative innovative antifungal antifungal treatments, treatments, we we reported reported in a in recent a recent paper paper the thedevelopment development of new of new hybrid hybrid fungicides fungicides obtained obtained by by combining combining the the pharmacop pharmacophorehore featuresfeatures ofof QoIQoI andand SDHISDHI [[18].18]. The first first-generation-generation hybrid hybrid compounds compounds (Figure (Figure 11,, 1a1a–c–c)) featuredfeatured thethe threethree ring-basedring-based structurestructure ofof azoxystrobinazoxystrobin ( 2(2)) and and contained contained the the methyl-( methyl-E(E)-)b-- methoxyacrylateβ-methoxyacrylate strobilurin strobilurin pharmacophore pharmacophore connected connected by aby proper a proper linker linker to the carbox-to the amidecarboxamide pharmacophore pharmacophore of three of commercial three commercial SDHI inhibitors, SDHI inhibitors, e.g., mepronil e.g., (mepronil3), fluopyram (3), (fluopyram4), and boscalid (4), and (5). Theboscalid pilot hybrid(5). The compounds pilot hybrid showed compounds good in showed vitro mycelium good in growth vitro inhibitionmycelium ofgrowth the rice inhibition blast pathogen, of the ricePyricularia blast pathogen, oryzae; however,Pyricularia they oryzae almost; however completely, they lostalmost their completely activity on lost strobilurin-resistant their activity on strobilurin strains. -resistant strains. Figure 1. First generation of dual compounds 1a–c [18], commercial strobilurins (2), and succinate dehydrogenaseFigure 1. First generation inhibitors of (SDHIs) dual compounds (3–6), chosen 1a– asc [18], templates. commercial strobilurins (2), and succinate dehydrogenase inhibitors (SDHIs) (3–6), chosen as templates. The major goal of this work was to modify the structure of the most promising candidate 1a in order to obtain highly active compounds, possibly capable of overcoming strobilurin