WO2020234497A1 - Chitin deacetylase inhibitors and use thereof as agricultural fungicides, arthopocides and nematicides - Google Patents
Chitin deacetylase inhibitors and use thereof as agricultural fungicides, arthopocides and nematicides Download PDFInfo
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- Chitin deacetylase inhibitors and their use as agricultural fungicides, arthropocides and nematicides
- Chitin is the main component of the exoskeleton of arthropods and of the cell walls of fungi; it is also present in the eggs and intestinal linings of nematodes. Chitin is considered a safe target for fungicides, insecticides or nematicides, since chitin is absent in plants and mammals 1 . Chitin is also a known inducer of immune responses in plants. As a consequence of the enzymatic activity of plant chitinases, small oligomers of chitin are released. These oligomers can be recognized by plant receptors such as CERK1, promoting the activation of the chitin-specific signaling cascade.
- pathogens have developed strategies to overcome chitin detection.
- One of these strategies is the conversion of chitin in the cell wall into chitosan by the action of chitin deacetylase (CDA - chitin deacetylase -).
- CDA - chitin deacetylase - This enzyme catalyzes the hydrolysis of the N-acetamido group in the N-acetylglucosamine units of chitin to convert it into chitosan, the deacetylated derivative of chitin, a poor substrate for chitinases and a compound with inductive activity significantly lower than chitin 2 ⁇ 3 .
- fungi the fungicides marketed for disease control in agricultural crops are under pressure.
- the best examples of fungal diseases in which chemicals play a key role in disease management are powdery mildews.
- powdery mildews are cereals, vines, and many horticultural crops and ornamental plants.
- the problem of resistance to fungicides in powdery mildews is well illustrated in the case of the cucurbit pathogen Podosphaera xanthii 4 .
- resistance to the most popular anti-powdery mildew fungicides has been described 5-7 , with multi-resistant isolates in the most intense cultivation areas 7 . Therefore, there is a pressing demand to identify and develop new phytosanitary products.
- CN105462996A refers to a technology based on gene silencing of a gypsy moth chitin deacetylase gene, a gene segment of a gypsy moth chitin deacetylase gene, and a dsRNA dsRNA of that gene segment and the application of that gene. DsRNA to interfere with the development process of the insect and cause an altered metamorphosis.
- CDA appears to be an important protein for nematodes as well. 9 In a paper using the model nematode Caenorhabditis elegans, disruption of CDA genes was shown to cause a delay in the nematode development time. They also demonstrated the presence of homologues in many other Nematoda species, including important plant and vertebrate parasitic nematodes, concluding that CDA may be a valid target for the development of intervention tools against parasitic nematodes.
- molecular topology is basically related to the association between molecules and graphs, in such a way that it allows describing molecular structures through graphotheoretical indices. Furthermore, it deals with the connectivity of atoms in molecules and not with geometric characteristics such as angles, distances, or three-dimensional structure, which is common in standard / conventional approaches. In this way, graph theory and the surrounding disciplines stand as basic tools of molecular topology. Following this approach, obtained excellent results in the design and selection of new drugs in different medical fields.
- CDA chitin deacetylase
- fungicide in this application refers to the toxic effect produced by the compound when brought into contact with a fungus. This effect may or may not be mediated by a plant.
- the present invention refers to a method for the identification of a compound with potential pesticidal activity against chitin-containing organisms, said compound being an inhibitor of the enzyme chitin deacetylase (CDA), comprising:
- step (a) further comprises obtaining for said compound a predictive value of the inhibitory activity Log (lnh%), where:
- N..N is the topological distance expressed as the number of edges between two consecutive nitrogen
- - JGI2 is the topological average load index of order 2;
- step (b) further comprises verifying if the value of Log (lnh%) obtained in (a) for said compound is between +1 and +2. If the value is between +1 and +2, said compound is identified as a potential inhibitor of the CDA enzyme.
- step (a) further comprises obtaining for said compound a value for the discriminant function DF2, in which:
- step (b) further comprises verifying whether the DF2 value obtained in (a) for said compound is between 0 and +6. If the value is between 0 and +6, said compound is identified as a potential inhibitor of the CDA enzyme.
- the method as defined above comprises after step (b):
- step (b) carrying out an experimental analysis of the compound identified as a potential inhibitor of the CDA enzyme according to step (b), which comprises putting said compound in contact with a pathogen, preferably a fungal pathogen, and more preferably a fungal pathogen in the presence of a plant and observe if there is growth of this.
- a pathogen preferably a fungal pathogen, and more preferably a fungal pathogen in the presence of a plant and observe if there is growth of this.
- the pathogen is a fungus located on a plant, even more particularly Podosphaera xanthii.
- the pest is an insect even more particularly Galleria mellonella.
- the method as defined above further comprises performing a docking experiment with a fragment of the CDA enzyme comprising the active site thereof and the compound identified as an inhibitor of the enzyme chitin deacetylase. .
- Said compound will show an affinity value lower than -7 kcal / mol, by virtue of its ability to interact with the active site of the CDA enzyme.
- the CDA enzyme used for the docking experiment is a fungal CDA enzyme, preferably the CDA from Colletotrichum lindemuthianum.
- the method defined above comprises a docking experiment with the entire CDA enzyme, of the compound identified as an inhibitor of the enzyme chitin deacetylase, in which said compound shows two or three hydrogen bonds with two or three different amino acids located within the active site of the CDA enzyme.
- the CDA enzyme used for the docking experiment is a fungal CDA enzyme, more preferably the CDA from Colletotrichum lindemuthianum.
- the CDA enzyme used for the docking experiment is an insect CDA enzyme, more preferably the Bombyx died CDA.
- the method defined above is useful to identify compounds to combat pests in which the pests are those organisms that contain chitin, such as fungi, arthropods or nematodes.
- the present invention also relates to the use of chemical compounds with pesticidal activity against a chitin-containing organism selected from fungi, arthropods and nematodes, identified by the method defined above.
- the invention refers to the use of a chemical compound with pesticidal activity against an organism that contains chitin selected from fungi, arthropods and nematodes, characterized in that:
- said compound also has a value of Log (lnh%) between +1 and +2;
- said compound also has a DF2 value between 0 and +6.
- the invention refers to the use of a chemical compound with pesticidal activity against an organism that contains chitin selected from fungi, arthropods and nematodes, as defined above, characterized in that said compound interacts with the active site of the CDA enzyme
- the chemical compound detected with pesticidal activity against a chitin-containing organism is selected from:
- Table 1 lists 20 compounds with pesticidal activity predicted by molecular topology, as well as their respective DF1, Log (inh%) and DF2 values.
- Particular embodiments refer to the use of the compounds detected following the method described above, to control plant diseases caused by phytopathogenic fungi, preferably biotrophic fungi, and more preferably powdery mildew and rusts, such as Podosphaera xanthii and other plant pathogenic necrotrophic fungi such as Botrytis cinerea among others.
- phytopathogenic fungi preferably biotrophic fungi
- powdery mildew and rusts such as Podosphaera xanthii and other plant pathogenic necrotrophic fungi such as Botrytis cinerea among others.
- pathogens include the Ascomycota and Basidiomycota classes, as well as pathogens of the Oomycetes class, on any cereal, vine, fruit tree, horticultural, fiber and / or ornamental crops.
- pathogens include: Ascomycetes, pathogens included in the order Erysiphales such as Blumeria graminis, Erysiphe necator, Erysiphe polygoni, Leveillula taurica, Podosphaera aphanis, and Podosphaera xanthii, and other pathogens including Alternar ⁇ a solani, Botrytis Columnarichumumumumumumumumumumumumumumumumumumumumumumumumumumumumthorn , Fusarium oxysporum, Gaeumannomyces graminis, Magnaporthe gr ⁇ sea, Monilinia fructicola, Mycosphaereiia fijensis, Phomopsis
- Uromyces viciae-fabae, Uromyces betae pathogens in the order Ustilaginales such as Ustilago maydis and Ustilago tritici, and other pathogens including Armillaria mellea, Rhizoctonia solani, and Sclerotium roifsir, Oomycetes, including pathogens Phytophthora, Phynophthora infestaytophyogens such as Phytophthora infestaytogens Pythium such as Pythium aphanidermatum, and pathogens of the Peronosporaceae family such as Plasmopara vit ⁇ cola, Pseudoperonospora cubensis and Bremia lactucae; and other genera and species closely related to these pathogens.
- the compounds of the invention are useful for the control of pests such as arthropods, particularly insects, mites and lice, of the house, garden, livestock and agriculture.
- Additional particular embodiments refer to the use of the compounds identified following the method described above, to control insects in larval stages, preferably of the infraclass Neoptera, which include, but are not limited to, the orders Lepidoptera, Homoptera, Isoptera, Diptera, Orthoptera, Hemiptera and Coleóptera. Most preferably, Drosophila meianogaster and Galleria mellonella, among others.
- other representative pests that are controlled by the compounds of the invention include members of the phylum Arthropoda, including mites of the suborders Mesostigmata, Sarcoptiformes, Trombidiformes, and Onchychopalpida; and lice of the orders Anoplura and Mallophaga.
- Particular embodiments relate to the use of the compounds identified following the method described above to inhibit the growth of the model nematode Caenorhabditis elegans.
- the compounds of the invention are useful for the control of parasitic nematodes, including, but not limited to, parasitic nematodes of plants of the order Tylenchida, especially species such as Meloidogyne spp., Heterodera spp., Rotylenchus spp. and Pratylenchus spp.
- the parasitic nematodes of mammals, including humans, livestock and pets are also controlled by the compounds of the invention, including the orders Ascaridia, Spirurida and Strongylida, and in particular species such as Trichinella spp., Trichuris spp. and Strongyloides spp.
- the inventors have developed a method for the identification of compounds to combat agricultural pests and other pests and parasites of non-agricultural origin. Furthermore, the effectiveness of these compounds against fungi, insects and nematodes has been confirmed.
- FIGURE 1 Flow diagram of the experimental tests used for the identification of CDA inhibitors.
- FIGURE 2 Fungicidal effects of some of the selected compounds against cucurbit powdery mildew (P. Xanthii) in the whole plant test.
- FIGURE 3 Insecticidal effects of some compounds identified by the molecular topology method in G. mellonella.
- FIGURE 4 Micrographs of the C. elegans toxicity test performed, showing the effects of some of the compounds identified by the molecular topology method on the development of nematodes.
- FIGURE 5 Physiological effects on the responses of plants to treatment with the compounds with the best fungicidal activity on melon cotyledons infected with P. xanthii.
- Example 1 Development of the molecular topology method and identification of compounds.
- DF1 (-10,295 x GATS4m) + (35,124 x GGI8) + 6,917
- GATS4m is the Geary-Iag4 / atomic mass-weighted autocorrelation index
- GGI8 is the 8th order topological load index.
- Active compounds (A) are those that have a DFi value in the range between 0 and +14. Therefore, this model is appropriate for classifying chemical compounds according to their ability to inhibit the CDA enzyme.
- GATS4m is the Geary autocorrelation index - Iag4 / weighted by atomic masses;
- T (N..N) is the topological distance expressed as the number of edges between two consecutive nitrogens.
- JGI2 indicates the topological average load index of order 2.
- a second screen is performed with the second function as described above. This time, a compound will be selected as a possible CDA inhibitor if it has a DF1 discriminant value between 0 and +14 and a predictive value of inhibitory activity (Log (lnh%) between +1 and +2. Therefore, the results of the laboratory tests helped us to obtain new equations that refined the mathematical models.
- Table 5 shows the list of compounds chosen after the second iteration of the molecular topology. Also included are their calculated DF1 and Log (lnh%) values, as well as the values of the topological indices that appear in both equations.
- F is the Fisher-Snedecor parameter
- Table 6 shows the list of the molecules detected in molecular topology approaches 1 and 2 (Table 2 and Table 4, respectively) classified according to DF2 values.
- a compound will be chosen as CDA inhibitor if, in addition to the conditions imposed by DF1 and Log (lnh%), they show a DF2 value between 0 and 6 and a docking affinity value lower than -7 kcal / mol. TABLE 6
- DF2 21.022 - (19.407 x GGI10) - (5.104 x Seige) - (7.911 x GATS3e), and
- Example 1.2 Experimental validation of the compounds identified by a molecular topology method as antioid fungicides.
- the discs were immersed in the solution of the corresponding compound (1 or 100 mM) or a 1% aqueous solution of acetone that was also included as a negative control, and incubated under the same conditions for 7 days.
- the surface of the leaf covered by the fungus was evaluated by digital analysis. For this analysis, the images were captured with a digital camera with a fixed distance from the discs of 20 cm. The images were analyzed using the free Java Image image processing software to calculate the area covered by powdery mildew symptoms on each disk.
- Table 7 shows the fungicidal effect of the compounds identified by the molecular topology method on the development of cucurbit powdery mildew (P. xanthii) in the leaf disk test.
- P. xanthii cucurbit powdery mildew
- Example 1.3 Molecular Dockinq from a fungal ADC to the identified compounds
- the area comprises the active site of CDA, in which the Zn atom and other essential amino acids for CDA activity are present.
- the docking results returned by AutoDock Vina indicated that the acid compound (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic acid had the best affinity results as a CDA inhibitor.
- This molecule forms several hydrogen bonds with amino acids in the active site of CDA.
- Table 8 The compounds tested in this test and the results obtained are shown in Table 8.
- Example 2 Fungicide sensitivity tests on plants and fruits
- the leaf disk test is a good test for a first screening of the efficacy of fungicidal compounds against powdery mildew.
- the predicted fungicidal activity of these compounds is presumably not associated with their toxicity but rather with their ability to activate chitin-triggered immunity. Since inoculum distribution may be crucial for the efficacy of this response, we decided to test the compounds in a plant assay using a dispersed inoculum. Therefore, a seedling assay was then performed using P. xanthii strain 2086 and only one concentration (100 mM). For the seedling test, 2-week-old melon seedlings were used. Plants were inoculated by spraying a suspension of P.
- xanthii conidia (1 x 10 4 conidia / ml) to the point of runoff. Twenty-four hours after inoculation, the leaves were sprayed with the compound solution (100 mM). A 1% acetone aqueous solution was also included as a negative control. The plants were kept in a growth chamber on a 16 h light / 8 h dark cycle at 25 ° C for 12 days. After this incubation, disease symptoms were assessed by digital analysis as indicated above.
- Table 9 shows the fungicidal effect of the compounds identified by the molecular topology method on the powdery mildew of cucurbits P. xanthii.
- the efficacy of the compound was determined by the Abbott formula and statistical differences were calculated using Fisher's least significant difference (LSD) method. Generally speaking, the efficacy of the compounds was increased compared to the leaf disk test. The compounds with the most significant fungicidal activity on P.
- xanthii according to the statistical analysis performed were: acid (1-methyl-2,4-dioxo-1, 4,6,7-tetrahydro [1, 3] thiazol [2,3f ] purin-3 (2H) yl) acetic acid, (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic acid,
- Figure 2 shows the fungicidal effect of some of the selected compounds against cucurbit powdery mildew (P. Xanthii) in the plant test. Melon leaves show a strong reduction in the number of powdery mildew colonies on the leaves treated with the selected compounds.
- Table 10 shows the fungicidal effect of the selected compounds in plant and fruit tests against three important fungal plant diseases, cucurbit powdery mildew (P. Xanthii), tomato gray rot (B. cinérea) and green rot of the citrus (P. digitatum).
- Figure 3 shows that the most dramatic effect is related to the formation of the moth that either did not form or resulted in the formation of abnormal adults with deformed wings, a phenotype previously associated with the silencing of a CDA gene in the moth beetle.
- potato Leptinotarsa decemlineata 16 potato Leptinotarsa decemlineata 16 .
- Table 12 shows the insecticidal effect of the compounds identified by the molecular topology method on the larvae of the wax moth G. mellonella. Generally speaking, the compounds did not affect pupal formation, but some of them had a significant effect on metamorphosis (moth formation).
- Toxicity against D. melanogaster was tested using the liquid food feeding assay 17 .
- 25 fly larvae were placed in 20 ml plastic containers containing 0.5 ml of liquid food and the corresponding compound at a concentration of 150 mM.
- a 1.5% aqueous acetone solution was also included as a negative control.
- the number of pupae formed was recorded, as well as the number of adults (flies) that resulted.
- Table 14 shows the insecticidal effect of the compounds identified by a molecular topology method on the larvae of the fruit fly D. melanogaster. Overall, most of the compounds significantly reduced fly formation.
- the C. elegans 18 plaque toxicity assay was used. To carry out the assay, a 75 ml drop of a 1 mM solution of the corresponding compound was added to each well on the agar surface, to reach a final concentration in the culture medium of 150 mM. As a negative control, a 10% acetone aqueous solution (1.5% final acetone concentration) was also included. The drops were allowed to dry at room temperature for 30 min. Subsequently, each well was inoculated with 40 ml of the washed and filtered nematode population. After five days of incubation at 20 ° C, each well was washed with 100 ml of M9 buffer. Next, ten 3 ml drops were examined under a light microscope to count the number of nematodes in the different larval stages (L1, L2-L3 and L4).
- Figure 4 shows some micrographs of the C. elegans toxicity test carried out with the effects of some compounds identified by the molecular topology method on the development of nematodes. Compared to the negative control (water), the compounds caused a clear reduction in nematode formation. TABLE 16
- Table 16 shows the nematicidal effect of the compounds identified by the molecular topology method on C. elegans. Most of the compounds showed a strong effect on the development of C. elegans, which is consistent with the phenotype of delay in development time associated with the disruption of genes encoding CDA 9 .
- CDA inhibitors Compounds with the best fungicidal potential were tested for their mode of action as CDA inhibitors. Two different experiments were performed to provide direct (enzymatic assay) and indirect (plant assay) evidence of inhibition of a fungal CDA.
- the plants were then kept in a growth chamber on a 16 h light / 8 h dark cycle at 25 ° C and subsequently examined for activation of defense responses and development of disease symptoms. Twelve days after inoculation, disease symptoms were assessed by digital analysis as above. On the other hand, 72 h after inoculation, the in situ accumulation of hydrogen peroxide (H 2 O 2 ) was studied by histochemical analysis, following the DAB 20 consumption method.
- H 2 O 2 hydrogen peroxide
- Figure 5 shows the physiological effects of melon cotyledon treatments with the compounds with the best fungicidal activity.
- the compounds induced a strong inhibition of fungal growth (Fig. 5A) as a consequence of the strong activation of the production of reactive oxygen species (ROS reactive oxygen species) (Fig. 5B).
- ROS reactive oxygen species ROS reactive oxygen species
- results show the relationship between the fungicidal activity of the compounds and the activation of chitin-activated immunity in the host plant, results that can be explained by the inhibition of CDA activity by the compounds and the subsequent perception of non-deacetylated chitin oligomers. by plant receptors.
- CDA proteins identified in P. xanthii were expressed in vitro in E. coli.
- Recombinant proteins with a polyhistidine tag at the N-terminus were produced following standard procedures.
- Enzyme activity was determined using the fluorescamine method, using colloidal chitin as substrate 12 .
- the enzymatic reaction was carried out in the absence or presence of the compounds at concentrations of 10 and 100 mM. The reaction mixtures were incubated for 45 min at 37 ° C. After incubation, reactions were stopped with 0.4M borate buffer (pH 9.0). After recording the data, the percentages of inhibition of enzyme activity were calculated.
- Table 18 shows the results of the CDA enzymatic activity inhibition experiments carried out with two CDA proteins from P. xanthii, PxCDAI and PxCDA2, expressed in E. coli and exposed to the compounds with the best fungicidal activity. At 100 mM, the compounds induced a strong inhibition of the enzymatic activity of CDA proteins in a range of 75 to 93%.
- Table 19 shows the docking analysis to a fungal CDA. Only compounds with fungicidal activity were able to form two hydrogen bonds with two different amino acid residues, Y145 and L146, resulting in a favorable binding site. Since Y145 is one of the catalytic residues of the active site of the fungal enzyme 21 , the docking results appear consistent with the previous data, confirming that these compounds are true inhibitors of the fungal CDA enzyme.
- Table 20 shows the docking analysis to the insect CDA.
- only compounds with insecticidal activity were capable of forming two or three hydrogen bonds with residues K508 and G512. These residues are part of the active site pocket of the CDA enzyme of B. mor ⁇ 22 , suggesting that these compounds may interfere with enzyme activity.
- Table 21 shows the results of fungal CDA coupling performed with different computer programs. Only compounds with the predicted fungicidal activity were able to form two or more hydrogen bonds with at least two different amino acid residues, thus resulting in a favorable binding site. In most cases, these amino acids are catalytic residues (HIS206, ASP169, TYR145 and other residues highlighted in bold in Table 21) of the active site of the fungal protein CDA 21 . Furthermore, the three compounds showed very favorable affinity values (DG> -6.5 kcal / mol), which provides information on their ability to couple to the catalytic site. The values of binding energy and hydrogen bonds with specific residues were comparable to those of EDTA and chitin trimer, resulting in consistent results. In conclusion, these molecular coupling results are consistent with previous molecular coupling results against a fragment of a fungal CDA protein that comprised the active site, thus confirming that these compounds are true inhibitors of the fungal CDA enzyme. TABLE 21
- Table 22 shows the insect CDA enzyme coupling analysis.
- the three compounds with fungicidal activity some specific interactions with the catalytic bag of the enzyme (PHE165 and HIS282) were detected, although only with the Glide software.
- the two compounds with insecticidal activity similar results were obtained with the three coupling software.
- These compounds formed three or four hydrogen bonds with catalytic residues such as ASP63, HIS121, and TRP284, which were also involved in binding to EDTA and the chitin trimer. Since these residues are part of the active site bag of the CDA enzyme of B. mor ⁇ 22 , the results of the coupling suggest that these compounds may interfere with the activity of the enzyme as inhibitors of the CDA enzyme of the insect, thus reinforcing its potential as insecticidal compounds.
- the docking results obtained from different computer programs provide computational evidence supporting the specific activity of these molecules as fungicidal or insecticidal compounds. This activity is mediated through their specific binding to the CDA enzyme of a fungus or an insect, thus enhancing their expected activity as specific CDA inhibitors.
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Abstract
The invention relates to a method for identifying a compound with potential pesticide activity against an organism containing chitin, the compound being an inhibitor of chitin deacetylase (CDA). The method comprises cheminformatics and experimental approximations that include molecular topology equations to examine the experimental chemical and analysis databases of the identified compounds to test their activities as pesticides. More specifically, the invention relates to the experimental analysis of a set of 20 compounds and their use as fungicides, arthopocides and/or nematicides. In addition, the specificity of these compounds as CDA inhibitors was confirmed using enzyme inhibition testing and docking. The method of the invention is useful for identifying CDA inhibitors useful as agricultural fungicides or for controlling other pests such as insects or nematodes that are harmful for crops and/or mammals.
Description
TÍTULO TITLE
Inhibidores de quitina deacetilasa y su uso como fungicidas agrícolas, artropocidas y nematicidas Chitin deacetylase inhibitors and their use as agricultural fungicides, arthropocides and nematicides
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
El control químico ha sido fundamental en la prevención de pérdidas debido a enfermedades de las plantas. La quitina es el principal componente del exoesqueleto de los artrópodos y de las paredes celulares de los hongos; también está presente en los huevos y en los revestimientos intestinales de los nematodos. La quitina se considera una diana segura para fungicidas, insecticidas o nematicidas, ya que la quitina está ausente en las plantas y en los mamíferos1. La quitina también es un conocido inductor de respuestas inmunes en las plantas. Como consecuencia de la actividad enzimática de las quitinasas vegetales, se liberan pequeños oligómeros de quitina. Estos oligómeros pueden ser reconocidos por receptores de plantas como CERK1 , promoviendo la activación de la cascada de señalización específica de quitina. En respuesta, los patógenos han desarrollado estrategias para superar la detección de quitina. Una de estas estrategias es la conversión de la quitina de la pared celular en quitosano por la acción de quitina deacetilasa (CDA - chitin deacetylase -). Esta enzima cataliza la hidrólisis del grupo N-acetamido en las unidades de N-acetilglucosamina de la quitina para convertirla en quitosano, el derivado deacetilado de la quitina, un sustrato pobre para quitinasas y un compuesto con actividad inductora notablemente menor que la quitina2·3. Chemical control has been essential in preventing losses due to plant diseases. Chitin is the main component of the exoskeleton of arthropods and of the cell walls of fungi; it is also present in the eggs and intestinal linings of nematodes. Chitin is considered a safe target for fungicides, insecticides or nematicides, since chitin is absent in plants and mammals 1 . Chitin is also a known inducer of immune responses in plants. As a consequence of the enzymatic activity of plant chitinases, small oligomers of chitin are released. These oligomers can be recognized by plant receptors such as CERK1, promoting the activation of the chitin-specific signaling cascade. In response, pathogens have developed strategies to overcome chitin detection. One of these strategies is the conversion of chitin in the cell wall into chitosan by the action of chitin deacetylase (CDA - chitin deacetylase -). This enzyme catalyzes the hydrolysis of the N-acetamido group in the N-acetylglucosamine units of chitin to convert it into chitosan, the deacetylated derivative of chitin, a poor substrate for chitinases and a compound with inductive activity significantly lower than chitin 2 · 3 .
En el caso particular de los hongos, los fungicidas comercializados para el control de enfermedades en cultivos agrícolas están bajo presión. Los mejores ejemplos de enfermedades fúngicas en las que los productos químicos desempeñan un papel clave en el manejo de enfermedades son los oídios. Entre los cultivos económicamente importantes afectados por los oídios se encuentran cereales, viña y muchos cultivos hortícolas y plantas ornamentales. El problema de la resistencia a los fungicidas en los oídios se ilustra perfectamente en el caso del patógeno de las cucurbitáceas Podosphaera xanthii 4. En el sur de España, se ha descrito resistencia a los fungicidas anti-oídio más populares5-7, con aislados multirresistentes en las áreas de cultivo más intenso7. Por lo tanto, existe una demanda acuciante para identificar y desarrollar nuevos productos fitosanitarios.
“Análisis morfológico y funcional de la interacción de Podosphaera xanthii - cucurbitáceas" es el título de la tesis doctoral de uno de los inventores de esta invención8. Este documento describe un estudio cuyo resultado identifica la quitina deacetilasa (CDA) como una proteína clave para la patogénesis, ya que el silenciamiento génico o su inhibición con diferentes ácidos carboxílicos activa la inmunidad disparada por quitina y bloquea el desarrollo fúngico, lo que la convierte en una diana ideal para el desarrollo de nuevos productos fitosanitarios. Sin embargo, no describe un método para detectar inhibidores como se expone en la presente invención. In the particular case of fungi, the fungicides marketed for disease control in agricultural crops are under pressure. The best examples of fungal diseases in which chemicals play a key role in disease management are powdery mildews. Among the economically important crops affected by powdery mildew are cereals, vines, and many horticultural crops and ornamental plants. The problem of resistance to fungicides in powdery mildews is well illustrated in the case of the cucurbit pathogen Podosphaera xanthii 4 . In southern Spain, resistance to the most popular anti-powdery mildew fungicides has been described 5-7 , with multi-resistant isolates in the most intense cultivation areas 7 . Therefore, there is a pressing demand to identify and develop new phytosanitary products. "Morphological and functional analysis of the interaction of Podosphaera xanthii - cucurbits" is the title of the thesis of one of the inventors of this invention 8 This document describes a study which identifies result deacetylase Chitin (CDA) as a key protein. pathogenesis, since gene silencing or its inhibition with different carboxylic acids activates the immunity triggered by chitin and blocks fungal development, which makes it an ideal target for the development of new phytosanitary products. However, it does not describe a method to detect inhibitors as set forth in the present invention.
La resistencia a insecticidas también es un problema muy grave para la agricultura y los agricultores también están demandando nuevas herramientas para controlar las plagas más importantes. CN105462996A se refiere a una tecnología basada en el silenciamiento génico de un gen de quitina deacetilasa de la polilla gitana, a un segmento génico de un gen de quitina deacetilasa de la polilla gitana, y a un ARNbc ARNbc de ese segmento génico y a la aplicación de ese ARNbc para interferir sobre el proceso de desarrollo del insecto y causar una metamorfosis alterada. Insecticide resistance is also a very serious problem for agriculture and farmers are also demanding new tools to control the most important pests. CN105462996A refers to a technology based on gene silencing of a gypsy moth chitin deacetylase gene, a gene segment of a gypsy moth chitin deacetylase gene, and a dsRNA dsRNA of that gene segment and the application of that gene. DsRNA to interfere with the development process of the insect and cause an altered metamorphosis.
De manera similar, CDA parece ser también una proteína importante para nematodos9 En un artículo que utiliza el nematodo modelo Caenorhabditis elegans se demostró que la interrupción de genes de CDA causaba un retraso en el tiempo de desarrollo del nematodo. También demostraron la presencia de homólogos en muchas otras especies de Nematoda, incluidos nematodos parásitos importantes de plantas y vertebrados, concluyendo que CDA puede ser una diana válida para el desarrollo de herramientas de intervención contra nematodos parásitos. Similarly, CDA appears to be an important protein for nematodes as well. 9 In a paper using the model nematode Caenorhabditis elegans, disruption of CDA genes was shown to cause a delay in the nematode development time. They also demonstrated the presence of homologues in many other Nematoda species, including important plant and vertebrate parasitic nematodes, concluding that CDA may be a valid target for the development of intervention tools against parasitic nematodes.
Uno de los métodos de diseño de fármacos asistidos por ordenador más prometedores es la topología molecular. Contrariamente al resto de los métodos cuantitativos de relación estructura-actividad (QSAR), la metodología permite una predicción rápida y precisa de muchas propiedades biológicas y fisicoquímicas. Definida como parte de la química matemática, la topología molecular se relaciona básicamente con la asociación entre moléculas y grafos, de modo que permite describir estructuras moleculares a través de índices grafoteóricos. Además, trata de la conectividad de los átomos en las moléculas y no de características geométricas tales como ángulos, distancias o estructura tridimensional, que es común en los enfoques estándar/convencionales. De esta manera, la teoría de grafos y las disciplinas circundantes se erigen como herramientas básicas de la topología molecular. Siguiendo este enfoque, se han
obtenido excelentes resultados en el diseño y selección de nuevos medicamentos en diferentes campos médicos. One of the most promising computer-aided drug design methods is molecular topology. Contrary to the rest of the quantitative structure-activity relationship (QSAR) methods, the methodology allows a fast and accurate prediction of many biological and physicochemical properties. Defined as part of mathematical chemistry, molecular topology is basically related to the association between molecules and graphs, in such a way that it allows describing molecular structures through graphotheoretical indices. Furthermore, it deals with the connectivity of atoms in molecules and not with geometric characteristics such as angles, distances, or three-dimensional structure, which is common in standard / conventional approaches. In this way, graph theory and the surrounding disciplines stand as basic tools of molecular topology. Following this approach, obtained excellent results in the design and selection of new drugs in different medical fields.
Partiendo del análisis estructura-actividad sobre el efecto de algunos ácidos carboxílicos conocidos sobre la proteína CDA fúngica, se elaboró una estrategia combinada de topología molecular y pruebas experimentales para identificar nuevos inhibidores de la quitina deacetilasa (CDA). Esta estrategia consistió en una selección mediante cribado virtual de más de 3.000.000 de moléculas pertenecientes a varias bases de datos y un análisis experimental adicional de las moléculas más interesantes, lo que permitió elaborar método útil para la identificación de moléculas para controlar plagas, tales como hongos, insectos o nematodos, basados en la inhibición de CDA. Starting from the structure-activity analysis on the effect of some known carboxylic acids on the fungal CDA protein, a combined strategy of molecular topology and experimental tests was developed to identify new inhibitors of chitin deacetylase (CDA). This strategy consisted of a selection by virtual screening of more than 3,000,000 molecules belonging to various databases and an additional experimental analysis of the most interesting molecules, which allowed the elaboration of a useful method for the identification of molecules to control pests, such as such as fungi, insects or nematodes, based on the inhibition of CDA.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
El término "fungicida" en esta solicitud se refiere al efecto tóxico producido por el compuesto al ponerlo en contacto con un hongo. Este efecto puede ser mediado, o no, por una planta. The term "fungicide" in this application refers to the toxic effect produced by the compound when brought into contact with a fungus. This effect may or may not be mediated by a plant.
Los términos restantes en esta memoria tienen el significado general comúnmente atribuido en el campo. The remaining terms in this specification have the general meaning commonly ascribed in the field.
La presente invención se refiere a un método para la identificación de un compuesto con potencial actividad pesticida frente a organismos que contienen quitina, siendo dicho compuesto un inhibidor de la enzima quitina deacetilasa (CDA), que comprende: The present invention refers to a method for the identification of a compound with potential pesticidal activity against chitin-containing organisms, said compound being an inhibitor of the enzyme chitin deacetylase (CDA), comprising:
a) obtener para dicho compuesto un valor que resulte de aplicar la función discriminante DF1 , que es: a) Obtain for said compound a value that results from applying the discriminant function DF1, which is:
DF1 = (-10.295 x GATS4m) + (35.124 x GGI8) + 6.917 donde: DF1 = (-10.295 x GATS4m) + (35.124 x GGI8) + 6.917 where:
- GATS4m: índice autocorrelación de Geary - Iag4 / ponderado por masas atómicas, - GATS4m: Geary's autocorrelation index - Iag4 / weighted by atomic masses,
- GGI8: índice topológico de carga de orden 8, - GGI8: topological load index of order 8,
b) comprobar si el valor DF1 obtenido en (a) para dicho compuesto está entre 0 y +14. Si el valor está entre 0 y +14, dicho compuesto se identifica como un inhibidor potencial de la enzima CDA.
De acuerdo con realizaciones particulares adicionales del método, tal como se ha definido anteriormente: b) check if the DF1 value obtained in (a) for said compound is between 0 and +14. If the value is between 0 and +14, said compound is identified as a potential inhibitor of the CDA enzyme. According to additional particular embodiments of the method, as defined above:
- la etapa (a) comprende adicionalmente obtener para dicho compuesto un valor predictivo de la actividad inhibitoria Log(lnh%), en donde: - step (a) further comprises obtaining for said compound a predictive value of the inhibitory activity Log (lnh%), where:
Log (lnh%) = 0.814 - 0.018 x T(N..N) + 9.552 x JGI2 donde: Log (lnh%) = 0.814 - 0.018 x T (N..N) + 9.552 x JGI2 where:
- T (N..N) es la distancia topológica expresada como número de aristas entre dos nitrógeno consecutivos, y - T (N..N) is the topological distance expressed as the number of edges between two consecutive nitrogen, and
- JGI2 es el índice topológico de carga promedio de orden 2; - JGI2 is the topological average load index of order 2;
- la etapa (b) comprende además verificar si el valor de Log (lnh%) obtenido en (a) para dicho compuesto está entre +1 y +2. Si el valor está entre +1 y +2, dicho compuesto se identifica como un inhibidor potencial de la enzima CDA. - step (b) further comprises verifying if the value of Log (lnh%) obtained in (a) for said compound is between +1 and +2. If the value is between +1 and +2, said compound is identified as a potential inhibitor of the CDA enzyme.
De acuerdo con realizaciones particulares adicionales del método, tal como se ha definido anteriormente: According to additional particular embodiments of the method, as defined above:
- la etapa (a) comprende adicionalmente obtener para dicho compuesto un valor para la función discriminante DF2, en la que: - step (a) further comprises obtaining for said compound a value for the discriminant function DF2, in which:
DF2 = 21.022 - (19.407 x GGI10) - (5.104 x SEige) - (7.911 x GATS3e) donde: DF2 = 21.022 - (19.407 x GGI10) - (5.104 x SEige) - (7.911 x GATS3e) where:
- GGI10: índice topológico de carga de orden 10, - GGI10: topological load index of order 10,
- Seige: Autovalor suma del valor propio de la electronegatividad. - Seige: Eigenvalue sum of the eigenvalue of electronegativity.
- GATS3e: Indice de autocorrelación de Geary -Iag3 / ponderado por electronegatividades atómicas de Sanderson; y. - GATS3e: Geary autocorrelation index -Iag3 / weighted by Sanderson atomic electronegativities; Y.
- la etapa (b) comprende además verificar si el valor DF2 obtenido en (a) para dicho compuesto está entre 0 y +6. Si el valor está entre 0 y +6, dicho compuesto se identifica como un inhibidor potencial de la enzima CDA. - step (b) further comprises verifying whether the DF2 value obtained in (a) for said compound is between 0 and +6. If the value is between 0 and +6, said compound is identified as a potential inhibitor of the CDA enzyme.
De acuerdo con realizaciones particulares adicionales, el método como se define anteriormente comprende después de la etapa (b): According to further particular embodiments, the method as defined above comprises after step (b):
- c) llevar a cabo un análisis experimental del compuesto identificado como un inhibidor potencial de la enzima CDA según la etapa (b), que comprende poner dicho compuesto
en contacto con un patógeno, preferiblemente un patógeno fúngico, y más preferiblemente un patógeno fúngico en presencia de una planta y observar si hay crecimiento de éste. - c) carrying out an experimental analysis of the compound identified as a potential inhibitor of the CDA enzyme according to step (b), which comprises putting said compound in contact with a pathogen, preferably a fungal pathogen, and more preferably a fungal pathogen in the presence of a plant and observe if there is growth of this.
De acuerdo con una realización más particular, el patógeno es un hongo ubicado en una planta, incluso más particularmente Podosphaera xanthii. Según otra realización más particular, la plaga es un insecto incluso más particularmente Gallería mellonella. According to a more particular embodiment, the pathogen is a fungus located on a plant, even more particularly Podosphaera xanthii. According to another more particular embodiment, the pest is an insect even more particularly Galleria mellonella.
De acuerdo con realizaciones particulares adicionales, el método como se define anteriormente comprende además realizar un experimento de docking (acoplamiento) con un fragmento de la enzima CDA que comprende el sitio activo de la misma y el compuesto identificado como un inhibidor de la enzima quitina deacetilasa. Dicho compuesto mostrará un valor de afinidad menor de -7 kcal / mol, en virtud de su capacidad para interactuar con el sitio activo de la enzima CDA. En particular, la enzima CDA utilizada para el experimento de docking es una enzima CDA fúngica, preferentemente la CDA de Colletotrichum lindemuthianum. According to additional particular embodiments, the method as defined above further comprises performing a docking experiment with a fragment of the CDA enzyme comprising the active site thereof and the compound identified as an inhibitor of the enzyme chitin deacetylase. . Said compound will show an affinity value lower than -7 kcal / mol, by virtue of its ability to interact with the active site of the CDA enzyme. In particular, the CDA enzyme used for the docking experiment is a fungal CDA enzyme, preferably the CDA from Colletotrichum lindemuthianum.
De acuerdo con realizaciones particulares adicionales, el método definido anteriormente comprende un experimento de docking con la enzima CDA completa, del compuesto identificado como un inhibidor de la enzima quitina deacetilasa, en el que dicho compuesto muestra dos o tres enlaces de hidrógeno con dos o tres aminoácidos diferentes localizados dentro del sitio activo de la enzima CDA. De acuerdo con una realización más particular, la enzima CDA usada para el experimento de docking es una enzima CDA fúngica, más preferiblemente la CDA de Colletotrichum lindemuthianum. Según otra realización más particular, la enzima CDA usada para el experimento de docking es una enzima CDA de insecto, más preferiblemente la CDA de Bombyx morí.According to additional particular embodiments, the method defined above comprises a docking experiment with the entire CDA enzyme, of the compound identified as an inhibitor of the enzyme chitin deacetylase, in which said compound shows two or three hydrogen bonds with two or three different amino acids located within the active site of the CDA enzyme. According to a more particular embodiment, the CDA enzyme used for the docking experiment is a fungal CDA enzyme, more preferably the CDA from Colletotrichum lindemuthianum. According to another more particular embodiment, the CDA enzyme used for the docking experiment is an insect CDA enzyme, more preferably the Bombyx died CDA.
El método definido anteriormente es útil para identificar compuestos para combatir plagas en el cual las plagas son aquellos organismos que contienen quitina, como hongos, artrópodos o nematodos. The method defined above is useful to identify compounds to combat pests in which the pests are those organisms that contain chitin, such as fungi, arthropods or nematodes.
La presente invención también se refiere al uso de compuestos químicos con actividad pesticida contra un organismo que contiene quitina seleccionado entre hongos, artrópodos y nematodos, identificados por el método definido anteriormente.
De acuerdo con una realización particular, la invención se refiere al uso de un compuesto químico con actividad pesticida contra un organismo que contiene quitina seleccionado entre hongos, artrópodos y nematodos, caracterizado porque: The present invention also relates to the use of chemical compounds with pesticidal activity against a chitin-containing organism selected from fungi, arthropods and nematodes, identified by the method defined above. According to a particular embodiment, the invention refers to the use of a chemical compound with pesticidal activity against an organism that contains chitin selected from fungi, arthropods and nematodes, characterized in that:
- dicho compuesto tiene un valor de DF1 entre 0 y +14, - said compound has a DF1 value between 0 and +14,
- preferiblemente dicho compuesto tiene además un valor de Log (lnh%) entre +1 y +2; - preferably said compound also has a value of Log (lnh%) between +1 and +2;
- más preferiblemente, dicho compuesto tiene además un valor de DF2 entre 0 y +6. - more preferably, said compound also has a DF2 value between 0 and +6.
Según una realización más particular, la invención se refiere al uso de un compuesto químico con actividad pesticida contra un organismo que contiene quitina seleccionado entre hongos, artrópodos y nematodos, como se define anteriormente, caracterizado por que dicho compuesto interacciona con el sitio activo de la enzima CDA de According to a more particular embodiment, the invention refers to the use of a chemical compound with pesticidal activity against an organism that contains chitin selected from fungi, arthropods and nematodes, as defined above, characterized in that said compound interacts with the active site of the CDA enzyme
- C. lindemuthianum y/o - C. lindemuthianum and / or
- B. morii - B. morii
en un experimento de docking caracterizado porque dicho compuesto muestra un valor de afinidad inferior a -7 kcal / mol en virtud de su capacidad para interactuar con el sitio activo de la enzima CDA. in a docking experiment characterized in that said compound shows an affinity value lower than -7 kcal / mol by virtue of its ability to interact with the active site of the CDA enzyme.
De acuerdo con realizaciones concretas, el compuesto químico detectado con actividad pesticida contra un organismo que contiene quitina se selecciona entre: According to specific embodiments, the chemical compound detected with pesticidal activity against a chitin-containing organism is selected from:
- ácido (1-metil-2,4-dioxo-1 ,4,6,7-tetrahidro[1 ,3]tiazol[2,3f]purin-3(2H)il)acético - (1-methyl-2,4-dioxo-1, 4,6,7-tetrahydro [1,3] thiazol [2,3f] purin-3 (2H) yl) acetic acid
- ácido (3-[(1 ,3-dimetil-2,4,6-trioxotetrahidro-5(2H)-pirimidinilen)metil]fenoxi)acético , - (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic acid,
- ácido 2-(2-metoxi-4-([(5Z)-2,4,6-trioxo-1-(prop-2-en-1-il)-1-3-diazinan-5- iliden]metil)fenoxi)acético, - 2- (2-methoxy-4 - ([(5Z) -2,4,6-trioxo-1- (prop-2-en-1-yl) -1-3-diazinan-5-ylidene] methyl acid ) phenoxy) acetic,
- 5-[4-(2-hidroxietoxi)-3-metoxibenziliden]-1 ,3-dimetil-2,4,6(1 H,3H,5H)-pirimidintriona, - 5- [4- (2-hydroxyethoxy) -3-methoxybenzylidene] -1, 3-dimethyl-2,4,6 (1H, 3H, 5H) -pyrimidinetrione,
- 7-[2-hidroxi-3-(4-morfolinil)propil]-1 ,3-dimetil-3,7-dihidro-1 H-purin-2,6-diona, - 7- [2-hydroxy-3- (4-morpholinyl) propyl] -1,3-dimethyl-3,7-dihydro-1 H-purine-2,6-dione,
2-amino-7-metil-5-oxo-4-[4-(trifluorometoxi)fenil]-4H,5H-pirano[4,3-b]pirano-3- carbonitrilo, 2-amino-7-methyl-5-oxo-4- [4- (trifluoromethoxy) phenyl] -4H, 5H-pyrano [4,3-b] pyran-3-carbonitrile,
- N-{4-[3-(2,6-dimetil-4-morfolinil)-2,5-dioxo-1-pirrolidinil]fenil}acetamida, - N- {4- [3- (2,6-dimethyl-4-morpholinyl) -2,5-dioxo-1-pyrrolidinyl] phenyl} acetamide,
- 2-(1 ,3-dimetil-2,6-dioxo-1 ,2,3,6-tetrahidro-7H-purin-7-il)-N-(2-metoxi-1- metiletil)acetamida, - 2- (1,3-dimethyl-2,6-dioxo-1, 2,3,6-tetrahydro-7H-purin-7-yl) -N- (2-methoxy-1-methylethyl) acetamide,
- N-ciclopropil-2-(1 ,3-dimetil-2,6-dioxo-1 ,2,3,6-tetrahidro-9H-purin-9-il)acetamida
- 5-imino-1-(2-metil-5-nitrofenil)-3-fenilidantoina, - N-cyclopropyl-2- (1,3-dimethyl-2,6-dioxo-1, 2,3,6-tetrahydro-9H-purin-9-yl) acetamide - 5-imino-1- (2-methyl-5-nitrophenyl) -3-phenylidantoin,
- 5-imino-1-(4-metil-5-nitrofenil)-3-fenilidantoina, - 5-imino-1- (4-methyl-5-nitrophenyl) -3-phenylidantoin,
- ácido 3-{2,5-dioxo-3-[(5Z)-4-oxo-5-(fenilmetiliden)-2-sulfaniliden-1 ,3-tiazolidin-3- il]pirrolidin-1-il}propanoico - 3- {2,5-dioxo-3 - [(5Z) -4-oxo-5- (phenylmethylidene) -2-sulfanylidene-1,3-thiazolidin-3-yl] pyrrolidin-1-yl} propanoic acid
- ácido 2-(10,12-dioxo-9-{[(1 ,3-tiazol-2-il)carbamoil]metil}-7-tia-9,11- diazatriciclo[6.4.0.02,6]dodeca-1 (8),2(6)-dien-11-il)acético, - 2- (10,12-dioxo-9 acid - {[(1, 3-thiazol-2-yl) carbamoyl] methyl} -7-thia-9,11 - diazatricyclo [6.4.0.0 2, 6] dodeca- 1 (8), 2 (6) -dien-11-yl) acetic,
- ácido 2-{4-[(1 ,3-dimetil-4,6-dioxo-2-sulfaniliden-1 ,3-diazinan-5- iliden)metil]fenoxi}acético, - 2- {4 - [(1,3-dimethyl-4,6-dioxo-2-sulfanylidene-1,3-diazinan-5-ylidene) methyl] phenoxy} acetic acid,
- ácido 2-({[(5E)-4,6-dioxo-1-[4-(propan-2-il)fenil]-2-sulfanil-1 ,4,5,6-tetrahidropirimidin-5- iliden]metil}amino)-3-(1 H-imidazol-4-il)propanoico , - 2 - ({[(5E) -4,6-dioxo-1- [4- (propan-2-yl) phenyl] -2-sulfanyl-1, 4,5,6-tetrahydropyrimidin-5-ylidene] acid methyl} amino) -3- (1 H-imidazol-4-yl) propanoic,
- ácido 2-[(5E)-4-oxo-5-{[2-(prop-2-en-1-iloxi)fenil]metiliden}-2-sulfaniliden-1 ,3- tiazolidin-3-il]pentanedioico, - 2 - [(5E) -4-oxo-5 - {[2- (prop-2-en-1-yloxy) phenyl] methylidene} -2-sulfanilidene-1,3-thiazolidin-3-yl] pentanedioic acid ,
- ácido 2-{1 -[(3,4-difluorofenil)metil]-3-oxopiperazin-2-il}acético, - 2- {1 - [(3,4-difluorophenyl) methyl] -3-oxopiperazin-2-yl} acetic acid,
- ácido 2-{3-[(2-cloro-6-fluorofenil)metil]-2,4,5-trioxoimidazolidin-1-il}acético, - 2- {3 - [(2-chloro-6-fluorophenyl) methyl] -2,4,5-trioxoimidazolidin-1-yl} acetic acid,
- ácido {2-[(1 ,3-dimetil-4,6-dioxo-2-tioxotetrahidro-5(2H)- pirimidiniliden)metil]fenoxi}acético y - {2 - [(1, 3-dimethyl-4,6-dioxo-2-thioxotetrahydro-5 (2H) -pyrimidinylidene) methyl] phenoxy} acetic acid and
- 3-benzil-1 ,7-dimetil-7,9-dihidro-1 H-purin-2,6,8(3H)-triona. - 3-benzyl-1,7-dimethyl-7,9-dihydro-1 H-purin-2,6,8 (3H) -trione.
En la Tabla 1 están detallados 20 compuestos con actividad pesticida predicha por topología molecular, así como sus respectivos valores de DF1 , Log(inh%) y DF2.
Table 1 lists 20 compounds with pesticidal activity predicted by molecular topology, as well as their respective DF1, Log (inh%) and DF2 values.
TABLA 1 TABLE 1
en in
Otros patógenos fúngicos incluyen las clases Ascomycota y Basidiomycota, así como patógenos de la clase Oomycetes, en cualquier cereal, viña, árbol frutal, cultivos hortícolas, de fibra y/o ornamentales. Estos patógenos incluyen: Ascomicetos, patógenos incluidos en el orden Erysiphales tales como Blumeria graminis, Erysiphe necator, Erysiphe polygoni, Leveillula táurica, Podosphaera aphanis y Podosphaera xanthii, y otros patógenos incluyendo Alternaría solani, Botrytis cinérea, Cercospora beticola, Colletotrichum graminicola, Fusarium graminearum, Fusarium oxysporum, Gaeumannomyces graminis, Magnaporthe grísea, Monilinia fructicola, Mycosphaereiia fijensis, Phomopsis vitícola, Pyrenophora teres, Rhizopus stolonifer, Rynchosporium secalis, Sclerotinia sclerotiorum, Septoria tritici, Venturia inaequalis y Verticillium dahliae ; Basidiomicetos, patógenos del orden Uredinales tales como Puccinia spp. ( Puccinia striiformis, Puccinia hordei, Puccinia graminis) y Uromyces spp. ( Uromyces viciae-fabae, Uromyces betae ), patógenos en el orden Ustilaginales tales como Ustilago maydis y Ustilago tritici, y otros patógenos incluidos Armillaria mellea, Rhizoctonia solani y Sclerotium roifsir, Oomicetos, incluidos patógenos Phytophthora tales como Phytophthora infestans y Phytophthora cinnamomi, patógenos Pythium tales como Pythium aphanidermatum, y patógenos de la familia Peronosporaceae tales como Plasmopara vitícola, Pseudoperonospora cubensis y Bremia lactucae ; y otros géneros y especies estrechamente relacionadas con estos patógenos. Other fungal pathogens include the Ascomycota and Basidiomycota classes, as well as pathogens of the Oomycetes class, on any cereal, vine, fruit tree, horticultural, fiber and / or ornamental crops. These pathogens include: Ascomycetes, pathogens included in the order Erysiphales such as Blumeria graminis, Erysiphe necator, Erysiphe polygoni, Leveillula taurica, Podosphaera aphanis, and Podosphaera xanthii, and other pathogens including Alternaría solani, Botrytis Columnarichumumumumumumumumumumumumumumumumumumumumumumumumumumumumthorn , Fusarium oxysporum, Gaeumannomyces graminis, Magnaporthe grísea, Monilinia fructicola, Mycosphaereiia fijensis, Phomopsis viticola, Pyrenophora teres, Rhizopus stolonifer, Rynchosporium secalis, Sclerotinia sclerotiorum, Septoria tritalis and Venturiaeicillium; Basidiomycetes, pathogens of the order Uredinales such as Puccinia spp. (Puccinia striiformis, Puccinia hordei, Puccinia graminis) and Uromyces spp. (Uromyces viciae-fabae, Uromyces betae), pathogens in the order Ustilaginales such as Ustilago maydis and Ustilago tritici, and other pathogens including Armillaria mellea, Rhizoctonia solani, and Sclerotium roifsir, Oomycetes, including pathogens Phytophthora, Phynophthora infestaytophyogens such as Phytophthora infestaytogens Pythium such as Pythium aphanidermatum, and pathogens of the Peronosporaceae family such as Plasmopara vitícola, Pseudoperonospora cubensis and Bremia lactucae; and other genera and species closely related to these pathogens.
Los compuestos de la invención son útiles para el control de plagas tales como, artrópodos, particularmente insectos, ácaros y piojos, de la casa, el jardín, el ganado y la agricultura. The compounds of the invention are useful for the control of pests such as arthropods, particularly insects, mites and lice, of the house, garden, livestock and agriculture.
Realizaciones particulares adicionales se refieren al uso de los compuestos identificados siguiendo el método descrito anteriormente, para controlar insectos en estadios de larvas, preferiblemente de la infraclase Neoptera, que incluyen, pero no se limitan, a los órdenes Lepidoptera, Homoptera, Isoptera, Díptera, Orthoptera, Hemiptera y Coleóptera. Más preferiblemente, Drosophila meianogaster y Gallería mellonella, entre otros.
En particular, otras plagas representativas que son controladas por los compuestos de la invención incluyen miembros del filo Arthropoda, incluyendo ácaros de los subórdenes Mesostigmata, Sarcoptiformes, Trombidiformes y Onchychopalpida; y piojos de los órdenes Anoplura y Mallophaga. Realizaciones particulares se refieren al uso de los compuestos identificados siguiendo el método descrito anteriormente para inhibir el crecimiento del nematodo modelo Caenorhabditis elegans. Additional particular embodiments refer to the use of the compounds identified following the method described above, to control insects in larval stages, preferably of the infraclass Neoptera, which include, but are not limited to, the orders Lepidoptera, Homoptera, Isoptera, Diptera, Orthoptera, Hemiptera and Coleóptera. Most preferably, Drosophila meianogaster and Galleria mellonella, among others. In particular, other representative pests that are controlled by the compounds of the invention include members of the phylum Arthropoda, including mites of the suborders Mesostigmata, Sarcoptiformes, Trombidiformes, and Onchychopalpida; and lice of the orders Anoplura and Mallophaga. Particular embodiments relate to the use of the compounds identified following the method described above to inhibit the growth of the model nematode Caenorhabditis elegans.
En particular, los compuestos de la invención son útiles para el control de nematodos parásitos, que incluyen, pero no se limitan a, nematodos parásitos de plantas del orden Tylenchida, especialmente especies tales como Meloidogyne spp., Heterodera spp., Rotylenchus spp. y Pratylenchus spp. En particular, los nematodos parásitos de mamíferos, incluidos seres humanos, ganado y mascotas también son controlados por los compuestos de la invención, incluidos los órdenes Ascaridia, Spirurida y Strongylida, y en particular especies tales como Trichinella spp., Trichuris spp. y Strongyloides spp. Los inventores han desarrollado un método para la identificación de compuestos para combatir plagas agrícolas y otras plagas y parásitos de origen no agrícola. Además, la efectividad de esos compuestos contra hongos, insectos y nematodos ha sido confirmada. In particular, the compounds of the invention are useful for the control of parasitic nematodes, including, but not limited to, parasitic nematodes of plants of the order Tylenchida, especially species such as Meloidogyne spp., Heterodera spp., Rotylenchus spp. and Pratylenchus spp. In particular, the parasitic nematodes of mammals, including humans, livestock and pets are also controlled by the compounds of the invention, including the orders Ascaridia, Spirurida and Strongylida, and in particular species such as Trichinella spp., Trichuris spp. and Strongyloides spp. The inventors have developed a method for the identification of compounds to combat agricultural pests and other pests and parasites of non-agricultural origin. Furthermore, the effectiveness of these compounds against fungi, insects and nematodes has been confirmed.
BREVE DESCRIPCIÓN DE LAS FIGURAS. BRIEF DESCRIPTION OF THE FIGURES.
FIGURA 1 Diagrama de flujo de las pruebas experimentales utilizadas para la identificación de inhibidores de CDA. FIGURE 1 Flow diagram of the experimental tests used for the identification of CDA inhibitors.
FIGURA 2 Efectos fungicidas de algunos de los compuestos seleccionados contra oídio de cucurbitáceas ( P . xanthii) en el ensayo de planta completa. FIGURE 2 Fungicidal effects of some of the selected compounds against cucurbit powdery mildew (P. Xanthii) in the whole plant test.
FIGURA 3 Efectos insecticidas de algunos compuestos identificados mediante el método de topología molecular en G. mellonella. FIGURE 3 Insecticidal effects of some compounds identified by the molecular topology method in G. mellonella.
FIGURA 4 Micrografías del ensayo de toxicidad de C. elegans realizado, mostrando los efectos de algunos de los compuestos identificados mediante el método de topología molecular sobre el desarrollo de nematodos.
FIGURA 5 Efectos fisiológicos sobre las respuestas de las plantas al tratamiento con los compuestos con la mejor actividad fungicida sobre cotiledones de melón infectados con P. xanthii. FIGURE 4 Micrographs of the C. elegans toxicity test performed, showing the effects of some of the compounds identified by the molecular topology method on the development of nematodes. FIGURE 5 Physiological effects on the responses of plants to treatment with the compounds with the best fungicidal activity on melon cotyledons infected with P. xanthii.
EJEMPLOS EXAMPLES
Ejemplo 1 : Desarrollo del método de topología molecular e identificación de compuestos. Example 1: Development of the molecular topology method and identification of compounds.
Se han identificado varios ácidos carboxílicos como inhibidores de CDA. Particularmente, EDTA, (GlcNAc)2, ácido láctico y ácido propiónico. Además, el mecanismo de acción del EDTA no está vinculado a su papel como agente quelante, sino a su efecto como inhibidor de la enzima. Además, todos ellos eran activos en concentraciones superiores a 10 Mm3. Para identificar nuevos compuestos con mayor actividad inhibitoria, se diseñó un enfoque basado en la topología molecular, Figura 1.Several carboxylic acids have been identified as inhibitors of CDA. In particular, EDTA, (GlcNAc) 2, lactic acid and propionic acid. Furthermore, the mechanism of action of EDTA is not linked to its role as a chelating agent, but to its effect as an enzyme inhibitor. Furthermore, all of them were active in concentrations higher than 10 Mm 3 . To identify new compounds with higher inhibitory activity, an approach based on molecular topology was designed, Figure 1.
Se desarrolló un enfoque basado en la topología molecular comenzando con las estructuras químicas de los compuestos químicos derivados de ácidos carboxílicos, con y sin actividad anti-CDA, previamente determinada por medios experimentales (Tabla 1) para la identificación de nuevas moléculas con mayor actividad inhibidora de CDA, Las estructuras químicas enumeradas en la Tabla 2 se convirtieron en grafos y luego se convirtieron en descriptores topológicos 2D utilizando la teoría de grafos. A continuación, se aplica el análisis discriminante lineal (LDA) para calcular una función discriminante (DF1)10. An approach based on molecular topology was developed starting with the chemical structures of the chemical compounds derived from carboxylic acids, with and without anti-CDA activity, previously determined by experimental means (Table 1) for the identification of new molecules with higher inhibitory activity. From CDA, the chemical structures listed in Table 2 were converted to graphs and then converted to 2D topological descriptors using graph theory. Next, linear discriminant analysis (LDA) is applied to calculate a discriminant function (DF1) 10 .
DF1 = (-10.295 x GATS4m) + (35.124 x GGI8) + 6.917 donde GATS4m es el índice de autocorrelación Geary - Iag4 / ponderado por masas atómicas; GGI8 es el índice topológico de carga de orden 8. Estos parámetros se calcularon utilizando el software DRAGON11. DF1 = (-10,295 x GATS4m) + (35,124 x GGI8) + 6,917 where GATS4m is the Geary-Iag4 / atomic mass-weighted autocorrelation index; GGI8 is the 8th order topological load index. These parameters were calculated using DRAGON 11 software.
Los compuestos activos (A) son aquellos que tienen un valor DFi en el rango entre 0 y +14. Por lo tanto, este modelo es apropiado para clasificar compuestos químicos de acuerdo con su capacidad para inhibir la enzima CDA. Active compounds (A) are those that have a DFi value in the range between 0 and +14. Therefore, this model is appropriate for classifying chemical compounds according to their ability to inhibit the CDA enzyme.
TABLA 2 TABLE 2
* N-Acetilglucosamina * N-Acetylglucosamine
A continuación, se analizaron varias bases de datos tales como SPECS, ChEMBL y eMolecules, aplicando la función DF1. Estas bases de datos comprenden más de 3.000.000 compuestos. Si el valor discriminante resultante está entre 0 y +14, ese compuesto se considerará como un inhibidor de CDA, mientras que, si el valor discriminante está fuera de ese rango, no se considerará como un inhibidor de CDA. En la Tabla 3 se enumeran los compuestos seleccionados de este cribado. Posteriormente, la capacidad inhibitoria de CDA de esos compuestos se probó experimentalmente en el laboratorio. Next, several databases such as SPECS, ChEMBL and eMolecules were analyzed, applying the DF1 function. These databases comprise more than 3,000,000 compounds. If the resulting discriminant value is between 0 and +14, that compound will be considered a CDA inhibitor, whereas if the discriminant value is outside that range, it will not be considered a CDA inhibitor. Compounds selected from this screen are listed in Table 3. Subsequently, the CDA inhibitory capacity of these compounds was experimentally tested in the laboratory.
TABLA 3 TABLE 3
GATS4m, GGI8 DF1 Prob(Act) Inh (%) exp a Valor de la función discriminante, DF1 , obtenido para cada compuesto (DF1 = (-10.295 x GATS4m) + (35.124 x GGI8) + 6.917); donde GATS4m es el índice de autocorrelación de Geary - Iag4 / ponderado por masas atómicas; GGI8, índice topológico de carga de orden 8. GATS4m, GGI8 DF1 Prob (Act) Inh (%) exp a Value of the discriminant function, DF1, obtained for each compound (DF1 = (-10,295 x GATS4m) + (35,124 x GGI8) + 6,917); where GATS4m is the Geary autocorrelation index - Iag4 / weighted by atomic masses; GGI8, topological load index of order 8.
b Probabilidad con la que el modelo lo clasifica como activo. b Probability with which the model classifies it as active.
c Inhibición experimental de CDA (%). La mayoría de los compuestos que tienen un DF1 entre 0 y 14 fueron experimentalmente activos contra P. xanthii. Tabla 2, lnh(%)(exp). A continuación, los resultados
experimentales se utilizaron para desarrollar un análisis de regresión multilineal10 para predecir la actividad inhibitoria de los nuevos candidatos. Se obtuvo así una segunda ecuación predictiva con mayor poder discriminante que DF1. c Experimental inhibition of CDA (%). Most of the compounds having a DF1 between 0 and 14 were experimentally active against P. xanthii. Table 2, lnh (%) (exp). Then the results Experiments were used to develop a multilinear regression analysis 10 to predict the inhibitory activity of the new candidates. Thus, a second predictive equation was obtained with a higher discriminating power than DF1.
La ecuación de correlación es: log(lnh%)= 0.814 - 0.018 x T(N..N) + 9.552 x JGI2 The correlation equation is: log (lnh%) = 0.814 - 0.018 x T (N..N) + 9.552 x JGI2
N=8; R2=0.803; Q2=0.657; SEE=0.112; F (2,5)=10.2; p=0.02,N = 8; R 2 = 0.803; Q 2 = 0.657; SEE = 0.112; F (2,5) = 10.2; p = 0.02,
N, Número de compuestos activos al ser testados N, Number of active compounds when tested
R2, coeficiente de determinación; R 2 , coefficient of determination;
Q2, coeficiente de predicción (determinado por validación cruzada); Q 2 , prediction coefficient (determined by cross validation);
SEE, Error estándar SEE, standard error
F, parámetro de Fisher-Snedecor F, Fisher-Snedecor parameter
p, significación estadística p, statistical significance
T (N..N) es la distancia topológica expresada como número de aristas entre dos nitrógenos consecutivos. T (N..N) is the topological distance expressed as the number of edges between two consecutive nitrogens.
JGI2 indica el índice topológico de carga promedio de orden 2. JGI2 indicates the topological average load index of order 2.
Estos parámetros se calcularon utilizando el software DRAGON11. These parameters were calculated using the DRAGON 11 software.
En la Tabla 4 se ilustran los compuestos identificados en el primer cribado, junto con sus valores experimentales. Las moléculas que mostraron alguna actividad experimental se utilizaron para desarrollar un análisis de regresión multilineal. Los descriptores utilizados en la Función 2 se señalan en la Tabla 3. En la última columna, Log (lnh%) Calcd, se puede observar el valor predicho de la actividad inhibitoria de CDA obtenida mediante el análisis de regresión multilineal (Función 2). Las moléculas que tienen un valor predicho de actividad inhibitoria (Log (lnh%) entre +1 y +2, fueron las que dieron resultados experimentales positivos.
TABLA 4 c valor inhibitorio experimental (log) Compounds identified in the first screening, along with their experimental values, are illustrated in Table 4. The molecules that showed some experimental activity were used to develop a multilinear regression analysis. The descriptors used in Function 2 are indicated in Table 3. In the last column, Log (lnh%) Calc d , the predicted value of the inhibitory activity of CDA obtained through multilinear regression analysis can be observed (Function 2) . The molecules that have a predicted value of inhibitory activity (Log (lnh%) between +1 and +2, were those that gave positive experimental results. TABLE 4 c experimental inhibitory value (log)
d valor inhibitorio calculado (log) d calculated inhibitory value (log)
Se realiza un segundo cribado con la segunda función como se describe anteriormente. Esta vez, se seleccionará un compuesto como posible inhibidor de CDA si tiene un valor discriminante DF1 entre 0 y +14 y un valor predictivo de actividad inhibitoria (Log (lnh%) entre +1 y +2.
Por lo tanto, los resultados de las pruebas de laboratorio nos ayudaron a obtener nuevas ecuaciones que refinaron los modelos matemáticos. La Tabla 5 muestra la lista de los compuestos elegidos después de la segunda iteración de la topología molecular. También se incluyen sus valores de DF1 y Log(lnh%) calculados, así como los valores de los índices topológicos que aparecen en ambas ecuaciones.
A second screen is performed with the second function as described above. This time, a compound will be selected as a possible CDA inhibitor if it has a DF1 discriminant value between 0 and +14 and a predictive value of inhibitory activity (Log (lnh%) between +1 and +2. Therefore, the results of the laboratory tests helped us to obtain new equations that refined the mathematical models. Table 5 shows the list of compounds chosen after the second iteration of the molecular topology. Also included are their calculated DF1 and Log (lnh%) values, as well as the values of the topological indices that appear in both equations.
TABLA 5 TABLE 5
Antes de comenzar las pruebas in vitro con los compuestos obtenidos en la segunda iteración por topología molecular, se llevó a cabo un experimento de docking. Los detalles se explican en el Ejemplo 1.3. Después de eso, los compuestos enumerados en la Tabla 4 se testaron experimentalmente. Before starting the in vitro tests with the compounds obtained in the second iteration by molecular topology, a docking experiment was carried out. Details are explained in Example 1.3. After that, the compounds listed in Table 4 were experimentally tested.
Aunque las últimas 8 moléculas de la Tabla 5 no tenían ninguna actividad en los resultados experimentales, se tuvieron en cuenta para obtener una nueva función discriminante (DF2), en la que esos 8 compuestos se introducen como inactivos. Although the last 8 molecules in Table 5 had no activity in the experimental results, they were taken into account to obtain a new discriminant function (DF2), in which those 8 compounds are introduced as inactive.
Finalmente, se obtiene una ecuación discriminante (DF2) final: Finally, a final discriminant equation (DF2) is obtained:
DF2 = 21.022 - (19.407 x GGI10) - (5.104 x SEige) - (7.911 x GATS3e) DF2 = 21.022 - (19.407 x GGI10) - (5.104 x SEige) - (7.911 x GATS3e)
N=35 l=0.450 F (3.31)=12.3 p<0.00001 donde N es el número de compuestos; N = 35 l = 0.450 F (3.31) = 12.3 p <0.00001 where N is the number of compounds;
F es el parámetro de Fisher - Snedecor; F is the Fisher-Snedecor parameter;
l es la lambda de Wilks; y l is Wilks lambda; Y
p es la significación estadística. donde: p is the statistical significance. where:
- GGI10: índice topológico de carga de orden 10, - GGI10: topological load index of order 10,
- Seige: Autovalor suma del valor propio de la electronegatividad. - Seige: Eigenvalue sum of the eigenvalue of electronegativity.
- GATS3e: Indice de autocorrelación Geary -Iag3 / ponderado por electronegatividades atómicas de Sanderson, - GATS3e: Geary autocorrelation index -Iag3 / weighted by Sanderson atomic electronegativities,
estos parámetros se obtuvieron utilizando el software DRAGON11, these parameters were obtained using the DRAGON 11 software,
La Tabla 6 muestra la lista de las moléculas detectadas en los enfoques de topología molecular 1 y 2 (Tabla 2 y Tabla 4, respectivamente) clasificadas según los valores de DF2. Table 6 shows the list of the molecules detected in molecular topology approaches 1 and 2 (Table 2 and Table 4, respectively) classified according to DF2 values.
Se elegirá un compuesto como inhibidor de CDA si, además de las condiciones impuestas por DF1 y Log (lnh%), muestran un valor de DF2 entre 0 y 6 y un valor de afinidad de docking inferior a -7 kcal / mol.
TABLA 6 A compound will be chosen as CDA inhibitor if, in addition to the conditions imposed by DF1 and Log (lnh%), they show a DF2 value between 0 and 6 and a docking affinity value lower than -7 kcal / mol. TABLE 6
Los compuestos subrayados y en cursiva son aquellos incorrectamente clasificados. a % inhibición de CDA para cada compuesto basado en ensayos experimentales; Compounds underlined and italicized are those incorrectly classified. a % inhibition of CDA for each compound based on experimental tests;
b Clasificación de los compuestos basada en los ensayos experimentales; b Classification of compounds based on experimental tests;
c Valor de la función discriminante, DF2 = 21.022 - (19.407 x GGI10) - (5.104 x Seige) - (7.911 x GATS3e), y c Value of the discriminant function, DF2 = 21.022 - (19.407 x GGI10) - (5.104 x Seige) - (7.911 x GATS3e), and
d Probabilidad de actividad basada en los resultados de DF2. d Activity probability based on DF2 results.
Ejemplo 1.2: Validación experimental de los compuestos identificados por un método de topología molecular como fungicidas antioídio. Example 1.2: Experimental validation of the compounds identified by a molecular topology method as antioid fungicides.
Los compuestos identificados mediante cribado de topología molecular se probaron primero para determinar la actividad fungicida en discos de cotiledones de calabacín contra dos aislados de P. xanthii, los aislados 2086 y SF60. Se usó un ensayo de disco de hoja descrito previamente para la prueba de sensibilidad a fungicidas5 con modificaciones menores. Antes de la aplicación de los tratamientos, los discos de cotiledón se inocularon en el centro con conidios de P. xanthii y se incubaron durante 24 h a 25 °C y un ciclo de luz/oscuridad de 16 h de luz / de 8 h de oscuridad. Después de esta incubación, los discos se sumergieron en la solución del compuesto correspondiente (1 o 100 mM) o una solución acuosa de acetona al 1 % que también se incluyó como control negativo, y se incubaron en las mismas condiciones durante 7 días. Después de la incubación, se evaluó la superficie de la hoja cubierta por el hongo mediante análisis digital. Para este análisis, las imágenes se capturaron con una cámara digital con una distancia fija desde los discos de 20 cm. Las imágenes se analizaron utilizando el software gratuito de procesamiento de imágenes Java Image para calcular la superficie cubierta por síntomas de oídio en cada disco. Compounds identified by molecular topology screening were first tested for fungicidal activity in zucchini cotyledon discs against two isolates of P. xanthii, isolates 2086 and SF60. Assay previously described leaf disc was used to test sensitivity to fungicides 5 with minor modifications. Before applying the treatments, the cotyledon discs were inoculated in the center with conidia of P. xanthii and incubated for 24 h at 25 ° C and a light / dark cycle of 16 h light / 8 h dark. . After this incubation, the discs were immersed in the solution of the corresponding compound (1 or 100 mM) or a 1% aqueous solution of acetone that was also included as a negative control, and incubated under the same conditions for 7 days. After incubation, the surface of the leaf covered by the fungus was evaluated by digital analysis. For this analysis, the images were captured with a digital camera with a fixed distance from the discs of 20 cm. The images were analyzed using the free Java Image image processing software to calculate the area covered by powdery mildew symptoms on each disk.
TABLA 7 TABLE 7
La Tabla 7 muestra el efecto fungicida de los compuestos identificados por el método de topología molecular sobre el desarrollo de oídio de cucurbitáceas (P. xanthii) en el ensayo del disco de hoja. Varios compuestos mostraron una eficacia superior al 50% en la reducción de los síntomas de la enfermedad en comparación con el control negativo (agua). Sin embargo, la respuesta fue diferente según la cepa fúngica y la concentración utilizada. Table 7 shows the fungicidal effect of the compounds identified by the molecular topology method on the development of cucurbit powdery mildew (P. xanthii) in the leaf disk test. Several compounds showed greater than 50% efficacy in reducing disease symptoms compared to the negative control (water). However, the response was different depending on the fungal strain and the concentration used.
Ejemplo 1.3: Dockinq molecular de una CDA fúnqica a los compuestos identificados Example 1.3: Molecular Dockinq from a fungal ADC to the identified compounds
Con el fin de confirmar la unión específica de los compuestos identificados en el cribado contra la enzima CDA, se realizó un experimento de docking molecular contra un fragmento de una proteína CDA fúngica que comprendía el sitio activo. Este filtro solo se aplicó a los compuestos que habían pasado los tres filtros anteriores. Aquellos compuestos con un valor inferior a -7 kcal/mol en el estudio de docking o fueron considerados activos. In order to confirm the specific binding of the compounds identified in the screening against the CDA enzyme, a molecular docking experiment was performed against a fragment of a fungal CDA protein that comprised the active site. This filter was only applied to compounds that had passed the previous three filters. Those compounds with a value lower than -7 kcal / mol in the docking study or were considered active.
Se realizó un experimento de docking con un fragmento de CDA de Colletotrichum lindemuthianum, que contiene el centro activo y donde ocurre la unión a los compuestos. Esta proteína fue elegida debido a su abundante descripción en la literatura12. La estructura proteica (2I W0) se obtuvo del Protein Databank (PDB). Para realizar el análisis de docking se utilizó el programa AutoDock Vina. A docking experiment was performed with a CDA fragment from Colletotrichum lindemuthianum, which contains the active center and where the binding to the compounds occurs. This protein was chosen due to its abundant description in the literature 12 . The protein structure (2I W0) was obtained from the Protein Databank (PDB). To perform the docking analysis, the AutoDock Vina program was used.
Las coordinadas aplicadas se detallan a continuación: The applied coordinates are detailed below:
receptor = cda.pdbqt receiver = cda.pdbqt
ligand = xxxx.pdbqt ligand = xxxx.pdbqt
- out = a//.pdbqt - out = a //. pdbqt
center_x = 13.3 center_x = 13.3
center_y = 12.6 center_y = 12.6
center_z = 11.3 center_z = 11.3
size_x = 36 size_x = 36
size_y = 36
size_z = 36 size_y = 36 size_z = 36
exhaustiveness = 8 exhaustiveness = 8
Estos son los valores que representan el espacio 3D en el que el ligando parece la mejor combinación para encajar en la proteína. El área comprende el sitio activo de CDA, en el que están presentes el átomo de Zn y otros aminoácidos esenciales para la actividad de CDA. Los resultados de docking devueltos por AutoDock Vina indicaron que el compuesto ácido (3-[(1 ,3-dimetil-2,4,6-trioxotetrahidro-5(2H)- pirimidinilen)metil]fenoxi)acético tenía los mejores resultados de afinidad como inhibidor de CDA. Esta molécula forma varios enlaces de hidrógeno con aminoácidos en el sitio activo de CDA. Los compuestos probados en este ensayo y los resultados obtenidos se muestran en la Tabla 8. These are the values that represent the 3D space in which the ligand appears to be the best match to fit the protein. The area comprises the active site of CDA, in which the Zn atom and other essential amino acids for CDA activity are present. The docking results returned by AutoDock Vina indicated that the acid compound (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic acid had the best affinity results as a CDA inhibitor. This molecule forms several hydrogen bonds with amino acids in the active site of CDA. The compounds tested in this test and the results obtained are shown in Table 8.
TABLA 8 TABLE 8
Ejemplo 2: Ensayos de sensibilidad a fungicidas sobre plantas v frutas Example 2: Fungicide sensitivity tests on plants and fruits
Ensayo de plántulas contra oídio de cucurbitáceas Seedling test against Cucurbit powdery mildew
El ensayo del disco de la hoja es un buen ensayo para un primer cribado de la eficacia de los compuestos fungicidas contra oídios. Sin embargo, la actividad fungicida predicha de estos compuestos no está presumiblemente asociada con su toxicidad sino con su capacidad para activar la inmunidad disparada por quitina. Como para la eficacia de esta respuesta la distribución del inoculo puede ser crucial, decidimos probar los compuestos en un ensayo de plantas utilizando un inoculo dispersado. Por lo tanto, después se realizó un ensayo de plántulas utilizando la cepa 2086 de P. xanthii y solo una concentración (100 mM). Para el ensayo de plántulas, se utilizaron plántulas de melón de 2 semanas de edad. Las plantas se inocularon pulverizando una suspensión de conidios de P. xanthii (1 x 104 conidios/ml) hasta el punto de escurrimiento. Veinticuatro horas después de la inoculación, las hojas se rociaron con la solución del compuesto (100 mM). También se incluyó una solución acuosa de acetona al 1 % como control negativo. Las plantas se mantuvieron en una cámara de crecimiento en un ciclo de 16 h de luz / 8 h de oscuridad a 25 °C durante 12 días. Después de esta incubación, los síntomas de la enfermedad se evaluaron mediante análisis digital como se indicó anteriormente.
The leaf disk test is a good test for a first screening of the efficacy of fungicidal compounds against powdery mildew. However, the predicted fungicidal activity of these compounds is presumably not associated with their toxicity but rather with their ability to activate chitin-triggered immunity. Since inoculum distribution may be crucial for the efficacy of this response, we decided to test the compounds in a plant assay using a dispersed inoculum. Therefore, a seedling assay was then performed using P. xanthii strain 2086 and only one concentration (100 mM). For the seedling test, 2-week-old melon seedlings were used. Plants were inoculated by spraying a suspension of P. xanthii conidia (1 x 10 4 conidia / ml) to the point of runoff. Twenty-four hours after inoculation, the leaves were sprayed with the compound solution (100 mM). A 1% acetone aqueous solution was also included as a negative control. The plants were kept in a growth chamber on a 16 h light / 8 h dark cycle at 25 ° C for 12 days. After this incubation, disease symptoms were assessed by digital analysis as indicated above.
TABLA 9 TABLE 9
La Tabla 9 muestra el efecto fungicida de los compuestos identificados por el método de topología molecular sobre el oídio de cucurbitáceas P. xanthii. La eficacia del compuesto se determinó mediante la fórmula de Abbott y las diferencias estadísticas se calcularon utilizando el método de la diferencia mínima significativa (LSD) de Fisher. En términos generales, la eficacia de los compuestos aumentó en comparación con el ensayo en disco de hoja. Los compuestos con la actividad fungicida más significativa sobre P. xanthii según el análisis estadístico realizado fueron: ácido (1-metil-2,4-dioxo-1 ,4,6,7-tetrahidro[1 ,3]tiazol[2,3f]purin-3(2H)il)acético, ácido (3-[(1 ,3-dimetil-2,4,6-trioxotetrahidro-5(2H)- pirimidinilen)metil]fenoxi)acético, Table 9 shows the fungicidal effect of the compounds identified by the molecular topology method on the powdery mildew of cucurbits P. xanthii. The efficacy of the compound was determined by the Abbott formula and statistical differences were calculated using Fisher's least significant difference (LSD) method. Generally speaking, the efficacy of the compounds was increased compared to the leaf disk test. The compounds with the most significant fungicidal activity on P. xanthii according to the statistical analysis performed were: acid (1-methyl-2,4-dioxo-1, 4,6,7-tetrahydro [1, 3] thiazol [2,3f ] purin-3 (2H) yl) acetic acid, (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic acid,
ácido 2-(2-metoxi-4-([(5Z)-2,4,6-trioxo-1-(prop-2-en-1-il)-1-3-diazinan-5- iliden]metil)fenoxi)acético, 2- (2-methoxy-4 - ([(5Z) -2,4,6-trioxo-1- (prop-2-en-1-yl) -1-3-diazinan-5-ylidene] methyl) acid phenoxy) acetic,
ácido 3-{2,5-dioxo-3-[(5Z)-4-oxo-5-(fenilmetiliden)-2-sulfaniliden-1 ,3-tiazolidin-3- il]pirrolidin-1-il}propanoico, 3- {2,5-dioxo-3 - [(5Z) -4-oxo-5- (phenylmethylidene) -2-sulfanylidene-1,3-thiazolidin-3-yl] pyrrolidin-1-yl} propanoic acid,
7-[2-hidroxi-3-(4-morfolinil)propil]-1 ,3-dimetil-3,7-dihidro-1 H-purin-2,6-diona, y 5-[4-(2-hidroxietoxi)-3-metoxibenziliden]-1 ,3-dimetil-2,4,6(1 H,3H,5H)- pirimidintriona. 7- [2-hydroxy-3- (4-morpholinyl) propyl] -1, 3-dimethyl-3,7-dihydro-1 H-purine-2,6-dione, and 5- [4- (2-hydroxyethoxy ) -3-Methoxybenzylidene] -1,3-dimethyl-2,4,6 (1H, 3H, 5H) -pyrimidinetrione.
Los compuestos con el mejor potencial como fungicidas de acuerdo con el ensayo de plántulas se probaron más a fondo en ensayos de plantas y frutas para determinar su potencial fungicida contra tres enfermedades principales causadas por hongos, el oídio de las cucurbitáceas (P. xanthii), la podredumbre gris del tomate ( Botrytis cinérea) y la podredumbre verde de los cítricos ( Penicillium digitatum). Compounds with the best potential as fungicides according to the seedling test were further tested in plant and fruit trials to determine their fungicidal potential against three main fungal diseases, Cucurbit powdery mildew (P. xanthii), the gray rot of tomato (Botrytis cinérea) and the green rot of citrus (Penicillium digitatum).
Ensayo de plantas frente a oídio de cucurbitáceas Plant test against cucurbit powdery mildew
Para el ensayo de plantas se usaron plantas de melón de 6 semanas de edad. Para este ensayo, la inoculación del patógeno, la aplicación de los compuestos (100 mM) y la evaluación de los síntomas, se realizaron como se describió anteriormente para el ensayo de plántulas. Melon plants 6 weeks old were used for the plant test. For this assay, inoculation of the pathogen, application of compounds (100 mM), and evaluation of symptoms were performed as described above for the seedling assay.
Ensayo de frutas frente a la podredumbre gris del tomate y la podredumbre verde de los cítricos
En estos ensayos se utilizaron tomates y naranjas comerciales13'14. Ocho horas después de la inoculación de las frutas con 30 ml de una solución de esporas (1x103 esporas/ml) del patógeno correspondiente, se aplicaron los tratamientos sumergiendo las frutas en las soluciones de los compuestos (100 mM) durante 1 min en el caso de tomates o 2 min en el caso de las naranjas. También se incluyó una solución acuosa de acetona al 1% como control negativo. Los frutos se incubaron en cajas y en oscuridad y a la temperatura adecuada (22 °C para los tomates o 25 °C para las naranjas) durante 5-6 días hasta el desarrollo de síntomas en los controles negativos. Los síntomas de la enfermedad (superficie de fruta cubierta por crecimiento fúngico) se evaluaron mediante análisis digital como se indicó anteriormente. Fruit test against gray rot of tomato and green rot of citrus In these trials tomatoes and oranges 13 trade '14 were used. Eight hours after inoculating the fruits with 30 ml of a spore solution (1x10 3 spores / ml) of the corresponding pathogen, the treatments were applied by immersing the fruits in the solutions of the compounds (100 mM) for 1 min in the in the case of tomatoes or 2 min in the case of oranges. A 1% acetone aqueous solution was also included as a negative control. The fruits were incubated in boxes and in the dark and at the appropriate temperature (22 ° C for tomatoes or 25 ° C for oranges) for 5-6 days until the development of symptoms in the negative controls. Symptoms of the disease (fruit surface covered by fungal growth) were evaluated by digital analysis as indicated above.
La Figura 2 muestra el efecto fungicida de algunos de los compuestos seleccionados contra el oídio de cucurbitáceas ( P . xanthii) en el ensayo de la planta. Las hojas de melón muestran una fuerte reducción en el número de colonias de oídio en las hojas tratadas con los compuestos seleccionados. Figure 2 shows the fungicidal effect of some of the selected compounds against cucurbit powdery mildew (P. Xanthii) in the plant test. Melon leaves show a strong reduction in the number of powdery mildew colonies on the leaves treated with the selected compounds.
TABLA 10 TABLE 10
La Tabla 10 muestra el efecto fungicida de los compuestos seleccionados en ensayos de plantas y frutas contra tres enfermedades fúngicas de plantas, de importancia, oídio de cucurbitáceas ( P . xanthii), podredumbre gris del tomate ( B . cinérea ) y podredumbre verde de los cítricos ( P . digitatum).
TABLA 11 Table 10 shows the fungicidal effect of the selected compounds in plant and fruit tests against three important fungal plant diseases, cucurbit powdery mildew (P. Xanthii), tomato gray rot (B. cinérea) and green rot of the citrus (P. digitatum). TABLE 11
Los tres compuestos mostraron efectos sobresalientes y significativos de supresión de la enfermedad sobre P. xanthii y B. cinérea de acuerdo con el análisis estadístico realizado (Tabla 11). Contra P. digitatum únicamente el ácido 2-(2-metoxi-4-([(5Z)-2,4,6- trioxo-1-(prop-2-en-1-il)-1-3-diazinan-5-iliden]metil)fenoxi)acético mostró un efecto inhibitorio significativo.. Ensayos de toxicidad sobre B. cinérea y P. digitatum The three compounds showed outstanding and significant disease suppression effects on P. xanthii and B. cinérea according to the statistical analysis performed (Table 11). Against P. digitatum only 2- (2-methoxy-4 - ([(5Z) -2,4,6-trioxo-1- (prop-2-en-1-yl) -1-3-diazinan- 5-ylidene] methyl) phenoxy) acetic showed a significant inhibitory effect. Toxicity tests on B. cinerea and P. digitatum
Para separar la actividad fungicida mediada por la planta por la actividad fungicida in vitro (toxicidad) de los compuestos utilizados en los ensayos de fruta, To separate the fungicidal activity mediated by the plant by the fungicidal activity in vitro (toxicity) of the compounds used in the fruit tests,
- ácido (1-metil-2,4-dioxo-1 ,4,6,7-tetrahidro[1 ,3]tiazol[2,3f]purin-3(2H)il)acético - (1-methyl-2,4-dioxo-1, 4,6,7-tetrahydro [1,3] thiazol [2,3f] purin-3 (2H) yl) acetic acid
- ácido (3-[(1 ,3-dimetil-2,4,6-trioxotetrahidro-5(2H)-pirimidinilen)metil]fenoxi)acético ,- (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic acid,
- ácido 2-(2-metoxi-4-([(5Z)-2,4,6-trioxo-1-(prop-2-en-1-il)-1-3-diazinan-5- iliden]metil)fenoxi)acético, - 2- (2-methoxy-4 - ([(5Z) -2,4,6-trioxo-1- (prop-2-en-1-yl) -1-3-diazinan-5-ylidene] methyl acid ) phenoxy) acetic,
la toxicidad de tales compuestos se ensayó in vitro contra B. cinérea y P. digitatum. En estos ensayos, los hongos se cultivaron en 1 mi de PDB (caldo de dextrosa de patata) en placas de 24 pocilios, que se complementaron con los diferentes compuestos para alcanzar concentraciones finales que oscilaron entre 0 y 200 mM. Los pocilios se inocularon con 30 ml de suspensiones de esporas (104 conidios/ml) y las placas se incubaron durante 72 h a 22 °C para B. cinérea o a 25 °C en el caso de P. digitatum.
No se observó ningún efecto inhibitorio en comparación con los controles negativos con los tres compuestos analizados en el rango de concentraciones analizadas. the toxicity of such compounds was tested in vitro against B. cinérea and P. digitatum. In these assays, the fungi were grown in 1 ml of PDB (potato dextrose broth) in 24-well plates, which were supplemented with the different compounds to reach final concentrations ranging from 0 to 200 mM. The wells were inoculated with 30 ml of spore suspensions (10 4 conidia / ml) and the plates were incubated for 72 h at 22 ° C for B. cinerea or at 25 ° C in the case of P. digitatum. No inhibitory effect was observed compared to negative controls with the three compounds tested in the range of concentrations tested.
Ejemplo 3: Ensayos de toxicidad en insectos Ensayo de toxicidad para Gallería mellonella Example 3: Toxicity tests on insects Toxicity test for Gallería mellonella
Para analizar el efecto de los diferentes compuestos sobre las larvas de la polilla de la cera G. mellonella 15, se inyectaron seis larvas con 20 mI de soluciones 150 mM de los diferentes compuestos. Como control negativo se incluyó una solución acuosa de acetona al 1.5%. Después de la incubación, se contó el número de pupas y polillas formadas. También se examinó la morfología de las polillas resultantes. To analyze the effect of the different compounds on the larvae of the wax moth G. mellonella 15 , six larvae were injected with 20 ml of 150 mM solutions of the different compounds. A 1.5% acetone aqueous solution was included as a negative control. After incubation, the number of pupae and moths formed was counted. The morphology of the resulting moths was also examined.
La Figura 3 muestra que el efecto más dramático está relacionado con la formación de la polilla que o bien no se formó o resultó en la formación de adultos anormales con alas deformadas, un fenotipo asociado previamente al silenciamiento de un gen CDA en el escarabajo de la patata Leptinotarsa decemlineata16.
Figure 3 shows that the most dramatic effect is related to the formation of the moth that either did not form or resulted in the formation of abnormal adults with deformed wings, a phenotype previously associated with the silencing of a CDA gene in the moth beetle. potato Leptinotarsa decemlineata 16 .
TABLA 12 TABLE 12
La Tabla 12 muestra el efecto insecticida de los compuestos identificados por el método de topología molecular sobre las larvas de la polilla de cera G. mellonella. En términos generales, los compuestos no afectaron a la formación de pupas, pero alguno de ellos tuvo un efecto significativo en la metamorfosis (formación de polillas). Table 12 shows the insecticidal effect of the compounds identified by the molecular topology method on the larvae of the wax moth G. mellonella. Generally speaking, the compounds did not affect pupal formation, but some of them had a significant effect on metamorphosis (moth formation).
TABLA 13 TABLE 13
Los compuestos con actividad insecticida significativa sobre G. mellonella según el análisis estadístico realizado (Tabla 13) fueron: - ácido (3-[(1 ,3-dimetil-2,4,6-trioxotetrahidro-5(2H)- pirimidinilen)metil]fenoxi)acético, The compounds with significant insecticidal activity on G. mellonella according to the statistical analysis performed (Table 13) were: - acid (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) - pyrimidinylene) methyl ] phenoxy) acetic,
5-imino-1-(2-metil-5-nitrofenil)-3-fenilidantoina y 5-imino-1- (2-methyl-5-nitrophenyl) -3-phenylidantoin and
- 5-imino-1-(4-metil-5-nitrofenil)-3-fenilidantoina. Ensayo de toxicidad para Drosophila melanogaster - 5-imino-1- (4-methyl-5-nitrophenyl) -3-phenylidantoin. Toxicity test for Drosophila melanogaster
La toxicidad contra D. melanogaster se probó utilizando el ensayo de alimentación de alimentos líquidos17. Para realizar el ensayo de toxicidad, se pusieron 25 larvas de mosca en recipientes de plástico de 20 mi que contenían 0,5 mi de alimento líquido y el compuesto correspondiente a una concentración de 150 mM. También se incluyó como control negativo una solución acuosa de acetona al 1 ,5%. Durante la incubación, se registró el número de pupas formadas, así como el número de adultos (moscas) resultante.
Toxicity against D. melanogaster was tested using the liquid food feeding assay 17 . To perform the toxicity test, 25 fly larvae were placed in 20 ml plastic containers containing 0.5 ml of liquid food and the corresponding compound at a concentration of 150 mM. A 1.5% aqueous acetone solution was also included as a negative control. During incubation, the number of pupae formed was recorded, as well as the number of adults (flies) that resulted.
TABLA 14 TABLE 14
La Tabla 14 muestra el efecto insecticida de los compuestos identificados por un método de topología molecular sobre las larvas de la mosca de la fruta D. melanogaster. En términos generales, la mayoría de los compuestos redujeron significativamente la formación de moscas. Table 14 shows the insecticidal effect of the compounds identified by a molecular topology method on the larvae of the fruit fly D. melanogaster. Overall, most of the compounds significantly reduced fly formation.
TABLA 15 TABLE 15
Si bien muchos de los compuestos tienen un fuerte efecto en la formación de moscas adultas (9 de 20), los compuestos con actividad insecticida significativa en la formación de pupas y moscas adultas según el análisis estadístico realizado (Tabla 15) fueron: ácido (3-[(1 ,3-dimetil-2,4,6-trioxotetrahidro-5(2H)-pirimidinilen)metil]fenoxi)acético, 7-[2-hidroxi-3-(4-morfolinil)propil]-1 ,3-dimetil-3,7-dihidro-1 H-purin-2,6-diona, Although many of the compounds have a strong effect on the formation of adult flies (9 of 20), the compounds with significant insecticidal activity in the formation of pupae and adult flies according to the statistical analysis performed (Table 15) were: acid (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic, 7- [2-hydroxy-3- (4-morpholinyl) propyl] -1, 3 -dimethyl-3,7-dihydro-1 H-purine-2,6-dione,
5-imino-1-(2-metil-5-nitrofenil)-3-fenilidantoina y 5-imino-1- (2-methyl-5-nitrophenyl) -3-phenylidantoin and
5-imino-1-(4-metil-5-nitrofenil)-3-fenilidantoina. 5-imino-1- (4-methyl-5-nitrophenyl) -3-phenylidantoin.
Ejemplo 4 Ensayo de toxicidad para Caenorhabditis elegans Example 4 Toxicity test for Caenorhabditis elegans
Para investigar la toxicidad sobre nematodos, se utilizó el ensayo de toxicidad en placas de C. elegans 18. Para realizar el ensayo, en cada pocilio se añadió una gota de 75 ml de una solución 1 mM del compuesto correspondiente sobre la superficie de agar, para alcanzar una concentración final en el medio de cultivo de 150 mM. Como control negativo, también se incluyó una solución acuosa de acetona al 10% (concentración final de acetona al 1 ,5%). Las gotas se dejaron secar a temperatura ambiente durante 30 min. Posteriormente, cada pocilio se inoculó con 40 mI de la población de nematodos lavada y filtrada. Después de cinco días de incubación a 20 °C, cada pocilio se lavó con 100 mI de tampón M9. A continuación, se examinaron diez gotas de 3 mI bajo un microscopio óptico para contar el número de nematodos en las diferentes etapas larvales (L1 , L2-L3 y L4). To investigate toxicity on nematodes, the C. elegans 18 plaque toxicity assay was used. To carry out the assay, a 75 ml drop of a 1 mM solution of the corresponding compound was added to each well on the agar surface, to reach a final concentration in the culture medium of 150 mM. As a negative control, a 10% acetone aqueous solution (1.5% final acetone concentration) was also included. The drops were allowed to dry at room temperature for 30 min. Subsequently, each well was inoculated with 40 ml of the washed and filtered nematode population. After five days of incubation at 20 ° C, each well was washed with 100 ml of M9 buffer. Next, ten 3 ml drops were examined under a light microscope to count the number of nematodes in the different larval stages (L1, L2-L3 and L4).
La Figura 4 muestra algunas micrografías del ensayo de toxicidad de C. elegans realizado con los efectos de algunos compuestos identificados por el método de topología molecular sobre el desarrollo de nematodos. En comparación con el control negativo (agua), los compuestos provocaron una clara reducción en la formación de nematodos.
TABLA 16 Figure 4 shows some micrographs of the C. elegans toxicity test carried out with the effects of some compounds identified by the molecular topology method on the development of nematodes. Compared to the negative control (water), the compounds caused a clear reduction in nematode formation. TABLE 16
ácido {2-[(1 ,3-dimetil-4,6-dioxo-2- tioxotetrahidro-5(2H)- pirimidiniliden)metil1fenoxi}acético {2 - [(1,3-dimethyl-4,6-dioxo-2-thioxotetrahydro-5 (2H) -pyrimidinylidene) methyl1phenoxy} acetic acid
a Los valores representan la estamación del número de individuos por mi. a The values represent the number of individuals by me.
b Los valores representan el porcentaje de eficacia de los compuestos según la fórmula de Abbott
b Values represent percent efficacy of compounds according to Abbott's formula
La Tabla 16 muestra el efecto nematicida de los compuestos identificados por el método de topología molecular sobre C. elegans. La mayoría de los compuestos mostraron un fuerte efecto sobre el desarrollo de C. elegans, que es consistente con el fenotipo de retraso en el tiempo de desarrollo asociado a la disrupción de genes codificantes de CDA9. Table 16 shows the nematicidal effect of the compounds identified by the molecular topology method on C. elegans. Most of the compounds showed a strong effect on the development of C. elegans, which is consistent with the phenotype of delay in development time associated with the disruption of genes encoding CDA 9 .
TABLA 17 TABLE 17
Todos los compuestos ensayados mostraron algún nivel de toxicidad al menos sobre el estado larvario. Los compuestos con la actividad nematicida más fuerte según el análisis estadístico realizado (Tabla 17) fueron: All the compounds tested showed some level of toxicity at least on the larval stage. The compounds with the strongest nematicidal activity according to the statistical analysis performed (Table 17) were:
- ácido (1-metil-2,4-dioxo-1 ,4,6,7-tetrahidro[1 ,3]tiazol[2,3f]purin-3(2H)il)acético, - (1-methyl-2,4-dioxo-1, 4,6,7-tetrahydro [1, 3] thiazol [2,3f] purin-3 (2H) yl) acetic acid,
5-imino-1-(2-metil-5-nitrofenil)-3-fenilidantoina, 5-imino-1- (2-methyl-5-nitrophenyl) -3-phenylidantoin,
ácido 2-(10, 12-dioxo-9-{[(1 ,3-tiazol-2-il)carbamoil]metil}-7-tia-9, 11 - diazatriciclo[6.4.0.02,6]dodeca-1 (8),2(6)-dien-11-il)acético, 2- (10, 12-dioxo-9 - {[(1, 3-thiazol-2-yl) carbamoyl] methyl} -7-thia-9, 11-diazatricyclo [6.4.0.0 2 , 6 ] dodeca-1 acid (8), 2 (6) -dien-11-yl) acetic,
ácido 2-{4-[(1 ,3-dimetil-4,6-dioxo-2-sulfaniliden-1 ,3-diazinan-5- iliden)metil]fenoxi}acético, 2- {4 - [(1,3-dimethyl-4,6-dioxo-2-sulfanylidene-1,3-diazinan-5-ylidene) methyl] phenoxy} acetic acid,
- ácido 2-({[(5E)-4,6-dioxo-1-[4-(propan-2-il)fenil]-2-sulfanil-1 ,4,5,6- tetrahidropirimidin-5-iliden]metil}amino)-3-(1 H-imidazol-4-il)propanoico y ácido 2-{1 -[(3,4-difluorofenil)metil]-3-oxopiperazin-2-il}acético - 2 - ({[(5E) -4,6-dioxo-1- [4- (propan-2-yl) phenyl] -2-sulfanyl-1, 4,5,6-tetrahydropyrimidin-5-ylidene] acid methyl} amino) -3- (1 H-imidazol-4-yl) propanoic acid and 2- {1 - [(3,4-difluorophenyl) methyl] -3-oxopiperazin-2-yl} acetic acid
Ejemplo 5: Determinación del modo de acción como inhibidores de CDA Example 5: Determination of mode of action as CDA inhibitors
Los compuestos con el mejor potencial fungicida se analizaron para determinar su modo de acción como inhibidores de CDA. Se realizaron dos experimentos diferentes para proporcionar evidencias directas (ensayo enzimático) e indirectas (ensayo en planta) de inhibición de una CDA fúngica. Compounds with the best fungicidal potential were tested for their mode of action as CDA inhibitors. Two different experiments were performed to provide direct (enzymatic assay) and indirect (plant assay) evidence of inhibition of a fungal CDA.
Ensayo en planta sobre tejidos con el gen CERK1 silenciado Plant assay on tissues with the silenced CERK1 gene
Si la inhibición de la CDA fúngica in planta causa la activación de la inmunidad activada por quitina y la supresión del crecimiento fúngico, la aplicación de inhibidores de CDA a los tejidos de las hojas en los que el gen del receptor de quitina CERK1 está silenciado, no debería tener ningún efecto sobre el crecimiento fúngico. El silenciamiento del gen CERK1 de melón se realizó utilizando cotiledones de melón de 2 semanas de edad y el ensayo ATM-HIGS (silenciamiento génico inducido por hospedador mediado por Agrobacterium)19. Veinticuatro horas después de la agroinfiltración, las plantas se inocularon con suspensión de conidios de P. xanthii (1 x 104conidios/ml) y 24 h más tarde, las hojas se pulverizaron con la solución del compuesto (100 mM). Las plantas se mantuvieron entonces en una cámara de crecimiento en un ciclo de 16 h de luz / 8 h de oscuridad a 25 °C y posteriormente se examinaron para detectar la activación de las respuestas de defensa y el desarrollo de los síntomas de la enfermedad. Doce días
después de la inoculación, los síntomas de la enfermedad se evaluaron mediante análisis digital como se indicó anteriormente. Por otro lado, 72 h después de la inoculación, se estudió la acumulación in situ de peróxido de hidrógeno (H2O2) mediante análisis histoquímico, siguiendo el método de consumo de DAB20. If inhibition of fungal CDA in planta causes activation of chitin-activated immunity and suppression of fungal growth, application of CDA inhibitors to leaf tissues in which the chitin receptor gene CERK1 is silenced, It shouldn't have any effect on fungal growth. Melon CERK1 gene silencing was performed using 2-week-old melon cotyledons and the ATM-HIGS (Agrobacterium-mediated host-induced gene silencing) assay 19 . Twenty-four hours after agroinfiltration, the plants were inoculated with suspension of P. xanthii conidia (1 x 10 4 conidia / ml) and 24 h later, the leaves were sprayed with the compound solution (100 mM). The plants were then kept in a growth chamber on a 16 h light / 8 h dark cycle at 25 ° C and subsequently examined for activation of defense responses and development of disease symptoms. Twelve days after inoculation, disease symptoms were assessed by digital analysis as above. On the other hand, 72 h after inoculation, the in situ accumulation of hydrogen peroxide (H 2 O 2 ) was studied by histochemical analysis, following the DAB 20 consumption method.
La Figura 5 muestra los efectos fisiológicos de los tratamientos de los cotiledones de melón con los compuestos con la mejor actividad fungicida. Como se mostró anteriormente con EDTA, los compuestos indujeron una fuerte inhibición del crecimiento del hongo (Fig. 5A) como consecuencia de la fuerte activación de la producción de especies reactivas de oxígeno (ROS reactive oxygen species) (Fig. 5B). Esta respuesta de la planta se puede suprimir con el silenciamiento del gen del receptor de quitina CERK1. En esas plantas, el crecimiento del hongo se restaura en presencia de los compuestos (Fig. 5A) y la producción de ROS se reduce claramente (Fig. 5B). Los resultados muestran la relación entre la actividad fungicida de los compuestos y la activación de la inmunidad activada por quitina en la planta huésped, resultados que pueden explicarse por la inhibición de la actividad CDA por los compuestos y la posterior percepción de oligómeros de quitina no deacetilados por los receptores de la planta. Figure 5 shows the physiological effects of melon cotyledon treatments with the compounds with the best fungicidal activity. As previously shown with EDTA, the compounds induced a strong inhibition of fungal growth (Fig. 5A) as a consequence of the strong activation of the production of reactive oxygen species (ROS reactive oxygen species) (Fig. 5B). This plant response can be suppressed by silencing the CERK1 chitin receptor gene. In these plants, fungal growth is restored in the presence of the compounds (Fig. 5A) and ROS production is clearly reduced (Fig. 5B). The results show the relationship between the fungicidal activity of the compounds and the activation of chitin-activated immunity in the host plant, results that can be explained by the inhibition of CDA activity by the compounds and the subsequent perception of non-deacetylated chitin oligomers. by plant receptors.
Ensayo de actividad quitina deacetilasa Chitin deacetylase activity assay
Para este ensayo dos proteínas CDA identificadas en P. xanthii (PxCDAI y PxCDA2) se expresaron in vitro en E. coli. Las proteínas recombinantes con una etiqueta de poli- histidina en el extremo N-terminal se produjeron siguiendo procedimientos estándar. La actividad enzimática se determinó utilizando el método de la fluorescamina, utilizando quitina coloidal como sustrato12. Para probar la actividad inhibidora de CDA de los compuestos seleccionados, la reacción enzimática se llevó a cabo en ausencia o presencia de los compuestos a concentraciones de 10 y 100 mM. Las mezclas de reacción se incubaron durante 45 min a 37 °C. Después de la incubación, las reacciones se detuvieron con tampón borato 0,4 M (pH 9,0). Después de registrar los datos, se calcularon los porcentajes de inhibición de la actividad enzimática.
TABLA 18 For this assay, two CDA proteins identified in P. xanthii (PxCDAI and PxCDA2) were expressed in vitro in E. coli. Recombinant proteins with a polyhistidine tag at the N-terminus were produced following standard procedures. Enzyme activity was determined using the fluorescamine method, using colloidal chitin as substrate 12 . To test the CDA inhibitory activity of the selected compounds, the enzymatic reaction was carried out in the absence or presence of the compounds at concentrations of 10 and 100 mM. The reaction mixtures were incubated for 45 min at 37 ° C. After incubation, reactions were stopped with 0.4M borate buffer (pH 9.0). After recording the data, the percentages of inhibition of enzyme activity were calculated. TABLE 18
La Tabla 18 muestra los resultados de los experimentos de inhibición de la actividad enzimática CDA llevados a cabo con dos proteínas CDA de P. xanthii, PxCDAI y PxCDA2, expresadas en E. coli y expuestas a los compuestos con la mejor actividad fungicida. A 100 mM, los compuestos indujeron una fuerte inhibición de la actividad enzimática de las proteínas CDA en un rango de 75 a 93%. Table 18 shows the results of the CDA enzymatic activity inhibition experiments carried out with two CDA proteins from P. xanthii, PxCDAI and PxCDA2, expressed in E. coli and exposed to the compounds with the best fungicidal activity. At 100 mM, the compounds induced a strong inhibition of the enzymatic activity of CDA proteins in a range of 75 to 93%.
Las secuencias de PxCDAI y PxCDA2 se han depositado en GenBank con el número de acceso KX495502 y KX495503. The sequences for PxCDAI and PxCDA2 have been deposited with GenBank under accession numbers KX495502 and KX495503.
Docking de proteínas CDA fúngica y de insecto a compuestos seleccionados Docking of fungal and insect CDA proteins to selected compounds
Para mapear las interacciones entre los mejores compuestos fungicidas e insecticidas y las proteínas CDA e identificar sitios de unión potenciales para estos compuestos, se realizaron experimentos de docking. Para estos experimentos, se utilizaron proteínas CDA completas del agente fúngico causal de la antracnosis del frijol Colletotrichum lindemunthianum y del gusano de la seda Bombyx morí, a diferencia del experimento de docking descrito en el Ejemplo 1.3 en el que solo se usó el sitio activo de C. lindemunthianum. Para ello se eligió la opción " blind docking " (acoplamiento ciego) en el que se empleaba la proteína CDA completa. Se utilizaron las estructuras proteicas correspondientes, 2IW0 y 5Z34, obtenidas del Protein Databank (PDB), y el servidor web
SwissDock. El docking se realizó utilizando el parámetro "Accurate" y para el resto de parámetros se usaron los predeterminados, sin una región de interés definida (acoplamiento ciego). Se utilizaron cinco compuestos en estos análisis, los tres seleccionados con el mejor potencial fungicida ácido (1-metil-2,4-dioxo-1 ,4,6,7-tetrahidro[1 ,3]tiazol[2,3f]purin-3(2H)il)acético, ácido (3-[(1 ,3-dimetil-2,4,6-trioxotetrahidro-5(2H)- pirimidinilen)metil]fenoxi)acético y To map the interactions between the best fungicidal and insecticidal compounds and CDA proteins and to identify potential binding sites for these compounds, docking experiments were performed. For these experiments, complete CDA proteins of the fungal agent responsible for the anthracnose bean Colletotrichum lindemunthianum and the silkworm Bombyx morí were used, unlike the docking experiment described in Example 1.3 in which only the active site of C. lindemunthianum. For this, the option "blind docking" was chosen, in which the complete CDA protein was used. The corresponding protein structures, 2IW0 and 5Z34, obtained from the Protein Databank (PDB), and the web server were used. SwissDock. Docking was carried out using the "Accurate" parameter and the default parameters were used for the rest of the parameters, without a defined region of interest (blind coupling). Five compounds were used in these analyzes, the three selected with the best acid fungicidal potential (1-methyl-2,4-dioxo-1, 4,6,7-tetrahydro [1, 3] thiazol [2,3f] purin- 3 (2H) yl) acetic acid, (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic acid and
ácido 2-(2-metoxi-4-([(5Z)-2,4,6-trioxo-1-(prop-2-en-1-il)-1-3-diazinan-5- iliden]metil)fenoxi)acético. y dos de los compuestos identificados con actividad insecticida notable 2- (2-methoxy-4 - ([(5Z) -2,4,6-trioxo-1- (prop-2-en-1-yl) -1-3-diazinan-5-ylidene] methyl) acid phenoxy) acetic. and two of the compounds identified with notable insecticidal activity
5-imino-1-(2-metil-5-nitrofenil)-3-fenilidantoina y 5-imino-1- (2-methyl-5-nitrophenyl) -3-phenylidantoin and
5-imino-1-(4-metil-5-nitrofenil)-3-fenilidantoina. 5-imino-1- (4-methyl-5-nitrophenyl) -3-phenylidantoin.
TABLA 19
TABLE 19
TABLA 20
TABLE 20
De manera similar, la Tabla 20 muestra el análisis de docking a la CDA de insectos. En este caso, solo los compuestos con actividad insecticida fueron capaces de formar dos o tres puentes de hidrógeno con los residuos K508 y G512. Estos residuos forman parte del bolsillo del sitio activo de la enzima CDA de B. morí22, lo que sugiere que estos compuestos pueden interferir con la actividad enzimática. Similarly, Table 20 shows the docking analysis to the insect CDA. In this case, only compounds with insecticidal activity were capable of forming two or three hydrogen bonds with residues K508 and G512. These residues are part of the active site pocket of the CDA enzyme of B. morí 22 , suggesting that these compounds may interfere with enzyme activity.
En conjunto, los resultados del docking proporcionan evidencia computacional que respalda la actividad específica de estas moléculas como compuestos fungicidas o insecticidas, a través de su unión específica a una enzima CDA fúngica o de insectos, lo que refuerza sus actividades como inhibidores específicos de CDA.
Análisis del Docking de proteínas CDA fúngica y de insecto a compuestos empleando diversos programas informáticos Taken together, the docking results provide computational evidence supporting the specific activity of these molecules as fungicidal or insecticidal compounds, through their specific binding to a fungal or insect CDA enzyme, reinforcing their activities as specific CDA inhibitors. Docking analysis of fungal and insect CDA proteins to compounds using various computer programs
Se llevó a cabo un estudio análogo al anterior, pero empleando con tres programas informáticos específicos de acoplamiento, AutoDock Vina23 Glide24 y SwissDock25, y el programa Quimera26 para la visualización de los datos cuando fuera necesario. Las proteínas receptoras se prepararon eliminando los ligandos predeterminados, las cargas de agua y añadiendo cargas polares de hidrógenos. El acoplamiento se realizó cubriendo toda la superficie de la proteína, sin asignar un conjunto específico de coordenadas (acoplamiento ciego). Además, se realizaron experimentos de acoplamiento del EDTA y del trímero de quitina (GlcNAc)3 a estas proteínas CDA como control. La puntuación de los cinco mejores subconjuntos de ligandos se calculó para los compuestos indicados en el apartado anterior, los resultados con la CDA entera de C. lindemunthianum se muestran en la Tabla 21 y los obtenidos con la CDA entera de B. morí en la Tabla 22. A study analogous to the previous one was carried out, but using three specific docking computer programs, AutoDock Vina 23 Glide 24 and SwissDock 25 , and the Chimera 26 program to visualize the data when necessary. Receptor proteins were prepared by removing predetermined ligands, water charges, and adding polar charges of hydrogens. The coupling was performed covering the entire surface of the protein, without assigning a specific set of coordinates (blind coupling). Furthermore, EDTA and chitin trimer (GlcNAc) 3 coupling experiments were performed to these CDA proteins as a control. The score of the five best subsets of ligands was calculated for the compounds indicated in the previous section, the results with the entire CDA of C. lindemunthianum are shown in Table 21 and those obtained with the entire CDA of B. morí in Table 22.
La Tabla 21 muestra los resultados de acoplamiento al CDA fúngico realizado con diferentes programas informáticos. Sólo los compuestos con la prevista actividad fungicida fueron capaces de formar dos o más enlaces de hidrógeno con al menos dos residuos de aminoácidos diferentes, resultando así un sitio de enlace favorable. En la mayoría de los casos, estos aminoácidos son residuos catalíticos (HIS206, ASP169, TYR145 y otros residuos destacados en negrita en la tabla 21) del sitio activo de la proteína fúngica CDA21. Además, los tres compuestos mostraron valores de afinidad muy favorables (DG > -6,5 kcal/mol), lo que proporciona información de su capacidad para acoplarse al sitio catalítico. Los valores de energía de enlace y los enlaces de hidrógeno con residuos específicos fueron comparables con los del EDTA y el trímero de quitina, lo que dio lugar a resultados coherentes. En conclusión, estos resultados del acoplamiento molecular son coherentes con los resultados anteriores de acoplamiento molecular contra un fragmento de una proteína CDA fúngica que comprendía el sitio activo, confirmando así que estos compuestos son verdaderos inhibidores de la enzima CDA de los hongos.
TABLA 21 Table 21 shows the results of fungal CDA coupling performed with different computer programs. Only compounds with the predicted fungicidal activity were able to form two or more hydrogen bonds with at least two different amino acid residues, thus resulting in a favorable binding site. In most cases, these amino acids are catalytic residues (HIS206, ASP169, TYR145 and other residues highlighted in bold in Table 21) of the active site of the fungal protein CDA 21 . Furthermore, the three compounds showed very favorable affinity values (DG> -6.5 kcal / mol), which provides information on their ability to couple to the catalytic site. The values of binding energy and hydrogen bonds with specific residues were comparable to those of EDTA and chitin trimer, resulting in consistent results. In conclusion, these molecular coupling results are consistent with previous molecular coupling results against a fragment of a fungal CDA protein that comprised the active site, thus confirming that these compounds are true inhibitors of the fungal CDA enzyme. TABLE 21
La Tabla 22 muestra el análisis de acoplamiento a la enzima CDA del insecto. En el caso de los tres compuestos con actividad se detectaron fungicida, algunas interacciones específicas con la bolsa catalítica de la enzima (PHE165 y HIS282) aunque sólo con el software Glide. Por el contrario, para los dos compuestos con actividad insecticida, se obtuvieron resultados similares con los tres programas informáticos de acoplamiento. Estos compuestos formaron tres o cuatro enlaces de hidrógeno con residuos catalíticos como ASP63, HIS121 y TRP284, que también estaban involucrados en la unión al EDTA y al trímero de quitina. Dado que estos residuos forman parte de la bolsa de sitio activo de la enzima CDA de B. morí22, los resultados del acoplamiento sugieren que estos compuestos pueden interferir con la actividad de la enzima como inhibidores de la enzima CDA del insecto, reforzando así su potencial como compuestos insecticidas. Table 22 shows the insect CDA enzyme coupling analysis. In the case of the three compounds with fungicidal activity, some specific interactions with the catalytic bag of the enzyme (PHE165 and HIS282) were detected, although only with the Glide software. In contrast, for the two compounds with insecticidal activity, similar results were obtained with the three coupling software. These compounds formed three or four hydrogen bonds with catalytic residues such as ASP63, HIS121, and TRP284, which were also involved in binding to EDTA and the chitin trimer. Since these residues are part of the active site bag of the CDA enzyme of B. morí 22 , the results of the coupling suggest that these compounds may interfere with the activity of the enzyme as inhibitors of the CDA enzyme of the insect, thus reinforcing its potential as insecticidal compounds.
En conjunto, los resultados de docking obtenido a partir de distintos programas informáticos proporcionan pruebas computacionales que apoyan la actividad específica de estas moléculas como compuestos fungicidas o insecticidas. Esta actividad es mediada a través de su unión específica a la enzima CDA de un hongo o un insecto, reforzando así su actividad prevista como inhibidores específicos de la CDA.
Taken together, the docking results obtained from different computer programs provide computational evidence supporting the specific activity of these molecules as fungicidal or insecticidal compounds. This activity is mediated through their specific binding to the CDA enzyme of a fungus or an insect, thus enhancing their expected activity as specific CDA inhibitors.
TABLA 22 TABLE 22
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Claims
1. Método para la identificación de un compuesto con potencial actividad pesticida frente a organismos que contienen quitina, siendo dicho compuesto un inhibidor de la enzima quitina deacetilasa (CDA), que comprende: 1. Method for the identification of a compound with potential pesticidal activity against chitin-containing organisms, said compound being an inhibitor of the enzyme chitin deacetylase (CDA), comprising:
a) obtener para dicho compuesto un valor para la función discriminante DF1 , en el que: a) obtain for said compound a value for the discriminant function DF1, in which:
DF1 = (-10.295 GATS4m) + (35.124 GGI8) + 6.917 donde: DF1 = (-10.295 GATS4m) + (35.124 GGI8) + 6.917 where:
- GATS4m es el índice de autocorrelación de Geary - Iag4 / ponderado por masas atómicas, - GATS4m is the Geary autocorrelation index - Iag4 / weighted by atomic masses,
- GGI8 es el índice topológico de carga de orden 8; y - GGI8 is the topological load index of order 8; Y
b) Comprobar si el valor DF1 obtenido en (a) para dicho compuesto está entre 0 y +14, en donde si dicho valor está entre 0 y +14 dicho compuesto se identifica como un inhibidor potencial de la enzima CDA. b) Check if the DF1 value obtained in (a) for said compound is between 0 and +14, where if said value is between 0 and +14 said compound is identified as a potential inhibitor of the CDA enzyme.
2. Método de acuerdo con la reivindicación 1 en el que: 2. Method according to claim 1 in which:
-el paso (a) comprende, además, obtener para dicho compuesto un valor predictivo de actividad inhibitoria de Log (lnh%), donde: -step (a) further comprises obtaining for said compound a predictive value of Log inhibitory activity (lnh%), where:
Log (lnh%) = 0.814 - 0.0184 x T(N..N) + 9.552 x JGI2 donde: Log (lnh%) = 0.814 - 0.0184 x T (N..N) + 9.552 x JGI2 where:
- T(N..N) es la distancia topológica, expresada como número de aristas entre dos átomos consecutivos de nitrógeno, - T (N..N) is the topological distance, expressed as the number of edges between two consecutive nitrogen atoms,
- JGI2 es el índice topológico de carga promedio de orden 2; y - JGI2 is the topological average load index of order 2; Y
-el paso b) adicionalmente consiste en comprobar si el valor de Log (lnh%) obtenido en (a) para dicho compuesto, está entre +1 and +2, en cuyo caso dicho compuesto es identificado como un potencial inhibidor de la enzima CDA. -step b) additionally consists of checking if the value of Log (lnh%) obtained in (a) for said compound is between +1 and +2, in which case said compound is identified as a potential inhibitor of the CDA enzyme .
3. Método de acuerdo con la reivindicación 2 en el que: 3. Method according to claim 2 in which:
-el paso (a) comprende además obtener para dicho compuesto un valor de la función discriminante DF2, en el que: -step (a) further comprises obtaining for said compound a value of the discriminant function DF2, in which:
DF2 = 21.022 - (19.407 x GGI10) - (5.104xSEige) - (7.911 x GATS3e)
en la que: DF2 = 21.022 - (19.407 x GGI10) - (5.104xSEige) - (7.911 x GATS3e) in which:
- GGI10 es el índice topológico de carga de orden 10, - GGI10 is the topological load index of order 10,
- SEige es el autovalor suma del valor propio de la electronegatividad, - SEige is the sum eigenvalue of the eigenvalue of electronegativity,
- GATS3e es el índice de autocorrelación de Geary -Iag3/ ponderado por electronegatividades atómicas de Sanderson; y - GATS3e is the Geary autocorrelation index -Iag3 / weighted by Sanderson atomic electronegativities; Y
- el paso b) comprende comprobar si el valor obtenido para dicho compuesto en el paso (a) está entre 0 y +6, en cuyo caso dicho compuesto es identificado como un potencial inhibidor de la enzima CDA. - step b) comprises checking whether the value obtained for said compound in step (a) is between 0 and +6, in which case said compound is identified as a potential inhibitor of the CDA enzyme.
4. Método de acuerdo con una cualquiera de las reivindicaciones anteriores que comprende después del paso (b): 4. Method according to any one of the preceding claims comprising after step (b):
- c) llevar a cabo un análisis experimental del compuesto identificado como un inhibidor potencial de la enzima CDA según el paso b), que comprende poner en contacto dicho compuesto con un patógeno y observar si hay crecimiento de este. - c) carrying out an experimental analysis of the compound identified as a potential inhibitor of the CDA enzyme according to step b), which comprises putting said compound in contact with a pathogen and observing if there is growth of this.
5. Método de acuerdo con la reivindicación 4 en el que en el que el patógeno puede ser seleccionado entre hongos e insectos, preferiblemente Podosphaera xanthii o Gallería mellonella. 5. Method according to claim 4 wherein the pathogen can be selected from fungi and insects, preferably Podosphaera xanthii or Galleria mellonella.
6. Método de acuerdo con una cualquiera de las reivindicaciones anteriores que comprende llevar a cabo un experimento docking a una enzima CDA con un compuesto identificado en el paso (b) como un inhibidor potencial de la enzima CDA por su habilidad para interaccionar con el sitio activo de la enzima CDA. 6. Method according to any one of the preceding claims, which comprises carrying out a docking experiment on a CDA enzyme with a compound identified in step (b) as a potential inhibitor of the CDA enzyme due to its ability to interact with the site. active enzyme CDA.
7. Método de acuerdo con la reivindicación 6 en el que se utiliza un fragmento que contiene el sitio activo de la enzima CDA de C. lindemuthianum para el experimento de docking. 7. Method according to claim 6, wherein a fragment containing the active site of the C. lindemuthianum CDA enzyme is used for the docking experiment.
8. Método de acuerdo con una cualquiera de las reivindicaciones anteriores en el que el organismo que contiene quitina está seleccionado entre hongos, artrópodos y nematodos.
8. Method according to any one of the preceding claims in which the chitin-containing organism is selected from fungi, arthropods and nematodes.
9. Uso de compuestos químicos con actividad pesticida contra un organismo que contiene quitina seleccionado entre hongos, artrópodos y nematodos detectado por el método definido en una cualquiera de las reivindicaciones 1 a 8. 9. Use of chemical compounds with pesticidal activity against an organism that contains chitin selected from fungi, arthropods and nematodes detected by the method defined in any one of claims 1 to 8.
10. Uso de un compuesto químico según la reivindicación 9 contra un organismo que contiene quitina seleccionado entre hongos, artrópodos y nematodos caracterizado por que dicho compuesto: 10. Use of a chemical compound according to claim 9 against a chitin-containing organism selected from fungi, arthropods and nematodes characterized in that said compound:
- i) tiene un valor de DF1 entre 0 y +14, - i) has a DF1 value between 0 and +14,
DF1 = (-10.295 x GATS4m) + (35.124 x GGI8) + 6.917 DF1 = (-10.295 x GATS4m) + (35.124 x GGI8) + 6.917
donde: where:
- GATS4m es el índice de autocorrelación de Geary - Iag4 / ponderado por masas atómicas, y - GATS4m is the Geary autocorrelation index - Iag4 / weighted by atomic masses, and
- GGI8 es el índice topológico de carga de orden 8. - GGI8 is the topological load index of order 8.
11. Uso de un compuesto químico de acuerdo con la reivindicación 10 contra un organismo que contiene quitina seleccionado entre hongos, artrópodos y nematodos caracterizado por que dicho compuesto: 11. Use of a chemical compound according to claim 10 against a chitin-containing organism selected from fungi, arthropods and nematodes, characterized in that said compound:
- ii) tiene un valor de Log (lnh%) entre +1 y +2, - ii) has a value of Log (lnh%) between +1 and +2,
Log (lnh%) = 0.814 - 0.018 x T (N..N) + 9.552 x JGI2 Log (lnh%) = 0.814 - 0.018 x T (N..N) + 9.552 x JGI2
donde: where:
- T (N..N) es la distancia topológica expresada como número de aristas entre dos nitrógeno consecutivos, y - T (N..N) is the topological distance expressed as the number of edges between two consecutive nitrogen, and
- JGI2 es el índice topológico de carga promedio de orden 2. - JGI2 is the topological average load index of order 2.
12. Uso de un compuesto químico según la reivindicación 11 contra un organismo que contiene quitina seleccionado entre hongos, artrópodos y nematodos caracterizado por que dicho compuesto: 12. Use of a chemical compound according to claim 11 against an organism that contains chitin selected from fungi, arthropods and nematodes, characterized in that said compound:
- iii) tiene un valor de DF2 entre 0 y +6,
DF2 = 21.022 - (19.407 x GGI10) - (5.104 x Seige) - (7.911 x GATS3e)donde: - iii) has a DF2 value between 0 and +6, DF2 = 21.022 - (19.407 x GGI10) - (5.104 x Seige) - (7.911 x GATS3e) where:
GGI10 es el índice topológico de carga de orden 10, GGI10 is the topological load index of order 10,
Seige es el autovalor suma del valor propio de la electronegatividad, y Seige is the sum eigenvalue of the eigenvalue of electronegativity, and
GATS3e es el índice de autocorrelación de Geary -Iag3/ ponderado por electronegatividades atómicas de Sanderson. GATS3e is the Geary autocorrelation index -Iag3 / weighted by Sanderson atomic electronegativities.
13. Uso de un compuesto químico según la reivindicación 12 contra un organismo que contiene quitina seleccionado entre hongos, artrópodos y nematodos caracterizado por que dicho compuesto interacciona con el sitio activo de la enzima CDA de: 13. Use of a chemical compound according to claim 12 against an organism that contains chitin selected from fungi, arthropods and nematodes, characterized in that said compound interacts with the active site of the CDA enzyme of:
- C. lindemuthianum y/o - C. lindemuthianum and / or
- B. morii - B. morii
en un experimento de docking. in a docking experiment.
14. Uso según una de las reivindicaciones 10 a 13 en el que el compuesto químico se selecciona entre: Use according to one of claims 10 to 13, in which the chemical compound is selected from:
- ácido (1-metil-2,4-dioxo-1 ,4,6,7-tetrahidro[1 ,3]tiazol[2,3f]purin-3(2H)il)acético - (1-methyl-2,4-dioxo-1, 4,6,7-tetrahydro [1,3] thiazol [2,3f] purin-3 (2H) yl) acetic acid
- ácido (3-[(1 ,3-dimetil-2,4,6-trioxotetrahidro-5(2H)-pirimidinilen)metil]fenoxi)acético , - (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic acid,
- ácido 2-(2-metoxi-4-([(5Z)-2,4,6-trioxo-1-(prop-2-en-1-il)-1-3-diazinan-5- iliden]metil)fenoxi)acético, - 2- (2-methoxy-4 - ([(5Z) -2,4,6-trioxo-1- (prop-2-en-1-yl) -1-3-diazinan-5-ylidene] methyl acid ) phenoxy) acetic,
- 5-[4-(2-hidroxietoxi)-3-metoxibenziliden]-1 ,3-dimetil-2,4,6(1 H,3H,5H)-pirimidintriona,- 5- [4- (2-hydroxyethoxy) -3-methoxybenzylidene] -1, 3-dimethyl-2,4,6 (1H, 3H, 5H) -pyrimidinetrione,
- 7-[2-hidroxi-3-(4-morfolinil)propil]-1 ,3-dimetil-3,7-dihidro-1 H-purin-2,6-diona, - 7- [2-hydroxy-3- (4-morpholinyl) propyl] -1,3-dimethyl-3,7-dihydro-1 H-purine-2,6-dione,
2-amino-7-metil-5-oxo-4-[4-(trifluorometoxi)fenil]-4H,5H-pirano[4,3-b]pirano-3- carbonitrilo, 2-amino-7-methyl-5-oxo-4- [4- (trifluoromethoxy) phenyl] -4H, 5H-pyrano [4,3-b] pyran-3-carbonitrile,
- N-{4-[3-(2,6-dimetil-4-morfolinil)-2,5-dioxo-1-pirrolidinil]fenil}acetamida, - N- {4- [3- (2,6-dimethyl-4-morpholinyl) -2,5-dioxo-1-pyrrolidinyl] phenyl} acetamide,
- 2-(1 ,3-dimetil-2,6-dioxo-1 ,2,3,6-tetrahidro-7H-purin-7-il)-N-(2-metoxi-1- metiletil)acetamida, - 2- (1,3-dimethyl-2,6-dioxo-1, 2,3,6-tetrahydro-7H-purin-7-yl) -N- (2-methoxy-1-methylethyl) acetamide,
- N-ciclopropil-2-(1 ,3-dimetil-2,6-dioxo-1 ,2,3,6-tetrahidro-9H-purin-9-il)acetamida - N-cyclopropyl-2- (1,3-dimethyl-2,6-dioxo-1, 2,3,6-tetrahydro-9H-purin-9-yl) acetamide
- 5-imino-1-(2-metil-5-nitrofenil)-3-fenilidantoina, - 5-imino-1- (2-methyl-5-nitrophenyl) -3-phenylidantoin,
- 5-imino-1-(4-metil-5-nitrofenil)-3-fenilidantoina,
- ácido 3-{2,5-dioxo-3-[(5Z)-4-oxo-5-(fenilmetiliden)-2-sulfaniliden-1 ,3-tiazolidin-3- il]pirrolidin-1-il}propanoico - 5-imino-1- (4-methyl-5-nitrophenyl) -3-phenylidantoin, - 3- {2,5-dioxo-3 - [(5Z) -4-oxo-5- (phenylmethylidene) -2-sulfanylidene-1,3-thiazolidin-3-yl] pyrrolidin-1-yl} propanoic acid
- ácido 2-(10,12-dioxo-9-{[(1 ,3-tiazol-2-il)carbamoil]metil}-7-tia-9, 11 - diazatriciclo[6.4.0.02,6]dodeca-1 (8),2(6)-dien-11-il)acético, - {[(1, 3-thiazol-2-yl) carbamoyl] methyl} -7-thia-9, 11 - - 2- (10,12-dioxo-9 acid diazatricyclo [6.4.0.0 2, 6] dodeca- 1 (8), 2 (6) -dien-11-yl) acetic,
- ácido 2-{4-[(1 ,3-dimetil-4,6-dioxo-2-sulfaniliden-1 ,3-diazinan-5- iliden)metil]fenoxi}acético, - 2- {4 - [(1,3-dimethyl-4,6-dioxo-2-sulfanylidene-1,3-diazinan-5-ylidene) methyl] phenoxy} acetic acid,
- ácido 2-({[(5E)-4,6-dioxo-1-[4-(propan-2-il)fenil]-2-sulfanil-1 ,4,5,6-tetrahidropirimidin-5- iliden]metil}amino)-3-(1 H-imidazol-4-il)propanoico , - 2 - ({[(5E) -4,6-dioxo-1- [4- (propan-2-yl) phenyl] -2-sulfanyl-1, 4,5,6-tetrahydropyrimidin-5-ylidene] acid methyl} amino) -3- (1 H-imidazol-4-yl) propanoic,
- ácido 2-[(5E)-4-oxo-5-{[2-(prop-2-en-1-iloxi)fenil]metiliden}-2-sulfaniliden-1 ,3- tiazolidin-3-il]pentanedioico, - 2 - [(5E) -4-oxo-5 - {[2- (prop-2-en-1-yloxy) phenyl] methylidene} -2-sulfanilidene-1,3-thiazolidin-3-yl] pentanedioic acid ,
- ácido 2-{1 -[(3,4-difluorofenil)metil]-3-oxopiperazin-2-il}acético, - 2- {1 - [(3,4-difluorophenyl) methyl] -3-oxopiperazin-2-yl} acetic acid,
- ácido 2-{3-[(2-cloro-6-fluorofenil)metil]-2,4,5-trioxoimidazolidin-1-il}acético, - 2- {3 - [(2-chloro-6-fluorophenyl) methyl] -2,4,5-trioxoimidazolidin-1-yl} acetic acid,
- ácido {2-[(1 ,3-dimetil-4,6-dioxo-2-tioxotetrahidro-5(2H)- pirimidiniliden)metil]fenoxi}acético y - {2 - [(1, 3-dimethyl-4,6-dioxo-2-thioxotetrahydro-5 (2H) -pyrimidinylidene) methyl] phenoxy} acetic acid and
- 3-benzil-1 ,7-dimetil-7,9-dihidro-1 H-purin-2,6,8(3H)-triona. - 3-benzyl-1,7-dimethyl-7,9-dihydro-1 H-purin-2,6,8 (3H) -trione.
15. Uso según una de las reivindicaciones 10 a 13 en el que el compuesto químico se selecciona entre aquellos que tienen un grupo purina: 15. Use according to one of claims 10 to 13 in which the chemical compound is selected from those having a purine group:
- ácido (1-metil-2,4-dioxo-1 ,4,6,7-tetrahidro[1 ,3]tiazol[2,3f]purin-3(2H)il)acético, - 7-[2-hidroxi-3-(4-morfolinil)propil]-1 ,3-dimetil-3,7-dihidro-1 H-purin-2,6-diona, - (1-methyl-2,4-dioxo-1, 4,6,7-tetrahydro [1,3] thiazol [2,3f] purin-3 (2H) yl) acetic acid, - 7- [2-hydroxy -3- (4-morpholinyl) propyl] -1,3-dimethyl-3,7-dihydro-1 H-purine-2,6-dione,
- 2-(1 ,3-dimetil-2,6-dioxo-1 ,2,3,6-tetrahidro-7H-purin-7-il)-N-(2-metoxi-1- metiletil)acetamida, - 2- (1,3-dimethyl-2,6-dioxo-1, 2,3,6-tetrahydro-7H-purin-7-yl) -N- (2-methoxy-1-methylethyl) acetamide,
- N-ciclopropil-2-(1 ,3-dimetil-2,6-dioxo-1 ,2,3,6-tetrahidro-9H-purin-9-il)acetamida y, - N-cyclopropyl-2- (1, 3-dimethyl-2,6-dioxo-1, 2,3,6-tetrahydro-9H-purin-9-yl) acetamide and,
- 3-benzil-1 ,7-dimetil-7,9-dihidro-1 H-purin-2,6,8(3H)-triona. - 3-benzyl-1,7-dimethyl-7,9-dihydro-1 H-purin-2,6,8 (3H) -trione.
16. Uso según una de las reivindicaciones 10 a 13 en el que el compuesto químico se selecciona entre aquellos que tienen un grupo pirimidina: 16. Use according to one of claims 10 to 13 in which the chemical compound is selected from those having a pyrimidine group:
- ácido (3-[(1 ,3-dimetil-2,4,6-trioxotetrahidro-5(2H)-pirimidinilen)metil]fenoxi)acético, - (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic acid,
- 5-[4-(2-hidroxietoxi)-3-metoxibenziliden]-1 ,3-dimetil-2,4,6(1 H,3H,5H)-pirimidintriona,
- ácido 2-({[(5E)-4,6-dioxo-1-[4-(propan-2-il)fenil]-2-sulfanil-1 ,4,5,6-tetrahidropirimidin- 5-iliden]metil}amino)-3-(1 H-imidazol-4-il)propanoico y - 5- [4- (2-hydroxyethoxy) -3-methoxybenzylidene] -1, 3-dimethyl-2,4,6 (1H, 3H, 5H) -pyrimidinetrione, - 2 - ({[(5E) -4,6-dioxo-1- [4- (propan-2-yl) phenyl] -2-sulfanyl-1, 4,5,6-tetrahydropyrimidin-5-ylidene] acid methyl} amino) -3- (1 H-imidazol-4-yl) propanoic and
- ácido {2-[(1 ,3-dimetil-4,6-dioxo-2-tioxotetrahidro-5(2H)- pirimidiniliden)metil]fenoxi}acético. - {2 - [(1,3-dimethyl-4,6-dioxo-2-thioxotetrahydro-5 (2H) -pyrimidinylidene) methyl] phenoxy} acetic acid.
17. Uso según una de las reivindicaciones 10 a 13 en el que el compuesto químico se selecciona entre aquellos que tienen un grupo imidazol: 17. Use according to one of claims 10 to 13 in which the chemical compound is selected from those having an imidazole group:
- 5-imino-1-(2-metil-5-nitrofenil)-3-fenilidantoina, - 5-imino-1- (2-methyl-5-nitrophenyl) -3-phenylidantoin,
- 5-imino-1-(4-metil-5-nitrofenil)-3-fenilidantoina y - 5-imino-1- (4-methyl-5-nitrophenyl) -3-phenylidantoin and
- ácido 2-{3-[(2-cloro-6-fluorofenil)metil]-2,4,5-trioxoimidazolidin-1-il}acético. - 2- {3 - [(2-chloro-6-fluorophenyl) methyl] -2,4,5-trioxoimidazolidin-1-yl} acetic acid.
18. Uso según una de las reivindicaciones 10 a 13 en el que el compuesto químico se selecciona entre aquéllos que tienen un grupo pirrolidina: 18. Use according to one of claims 10 to 13 in which the chemical compound is selected from those having a pyrrolidine group:
- N-{4-[3-(2,6-dimetil-4-morfolinil)-2,5-dioxo-1-pirrolidinil]fenil}acetamida y - N- {4- [3- (2,6-dimethyl-4-morpholinyl) -2,5-dioxo-1-pyrrolidinyl] phenyl} acetamide and
- ácido 3-{2,5-dioxo-3-[(5Z)-4-oxo-5-(fenilmetiliden)-2-sulfaniliden-1 ,3-tiazolidin-3- il]pirrolidin-1-il}propanoico. - 3- {2,5-dioxo-3 - [(5Z) -4-oxo-5- (phenylmethylidene) -2-sulfanylidene-1,3-thiazolidin-3-yl] pyrrolidin-1-yl} propanoic acid.
19. Uso según una de las reivindicaciones 14 a 18 para controlar una plaga seleccionada entre hongos, artrópodos, nematodos y cualquier combinación de ellos. Use according to one of claims 14 to 18 to control a pest selected from fungi, arthropods, nematodes and any combination of them.
20. Uso según una de las reivindicaciones 10 a 13 para controlar una plaga seleccionada entre hongos, artrópodos, nematodos y cualquier combinación de ellos, en el que el compuesto se selecciona entre: 20. Use according to one of claims 10 to 13 to control a pest selected from fungi, arthropods, nematodes and any combination of them, in which the compound is selected from:
- 2-amino-7-metil-5-oxo-4-[4-(trifluorometoxi)fenil]-4H,5H-pirano[4,3-b]pirano-3- carbonitrilo, - 2-amino-7-methyl-5-oxo-4- [4- (trifluoromethoxy) phenyl] -4H, 5H-pyrano [4,3-b] pyran-3-carbonitrile,
- ácido 2-(10,12-dioxo-9-{[(1 ,3-tiazol-2-il)carbamoil]metil}-7-tia-9, 11 - diazatriciclo[6.4.0.02,6]dodeca-1 (8),2(6)-dien-11-il)acético, - {[(1, 3-thiazol-2-yl) carbamoyl] methyl} -7-thia-9, 11 - - 2- (10,12-dioxo-9 acid diazatricyclo [6.4.0.0 2, 6] dodeca- 1 (8), 2 (6) -dien-11-yl) acetic,
ácido 2-{4-[(1 ,3-dimetil-4,6-dioxo-2-sulfaniliden-1 ,3-diazinan-5- iliden)metil]fenoxi}acético,
- ácido 2-(2-metoxi-4-([(5Z)-2,4,6-trioxo-1-(prop-2-en-1-il)-1-3-diazinan-5- iliden]metil)fenoxi)acético, 2- {4 - [(1,3-dimethyl-4,6-dioxo-2-sulfanylidene-1,3-diazinan-5-ylidene) methyl] phenoxy} acetic acid, - 2- (2-methoxy-4 - ([(5Z) -2,4,6-trioxo-1- (prop-2-en-1-yl) -1-3-diazinan-5-ylidene] methyl acid ) phenoxy) acetic,
- ácido 2-[(5E)-4-oxo-5-{[2-(prop-2-en-1-iloxi)fenil]metiliden}-2-sulfaniliden-1 ,3- tiazolidin-3-il]pentanedioico y - 2 - [(5E) -4-oxo-5 - {[2- (prop-2-en-1-yloxy) phenyl] methylidene} -2-sulfanilidene-1,3-thiazolidin-3-yl] pentanedioic acid Y
- ácido 2-{1-[(3,4-difluorofenil)metil]-3-oxopiperazin-2-il}acético. - 2- {1 - [(3,4-difluorophenyl) methyl] -3-oxopiperazin-2-yl} acetic acid.
21. Uso según una cualquiera de las reivindicaciones 15 a 19 en el que las plagas son hongos seleccionados entre las clases Ascomycota y Basidiomycota, preferentemente Blumeria graminis, Erysiphe necator, Erysiphe polygoni, Leveillula táurica, Podosphaera aphanis, Podosphaera xanthii, Alternaría solani, Botrytis cinérea, Penicillium digitatum, Cercospora beticola, Colletotrichum graminicola, Fusarium graminearum, Fusarium oxysporum, Gaeumannomyces graminis, Magnaporthe grísea, Monilinia fructicola, Mycosphaerella fijensis, Phomopsis vitícola, Pyrenophora teres, Rhizopus stolonifer, Rynchosporium secalis, Sclerotinia sclerotiorum, Septoria tritici, Venturia inaequalis, Verticillium dahliae; Puccinia striiformis, Puccinia hordei, Puccinia graminis, Uromyces viciae-fabae, Uromyces betae, Ustilago maydis, Ustilago tritici, Armillaria mellea, Rhizoctonia solani, Sclerotium rolfsii, y patógenos Oomicetos, preferentemente Phytophthora infestaos, Phytophthora cinnamomi, Pythium aphanidermatum, Plasmopara vitícola, Pseudoperonospora cubensis y Bremia lactucae.. 21. Use according to any one of claims 15 to 19 in which the pests are fungi selected from the classes Ascomycota and Basidiomycota, preferably Blumeria graminis, Erysiphe necator, Erysiphe polygoni, Leveillula taurica, Podosphaera aphanis, Podosphaera xanthii, Alternaria solani, Botrytis cinérea, Penicillium digitatum, Cercospora beticola, Colletotrichum graminicola, Fusarium graminearum, Fusarium oxysporum, Gaeumannomyces graminis, Magnaporthe grísea, Monilinia fructicola, Mycosphaerella fijensis, Phomopsis viticola, Pyrenopusiciifer, Septlisotinia, storithuria, Rhynophora, storithuria, Storidisporus inactium Verticillium dahliae; Puccinia striiformis, Puccinia hordei, Puccinia graminis, Uromyces viciae-fabae, Uromyces betae, Ustilago maydis, Ustilago tritici, Armillaria mellea, Rhizoctonia solani, Sclerotium rolfsii, and pathogens Oomycetes, preferably Phytophthora aphudo-phythoracinus, Pythonium vitreous, Pythonium-phoracinus, Plasma vitriolumidum, Pythonium-phoracinum. cubensis and Bremia lactucae ..
22. Uso según una cualquiera de las reivindicaciones 15 a 20 en el que las plagas son insectos, seleccionados entre los órdenes Lepidoptera, Homoptera, Isoptera, Díptera, Orthoptera, Hemiptera y Coleóptera, preferentemente G. mellonella o D. meianogaster. 22. Use according to any one of claims 15 to 20 in which the pests are insects, selected from the orders Lepidoptera, Homoptera, Isoptera, Diptera, Orthoptera, Hemiptera and Coleoptera, preferably G. mellonella or D. meianogaster.
23. Uso según una cualquiera de las reivindicaciones 15 a 20 en el que las plagas son ácaros seleccionados entre los subórdenes Mesostigmata, Sarcoptiformes, Trombidiformes y Onchychopalpida. 23. Use according to any one of claims 15 to 20 in which the pests are mites selected from the suborders Mesostigmata, Sarcoptiformes, Trombidiformes and Onchychopalpida.
24. Uso según una cualquiera de las reivindicaciones 15 a 20 en el que las plagas son piojos seleccionados entre los subórdenes Anoplura y Mallophaga.
24. Use according to any one of claims 15 to 20 in which the pests are lice selected from the suborders Anoplura and Mallophaga.
25. Uso según una cualquiera de las reivindicaciones 15 a 20 en el que las plagas son nematodos parásitos de plantas seleccionados entre el orden Tylenchida, preferentemente Meloidogyne spp., Heterodera spp., Rotylenchus spp. y Pratylenchus spp.; y nematodos parásitos de mamíferos de los órdenes Ascaridia, Spirurida y Strongylida, preferentemente Trichinella spp., Trichuris spp. y Strongyloides spp.
25. Use according to any one of claims 15 to 20 in which the pests are parasitic nematodes of plants selected from the order Tylenchida, preferably Meloidogyne spp., Heterodera spp., Rotylenchus spp. and Pratylenchus spp .; and parasitic nematodes of mammals of the orders Ascaridia, Spirurida and Strongylida, preferably Trichinella spp., Trichuris spp. and Strongyloides spp.
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| ES201930440A ES2794348B2 (en) | 2019-05-17 | 2019-05-17 | Chitin deacetylase inhibitors and their use as arthropocidal agricultural fungicides and nematicides |
| ESP201930440 | 2019-05-17 |
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| WO2020234497A1 true WO2020234497A1 (en) | 2020-11-26 |
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| ES (1) | ES2794348B2 (en) |
| WO (1) | WO2020234497A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113100233A (en) * | 2021-04-19 | 2021-07-13 | 中国农业科学院植物保护研究所 | Application of arylhydroxamic acid and derivatives thereof as chitin deacetylase inhibitor and plant antifungal agent |
| CN114317568A (en) * | 2021-12-31 | 2022-04-12 | 山西大学 | Synthetic method and application of dsRNA of cricket CDA1 and CDA2 genes |
-
2019
- 2019-05-17 ES ES201930440A patent/ES2794348B2/en active Active
-
2020
- 2020-05-15 WO PCT/ES2020/070314 patent/WO2020234497A1/en active Application Filing
Non-Patent Citations (3)
| Title |
|---|
| JESUS MARTINEZ CRUZ: "Analisis morfologico y funcional de la interaccion Podosphaera xanthii-cucurbitaceas. Publications and scientific divulgation", THESIS, 2016, UMA. UNIVERSIDAD DE MÁLAGA, pages 1 - 20 , 463-469, XP055761774 * |
| LAIA GRIFOLL-ROMERO, SERGI PASCUAL ID , HUGO ARAGUNDE, XEVI BIARNÉS AND ANTONI PLANAS: "Chitin Deacetylases: Structures, Specificities, and Biotech Applications", POLYMERS, vol. 10, no. 4, 8 January 2020 (2020-01-08), pages 1 - 29, XP055761777, DOI: 10.3390/polym10040352 * |
| YONG ZHAO; RO-DONG PARK; RICCARDO A A MUZZARELLI: "Chitin deacetylases: Properties and applications", MARINE DRUGS 2010 MOLECULAR DIVERSITY PRESERVATION INTERNATIONAL, vol. 8, 31 December 2009 (2009-12-31), pages 24 - 46, XP055058962, ISSN: 1660-3397, DOI: 10.3390/md8010024 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113100233A (en) * | 2021-04-19 | 2021-07-13 | 中国农业科学院植物保护研究所 | Application of arylhydroxamic acid and derivatives thereof as chitin deacetylase inhibitor and plant antifungal agent |
| CN114317568A (en) * | 2021-12-31 | 2022-04-12 | 山西大学 | Synthetic method and application of dsRNA of cricket CDA1 and CDA2 genes |
| CN114317568B (en) * | 2021-12-31 | 2023-12-26 | 山西大学 | Method for synthesizing dsRNA of cricket CDA1 and CDA2 genes and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| ES2794348B2 (en) | 2021-04-14 |
| ES2794348A1 (en) | 2020-11-17 |
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