GWT-1 protein in glycosylphosphatidylinositol-anchor biosynthesis

Aminopyrifen, 4-phenoxybenzyl 2-amino-6-methylnicotinate, strongly inhibited the mycelial growth of a wild-type Neurospora crassa strain on Vogel's minimal medium containing 1.2% sucrose, with a 0.001 mg/L concentration required for 50% growth inhibition. Similar to micafungin, an inhibitor of beta-1, 3-glucan synthetase, aminopyrifen further inhibited the growth of N. crassa deletion mutants of MAP kinase cascade genes, such as mak-1 and mak-2, than the wild-type strain, suggesting that aminopyrifen perturbs cell wall-related processes. Furthermore, we found that three chitin synthase gene mutants (chs-1, chs-5, and chs-7) were highly sensitive to both chemicals; however, aminopyrifen, but not micafungin, induced a swollen germ tube from the conidia of chs-5 and chs-7 mutants on Vogel's medium containing 1.2% sucrose. To elucidate the target protein of aminopyrifen, we isolated mutants resistant to aminopyrifen after UV treatment of conidia of the wild-type strain or the chs-5 strain. The resistance mutations were localized to the gwt-1 gene that encodes an acyltransferase, GWT-1, which participates in the biosynthesis of the glycosylphosphatidylinositol (GPI) precursor, and were found to result in S180F and V178A alterations in the protein. These results strongly suggest that aminopyrifen works as an inhibitor targeting GWT-1, a protein involved in GPI-anchor biosynthesis[1].

Aminopyrifen is a novel 2-aminonicotinate fungicide with unique chemistry and a novel mode of action. The fungicide showed high antifungal activity mainly against Ascomycetes and its related anamorphic fungi under in vitro and pot conditions (EC50 values: 0.0039-0.23 mg/L and 1.2-12 mg/L, respectively). The active ingredient strongly inhibited germ-tube elongation of Botrytis cinerea below 0.1 mg/L and invasion into a plant. The compound exhibited no cross-resistance to commercial fungicides in B. cinerea. The antifungal agent showed high preventive efficacy and translaminar action. In the field, aminopyrifen controlled gray mold and powdery mildew at 150 mg/L. Our findings suggest that aminopyrifen is useful for protecting crops from various plant pathogens[2].

In vitro experiments[2]
Potato dextrose agar medium containing fungicide was prepared within a Petri dish 90 mm in diameter. Each pathogen was precultured in appropriate conditions. The mycelial disc (4 mm in diameter) was placed on the medium. After incubation at 20°C 2–10 days for the pathogens, with the exception of Venturia inaequalis, which was incubated at 13°C for 24–43 days under lighted condition, the diameter of the radial mycelial growth was measured. Inhibitory activity was calculated as the percent of inhibition as compared with the untreated plot. Tested fungi and pseudofungus were Botrytis cinerea (strain name: ma4-3), Colletotrichum acutatum (strain name: An8), Fusarium oxysporum f. sp. spinaciae (strain name: M4), Glomerella cingulata (strain name: An1), Monilinia fructicola (strain name: F9), Sclerotium rolfsii (strain name: M28), Sclerotinia sclerotiorum (strain name: SR4), Venturia inaequalis (strain name: F20), Verticillium dahliae (strain name: MAFF731077), Rhizoctonia solani AG-1 IA (strain name: Rs0508), and Pythium aphanidermatum (strain name: MAFF725006). Tests were repeated 2 or 3 times.

Inhibition of invasion into cucumber epidermal cells[2]
Effect of aminopyrifen on infection process of B. cinerea on cucumber cotyledons was examined. Solutions of aminopyrifen SC and M006 were sprayed on the whole plant. One day after treatment, 5 µL of conidial suspensions (1.0×105 spores/mL) were dropped onto the upper side of cucumber cotyledons, and inoculated plants were kept in a humid condition of 20°C. The inoculated part of the cucumber cotyledon was cut into 5 mm squares 6, 24, and 48 hr after inoculation. The tissue was fixed and decolorized by immersing into a formalin-acetic acid-alcohol solution (36.0–38.0% formaldehyde: 99.7% acetic acid: 99% ethanol=1 : 1 : 1). The observation was conducted under a light microscope after staining with 0.1% methyl blue.

In vivo pot tests[2]
Cucumber diseases[2]
Solutions of tested fungicides were applied to whole plants using a spray gun. One day after treatment, conidial suspensions (1.0×106 spores/mL) of Colletotrichum orbiculare (strain name: Dai) or sporangial suspensions (1.0×105 sporangia/mL) of Pseudoperonospora cubensis (strain name: AK) were sprayed onto the abaxial surface of leaves at 0.5 mL per pot using a spray gun. Inoculated plants of C. orbiculare or P. cubensis were kept at 20°C in the dark in a humid chamber for 48 hr or 24 hr, respectively. Those plants were transferred to a greenhouse. Disease severity was assessed at 7 days after inoculation based on criteria shown in 3.2.5. Tests were repeated 2 or 3 times.
Tested fungicides were applied to whole plants. One day after treatment, conidial suspensions (1.0×105 spores/mL) of Podosphaera xanthii (strain name: AK) were inoculated onto the adaxial surface of leaves at 0.5 mL per pot using a spray gun. Inoculated plants were kept in the greenhouse, and disease severity was assessed 10 days after inoculation based on criteria shown in 3.2.5. Tests were repeated 3 times.

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Tomato disease[2]
Fungicide solutions were applied to the whole plants. One day after treatment, sporangial suspensions (1.0×105 sporangia/mL) of Phytophthora infestans (strain name: PiN5-2) were sprayed onto the adaxial surface of leaves at 0.5 mL per pot using a spray gun. Inoculated plants were kept at 20°C in a humid chamber for 24 hr. After incubation, plants were transferred to the greenhouse. Disease severity was assessed 7 days after inoculation based on criteria shown in 3.2.5. Tests were repeated 2 times.

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Wheat diseases Diluted solutions of tested fungicides were sprayed on whole plants at a rate of 1000 L/ha using an atomizer. One day after treatment, the first leaf was fragmented into 30 mm lengths (5 fragments per pot), and the fragments were placed on wet paper in a plastic box (235×325×50 mm3). Infected leaves with large amounts of conidia of Blumeria graminis f. sp. tritici (strain name: AK) were tapped from 300 mm above the box. After inoculation, the fragments were transferred to 1.5% agar medium in a 90 mm Petri dish and incubated in a growth chamber under conditions of 20°C, 50% relative humidity, and 5000 lx (16 hr/day). Disease severity was assessed 7 days after treatment based on criteria shown in 3.2.5. Tests were repeated 3 times.
Solutions of tested fungicides were applied to seedlings at a rate of 1000 L/ha using an atomizer. One day after treatment, conidial suspensions (1.0×105 spores/mL) of Puccinia recondita (strain name: AK) were sprayed onto whole plants using a spray gun. Inoculated plants were kept at 20°C in a humid chamber for 48 hr. After incubation, those plants were transferred to the greenhouse. Disease severity was assessed 7 days after inoculation based on criteria shown in 3.2.5. Tests were repeated 3 times.

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Rice disease[2]
Fungicide solutions were applied to whole plants. One day after treatment, conidial suspensions (1.0×105 spores/mL) of Pyricularia oryzae (strain name: 05044) were inoculated onto the leaves using a spray gun. After inoculation, tested plants were kept at 20°C in a humid chamber for 48 hr. The plants were transferred to the greenhouse after incubation. The number of lesions on secondary leaves was counted 7 days after inoculation. Efficacy was calculated by comparing the number of lesions of the treated pot with that of the untreated pot. Tests were repeated 2 times.

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Biological properties against cucumber gray mold in pots[2]
Cucumber cotyledons were used for tests of preventive and curative activity and translaminar action. For a preventive test, the solutions of aminopyrifen SC were applied to the seedlings using a spray gun. Spore suspensions (1.0×106 spores/mL; strain name: ma-4-3) were inoculated onto the abaxial surface of the leaves one day or 8 days after treatment. In the test of translaminar action, fungicide solutions were sprayed on the adaxial surface of the leaves. The spore suspensions were inoculated onto the abaxial surface of the leaves one day after application. For a curative test, the spore suspensions were inoculated onto the abaxial surface of the leaves. Fungicide solutions were sprayed onto the whole plants 21.5 hr after inoculation. Evaluation was performed based on criteria shown in 3.2.5. Tests were repeated 2 or 3 times.

[1]. Pestic Biochem Physiol. 2019 May;156:1-8.
[2]. J Pestic Sci. 2021 May 20;46(2):198-205.

GWT-1 inhibitors were well studied for pharmaceuticals.) For instance, BIQ (1-[(4-butylphenyl)methyl]isoquinoline)) and manogepix (3-(3-{4-[(pyridin-2-yloxy)methyl]benzyl}isoxazol-5-yl)pyridin-2-amine)) are active against pathogens including Candida, Cryptococcus, and Aspergillus sp. The former is a quinoline compound, and the latter is a pyridine and isoxazole derivative and the prodrug of the compound, fosmanogepix, which is under clinical development. However, aminopyrifen is a novel compound with pyridine and ester moieties for agricultural use; its structure is unique from those of pharmaceutical compounds.
In addition to aminopyrifen’s unique mode of action, this fungicide showed high activity against various economically important pathogenic fungi (Table 1 and 2)) Furthermore, aminopyrifen exhibited high preventive, residual efficacies and also translaminar action, which contribute to efficient disease control in the field (Fig. 3). In fact, this fungicide was highly applicable as preventive use in the field against gray mold and powdery mildew (Fig. 4). In agricultural situations, farmers face problems of crop losses arising from various diseases and the appearance of pathogens resistant to fungicides. A novel fungicide, aminopyrifen, is anticipated to be one crop protection tool contributing to stable crop production.[2]

C20H18N2O3

334.37

334.131

C, 71.84; H, 5.43; N, 8.38; O, 14.35

1531626-08-0

91810812

Typically exists as solid at room temperature

4.3

1

5

6

25

417

0

PWWPULQZEAPTTB-UHFFFAOYSA-N

InChI=1S/C20H18N2O3/c1-14-7-12-18(19(21)22-14)20(23)24-13-15-8-10-17(11-9-15)25-16-5-3-2-4-6-16/h2-12H,13H2,1H3,(H2,21,22)

(4-phenoxyphenyl)methyl 2-amino-6-methylpyridine-3-carboxylate

Aminopyrifen; Aminopyrifen [ISO]; D4FFS38EC4; 1531626-08-0; UNII-D4FFS38EC4; (4-Phenoxyphenyl)methyl 2-amino-6-methyl-3-pyridinecarboxylate; 4-PHENOXYBENZYL 2-AMINO-6-METHYLNICOTINATE; (4-phenoxyphenyl)methyl 2-amino-6-methylpyridine-3-carboxylate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)