Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
1998-11-19
2002-06-04
Woodward, Michael P. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S194000, C424S094500, C504S117000
Reexamination Certificate
active
06399319
ABSTRACT:
This application is the U.S. filing under 35 USC 371 of PCT/EP97/02476, filed May 15, 1997.
The present invention relates to a novel method which is useful in the field of the identification of fungicides.
Protein phosphorylation has long been recognized as an important event in the regulation of cell differentiation as well as in the generation and the transduction of signals in cells. In spite of this knowledge, selection of antifungals has never been based on the protein kinase inhibitory effect.
The present invention consists in a procedure for the identification of fungal protein kinase inhibitors. The procedure consists in the cultivation of the target fungus up to the logarithmic growth phase, in the isolation of a protein kinase and in the protein kinase activity measurement with various concentrations of inhibitors from a concentrated stock solution.
An objective of the present invention is an assay for detecting substances having fungicidal activities which comprises
a) a protein kinase as enzyme in a buffer system,
b) ATP as a phosphate donor, and
c) a phosphate acceptor,
together with an appropriate solvent and a device for measuring the phosphorylation rates.
In particular prefered is an assay for detecting substances having fungicidal activities which comprises
a) determining the activity of a protein kinase in the presence of ATP as a phosphate donor and a phosphate acceptor, together with an appropriate solvent and a device for measuring the phosphorylation rates and
b) selecting substances which inhibit the protein kinase.
A further aspect of the invention is a method for identifying potential fungicides which comprises testing a candidate compound in a protein kinase inhibition assay. Prefered is a method for identifying potential fungicides which comprises
(a) testing a candidate compound in a protein kinases inhibition assay, and
(b) if the candidate compound is active in the protein kinase inhibition assay, testing the compound for activity against fungi.
A further aspect of the invention is a fungicide that has been identified by the protein kinase inhibition assay. Comprised is a compound active in a protein kinase inhibition assay according to the invention as a fungicide.
Another aspect of the invention is the use of a compound, which has been identified with a protein kinase inhibition assay, as a fungicide.
A further aspect of the invention is the use of a compound, which inhibits a protein kinase, as a fungicide.
A further aspect of the invention is a fungicide composition comprising a protein kinase inhibitor as the active ingredient in combination with a carrier and/or a second fungicide.
Another aspect of the invention is a fungicide method which comprises applying a fungi inhibiting amount of a protein kinase inhibitor to the locus it is desired to protect from fungi.
It is known that usnic acid amide inhibits protein kinase C (Phytochemistry 31,2999-3001,1992), that certain medicaments shows protein kinase C inhibition (EP-A-446798) and that a serine protein kinase confers fungicide resistance (Applied and Environmental Microbiology, 61,2341-2345,1995).
It was found that potential fungicides inhibit the protein kinase activity. The inhibition is observed by the measurement of the protein kinase activity as a result of the phosphorylation reaction with a substrate in the presence of the test compound which is being evaluated for fungicidal properties. Protein kinase inhibition assays are utilized in a method for identifying new fungicides.
A test compound can be characterized as having fungicidal properties when one or more of the protein kinases fractions (PK-I, PK-II, PK-III etc) decreases in the activity by the phosphorylation reaction of a substrate compared to the activity observed in the same reaction without the test compound.
Preferred is the use of a test compound in an protein kinase inhibition assay wherein the protein kinase is a protein kinase III from
Neurospora crassa.
The method consists in the isolation of a protein kinase from a fungus, e.g.
Neurospora crassa.
The fungus is cultivated using standard methods, preferably as inoculum in Petri dishes, up to the logarithmic growth phase.
Protein kinase is isolated using standard methods. The protein kinase extract is then resolved into fractions of a suitable volume, preferably of 2 ml. Further purification is carried out, for example by centrifugation, gel filtration, ion exchange chromatography, affinity chromatography, ammonium sulfate precipitation or combinations thereof.
The collected fractions are submitted to the phosphorylation reaction with a dye-labeled substrate or a non labeled substrate, e.g. casein, histon, protamine-sulfate, protamine free base, kemptide; preferably selected from protamine base or protamine-salt, more preferably with the non labeled protamine-sulfate.
Suitable phosphorylation agents are [&ggr;-
33
P]ATP, [&ggr;-
32
P]ATP, ATP, preferably radioactive [&ggr;-
33
P]ATP.
The reaction is carried out in a suitable polar solvent, e.g. water, ethanol, dimethylsulfoxide, acetone, preferably in dimethylsulfoxide.
The amount of phosphorylated products is determined by conventional methods, e.g. colorimetry, scintillation counting, preferably by scintillation counting by use of [&ggr;-
33
P]ATP.
Fractions containing a protein kinase (PK-I, PK-II, PK-III etc) are used in a phosphorylating reaction in the presence of a testing compound under evaluation for fungicidal properties. The method according to the present invention is able to confirm the presence of fungicidal properties when the compound tested has an inhibitory effect on protein kinase activity. If inhibition of the protein kinase activity is observed, the test compound is very probably a fungicide, and to be further tested on pathogens in plants by greenhouse screenings.
This selection test method is useful as a fast screening of compounds that potentially have fungicidal properties.
A preferred assay involves measuring the protein phosphorylating activity by using radioactive [&ggr;-
33
P]ATP and protamine-sulfate as substrate. The phosphorylated products are isolated and quantified by adsorption on P81 chromatography paper and radioactivity measurement in a scintillation counter.
Another aspect of the invention is a fungicide method which comprises applying a fungi inhibiting amount of a protein kinase inhibitor to the locus it is desired to protect from fungi. As used herein, the term “fungi inhibiting amount of protein kinase” refers to an amount of protein kinase inhibitor sufficient to kill or inhibit the fungi it is desired to control. When employed in the treatment of plant fungal diseases, the protein kinase inhibitors are applied to the plants in a disease inhibiting and phytologically acceptable amount. The term “disease inhibiting and phytologically acceptable amount” as used herin, refers to an amount of a compound of the invention which kills or inhibits the plant disease for which control is desired, but is not significantly toxic to the plant.
One aspect of the invention is a fungicide composition comprising a protein kinase inhibitor as the active ingredient in combination with a carrier. When used on plants, protein kinase inhibitors are applied in the form of compositions which are important embodiments of the invention, and which comprise a protein kinase inhibitor as active ingredient in combination with a phytologically-acceptable carrier.
The compositions can be applied to the crop area or plant to be treated, simultaneously or in succession with further compounds. These further compounds can be e.g. fertilisers or micronutrient donors or other preparations which influence the growth of plants. They can also be selective herbicides as well as insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures of several of these preparations, if desired together with further carriers, surfactants or application promoting adjuvants customarily employed in the art of formulation.
The protein kinase inhibitors can be m
Meyer Thomas
Pillonel Christian
Moran Marjorie A
Syngenta Crop Protection Inc.
Teoli, Jr. William
Woodward Michael P.
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