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Title:
INHIBITORS OF PEPSIN OR PEPSIN-LIKE PROTEINASES AS ACARICIDES
Document Type and Number:
WIPO Patent Application WO/1997/007681
Kind Code:
A1
Abstract:
The invention provides novel methods of controlling mite infestations of plants. The novel methods employ the step of applying an effective amount of a compound that can inhibit the proteolytic activity of pepsin and pepsin-like proteinase. A preferred compound for use in the subject methods is pepstatin A. Another aspect of the invention is to provide formulations for use in applying to plants so as to control mites. The formulations comprise one or more inert carrier and a compound that can inhibit the proteolytic activity of pepsin. A preferred acaricidal compound for inclusion in the subject formulations is pepstatin A. Another aspect of the invention is to provide improved methods for the screening of compounds that have acaricidal activity. The screening method of the invention limits the range of compounds for screening to those compounds capable of inhibiting the proteolytic activity is pepsin and pepsin-like proteinases.

Inventors:
YU, Ida, K.
Application Number:
PCT/US1996/014277
Publication Date:
March 06, 1997
Filing Date:
August 23, 1996
Export Citation:
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Assignee:
NZYM, INC.
International Classes:
A01N37/46; A01N61/00; (IPC1-7): A01N61/00; A01N37/46
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Claims:
CLAIMS
1. What is claimed is: A method of treating plants infected with mites, said method comprising the step of applying an effective amount of a compound that inhibits the proteolytic activity of pepsin or pepsinlike proteinase.
2. A method according to Claim 1, wherein the mite is a Tetranychus species.
3. A method according to Claim 2, wherein the mite is Tetranychus urticae .
4. A method according to claim 1, wherein the compound is pepstatin A.
5. A method according to claim 2, wherein the compound is pepstatin A.
6. A method according to claim 3, wherein the compound is pepstatin A.
7. A formulation for application to plants, said formulation comprising, a compound that can inhibit the proteolytic activity of pepsin, and an inert carrier.
8. A formulation according to claim 7, wherein the compound is pepsin.
9. A method of screening for compounds having acaricidal activity against mites, said method comprising the steps, exposing a compound that can inhibit proteolytic activity of pepsin to a plurality of mites belonging to a species of mite, and measuring the survival rate of the mites.
10. A method according to claim 10, wherein the mite species belongs to the genes Tetranychus .
11. A method according to claim 11, wherein the i mite is Tetranychus urticae.
12. A method according to claim 1, wherein the mite is a life stage selected from the group consisting of adult and larva.
Description:
INHIBITORS OF PEPSIN OR PEPSIN-LIKE PROTEINASES AS ACARICIDES.

Field of invention

This invention is in the field of agricultural acaricides, more particularly, in the field of acaricide specific for Tetranychus species.

Background

Many species of mites are known to damage plants of agricultural importance. Mites that are known to be significant agricultural pests include those that belong to the families Tetranychidae, Tyroglyphidae, Glycyphagidae, Eriophydae, and Panonychidae. Mites belonging to the family Tetranychidae, such as Tetranychus urticae and Tetranychus cii-JiaJarinus, are of particular commercial significance. It is of particular interest to prepare compounds that have acaricidal activity so as to prevent and reduce mite infestations. It is also of interest to provide improved methods of screening for compounds that have acaricidal activity.

Summary of the invention

One aspect of the invention is to provide for novel methods of controlling mite infestations of plants. The novel methods employ the step of applying an effective amount of a compound that can inhibit the proteolytic activity of pepsin and pepsin-like proteinase. A preferred compound for use in the subject methods is pepstatin A.

Another aspect of the invention is to provide formulations for use in applying to plants so as to control mites. The formulations comprise one or more inert carrier and a compound that can inhibit the proteolytic activity of pepsin. A preferred acaricidal compound for use inclusion in the subject formulations is pepstatin A. Another aspect of the invention is to provide improved methods for the screening of compounds that have acaricidal activity. The screening method of the invention limits the

range of compounds for screening to those compounds capable of inhibiting the proteolytic activity of pepsin and pepsin¬ like proteinase.

Description of the Specific τmι*.afl_ments

The invention provided herein exploits the discovery that many compounds capable of inhibiting the proteolytic activity of pepsin have acaricidal activity against mites, especially the mite Tetranychus urticae . One aspect of the invention is to provide novel methods of controlling mite infestations of plants. The term "controlling" as used with respect to mites, refers both to the prevention of mite infestations and to the reduction of the number of mites already present on a given plant suffering from a mite infestation. Thus compounds for use in the subject methods have acaricidal activity, i.e., the compounds can kill mites in relatively low concentrations. The compounds for use in the subject methods may have acaricidal activity against one or more of the three stages of the mite life cycle, i.e., the egg stage, the larval stage, and the adult stage. The acaricidal compounds for use in the subject method are inhibitors of the proteolytic activity of the enzyme pepsin. The term "pepsin" as used herein refers to pepsin as produced in the stomach of any of a variety of mammals. Thus the compounds for use in the methods of the invention may inhibit bovine pepsin, ovine pepsin, porcine pepsin, and pepsin-like enzymes. The term "pepsin-like" enzyme as used herein refers to a proteinase that has three-dimensional structural homology to the enzyme pepsin, particularly the two aspartic acid residues present at the active site in pepsin. The term "pepsin inhibitor" as used herein refers to a compounds that interacts with pepsin and can significantly lower the turnover rate of a pepsin catalyzed proteolytic reaction. Many pepsin inhibitors are known to the person of ordinary skill in the art and can be found by referring to numerous publications such as the Handbook of Enzvme Inhibitors, 2nd Edition (ISBN 156081-219-

2) , and the like. Additional pepsin inhibitors of interest are described in, among other places, US Patent 4,980,283, US Patent 4,874,745, US Patent 4,904,583, US Patent 4,874,745, US Patent 4,746,648, US Patent 4,725,580, US Patent 4,185,096, US Patent 3,975,366, US Patent 3,963,579, US Patent 3,869,347, US Patent 3,840,516, and US Patent 3,740,319. Additionally, the invention contemplates the use of pepsin inhibitors that have not yet been identified or synthesized in the methods of the invention. The three dimensional structure of pepsin has been established and the person of ordinary skill in the art may design novel compounds that act as pepsin inhibitor by use of molecular modeling computer software. Additionally, compounds suspected of being pepsin inhibitors may readily be evaluated for their proteolytic inhibitory properties by measuring the effect of the suspected pepsin inhibitor on a pepsin catalyzed reaction. Preferred pepsin inhibitors for use in the subject methods are pepsin inhibitors that lack significant toxicity for humans and for plants to be treated by the subject methods. Not all inhibitors of pepsin proteolytic activity have acaricidal activity; however, pepsin inhibitors having acaricidal may readily be detected by screening pepsin inhibitors of interest in bioassays for toxicity to mites. Typically, such assays involve directly applying pepsin inhibitors of interest to either mite adults, larvae, or eggs and measuring the survival rate, or allowing mite adults or larvae to ingest pepsin inhibitors of interest and then measuring survival rates. Preferred compounds for use in the subject methods are pepstatin A and analogs thereof having anti-pepsin activity. Although the methods of the invention may be used to control many different species of mites, the subject methods are particularly effective against Tetranychus urticae .

The methods of the invention comprise the step of applying a pepsin inhibitor having acaricidal activity to a plant of interest. The acaricide may be applied by a variety of means such as spraying a liquid, dusting a powder and the

like, well known to the person of ordinary skill in the art of crop protection. The particular method of application selected will be dependent upon a number of factors such as the type of plant, the formulation selected, the arrangement of plants in the field, weather conditions, and the like. The actual amount of acaracide applied to each plant may be varied so as to achieve the desired degree of pest control. The optimal dosage for a specific plant, specific mite species, and specific pepsin inhibitor, under a given set of environmental conditions may be determined through routine experimentation in which the dosage is systematically varied.

The methods of the invention preferably use the acaricidal pepsin inhibitor in a formulation adapted for application to plants. The acaricidal pepsin inhibitors for use in the subject methods will generally be used in formulation with a liquid or solid diluent or with an organic solvent. The invention specifically provides for numerous formulations comprising pepsin inhibitors for use as acaricides on plants and a inert carrier, such as a diluent. The term "inert" is used to indicate that the carrier does not have significant acaricidal activity. The formulations of the inventions comprise an acaricidal pepsin inhibitor and a diluent or surfactant, which may not act as an inert carrier. The formulations may further comprise additional compounds that have acaricidal activity. Useful formulations can be prepared in conventional ways. They include dusts, granules, pellets, solutions, emulsions, wettable powders, emuisifiable concentrates and the like. Many of these may be applied directly. Sprayable formulations can be extended in suitable media and used at spray volumes of from about one to several hundred liters per hectare. High strength compositions are primarily used as intermediates for further formulation. The formulations, broadly, contain about 1% to 99% by weight of active ingredient(s) and at least one of a) about 0.1% to 35% surfactant(s) and b) about 5% to 99% solid or liquid inert diluent(s) . More specifically, they will

contain these ingredients in the following approximate proportions:

Active Percent by Weight Ingredient

Diluent(s) Surfactant(s)

Wettable Powders 20-90 0-74 1-10

Oil Suspensions, 5-50 40-95 0-35 Emulsions, Solutions, (including Emuisifiable Concentrates)

Aqueous Suspensions 10-50 40-84 1-20

Dusts 1-25 70-99 0-5

Granules and Pellets 1-95 5-99 0-15

High Strength 90-99 0-10 0-2 Compositions

Lower or higher levels of active ingredient can, of course, be present depending on the intended use and the physical properties of the compound. Higher ratios of surfactant to active ingredient are sometimes desirable, and are achieved by incorporation into the formulation or by tank mixing.

Typical solid diluents are described in Watkins et al. , "Handbook of Insecticide Dust Diluents and Carriers," 2nd Ed., Dorland Books, Caldwell, N.J. The more absorptive diluents are preferred for the wettable powders and the denser ones for dusts. Typical liquid diluents and solvents are described in Marsden, "Solvents Guide," 2nd Ed., Interscience, New York, N.Y., 1950. Solubility under 0.1% is preferred for suspension concentrates; solution concentrates are preferably stable against phase separation at 0°C. "McCutcheon's Detergents and Emulsifiers Annual," MC Publishing Corp., Ridgewood, N.J., as well as Sisely and Wood, "Encyclopedia of Surface Active Agents," Chemical Publ. Co., Inc., New York, N.Y., 1964, list surfactants and recommended uses. All formulations can contain minor amounts

of additives to reduce foam, caking, corrosion, microbiological growth, etc. Additives to protect the active compounds against light induced degradation, e.g., photoprotectants, UV screening compounds, and the like are also preferably included in the subject formulations. Preferably, ingredients should be approved by the U.S. Environmental Protection Agency for the use intended.

The methods of making such compositions are well known. Solutions are prepared by simply mixing the ingredients. Fine solid compositions are made by blending and, usually, grinding as in a hammer or fluid energy mill. Suspensions are prepared by wet milling (see, for example, Littler, U.S. Pat. No. 3,060,084). Granules and pellets may be made by spraying the active material upon prefoamed granular carriers or by agglomeration techniques. See J. E. Browning,

"Agglomeration," Chemical Engineering, Dec. 4, 1967, pp. 147ff and "Perry's Chemical Engineer's Handbook," 4th Ed., McGraw-Hill, New York, N.Y., 1963, pp. 8-59ff.

For more information regarding the art of formulation, see for example:

H. M. LOUX, U.S. Pat. No. 3,235,361, Feb. 15, 1966, Column 6, Line 16 through Column 7, Line 19 and Examples 10 through 41.

R. W. Luckenbaugh, U.S. Pat. No. 3,309,192, Mar. 14, 1967, Column 5, line 43 through Column 7, Line 62 and

Examples 8, 12, 15, 39, 41, 52, 53, 58, 132, 138-140, 162- 164, 166, 167, 169-182.

H. Gysin and E. Knusli, U.S. Pat. No. 2,891,855, June 23, 1959, Column 3, Line 66 through Column 5, Line 17 and Examples 1-4.

G. C. Klingman, "Weed Control as a Science," John Wiley and Sons, Inc., New York, N.Y., 1961, pp.81-96.

J. D. Fryer and S. A. Evans, "Weed Control Handbook," 5th Ed. Blackwell Scientific Publications, Oxford, 1968, pp. 101-103.

Another aspect of the invention is to provide novel methods of screening for compounds that have acaricidal activity, especially acaricidal activity against Tetranychus species, particularly Tetranychus urticae . Conventional methods of screening compounds for acaricidal activity involve the use of relatively simple bioassays in which the toxicity of potential acaricidal is measured against mite eggs, larvae, or adults. A significant disadvantage of these previous screening methods is that the range of potential compounds for screening is virtually infinite. The improved methods of screening for acaricidal compounds described herein limit the potential range of compounds for screening to those compounds that are capable of inhibiting the proteolytic activity of pepsin. The improved screening method of the invention comprises the steps of limiting the compounds for screening to pepsin inhibitors and then applying selected pepsin inhibitors to mites (eggs, larvae, adults) , either directly or through ingestion, and measuring the survival rate of the mites. The invention may be better understood by referring to the following examples. The following examples are offered for the purpose of illustrating the invention and should not be interpreted as a limitation of the invention.

EXAMPLES

Several compounds having proteinase inhibitory properties were tested for acaricidal activity. 100 ppm aqueous solutions were applied to eggs, larvae, and adults of the following species: Aphis craccivora, (Cowpea Aphid) ; Nilaparvata lugens, (Rice Plant Hopper) ; Tetranychus urticae , (Two Spotted Spider Mide) ; Spodoptera Littoralls , (Cotton Leafworm) ; Heliothis virescens , (Tobacco Budword) ; Anthonomus granάis , (Cotton Boll Weevil); and Diabrotica balteata , (Banded Cucumber Beetle) . Activity was only observed against Tetranychus urticae . Pepstatin A was effective against larvae and adults. Soybean trypsin inhibitor, lima bean trypsin inhibitor, and pentamidine isothionate were effective

against larvae and adults. Aprotinin was effective against adults only.

INCORPORATION BY REFERENCE All patents, patents applications, and publications cited are incorporated herein by reference.

EQUIVALENTS

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. Indeed, various modifications of the above- described makes for carrying out the invention which are obvious to those skilled in the field of organic chemistry or related fields are intended to be within the scope of the following claims.