Patent Application
20070078185
The invention relates to methods for controlling invertebrate pests.
Biogenic amines play pivotal roles in the control of animal behavior. The biogenic amine octopamine can act as a neurotransmitter in invertebrates and is considered the invertebrate counterpart to norepinephrine. Octopamine has been implicated in several physiological processes, including light emission by fireflies (Nathanson, J. A., Science 203:65-68 (1979)), foraging behavior (Barron et al., J Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 188:603-610 (2002)), the sting response of honey bees (Burrell et al., J Insect. Physiol. 41:671-680 (1995)), the fight or flight response of locusts (Orchard et al., A. Rev. Ent. 38:227-249 (1993)), associative learning by fruit flies and honey bees (Hammer et al., Learn. Mem. 5:146-156 (1998); and Schwaerzel et al., J Neurosci 23:10495- 10502 (2003)), and ovary muscle contraction in locusts and fruit flies (Orchard et al., J. NeurobioL 16:171-181 (1985); Lee et al., Dev. Biol. 264:179-190 (2003); and Monastirioti, M., Dev. Biol. 264:38-49 (2003)).
Octopamine biosynthesis requires tyrosine decarboxylase to convert tyrosine into tyramine and tyramine beta-hydroxylase to convert tyramine into octopamine. The physiological role of tyramine, the biosynthetic precursor of octopamine, has been relatively unexplored (Roeder et al., Arch. Insect Biochem. Physiol. 54:1-13 (2003)). Tyramine was initially thought to be simply a precursor octopamine. However, the identification of G-protein coupled receptors in Drosophila (Saudou et al., EMBO J. 9:3611-3617 (1990)), the locust (Vanden Broeck et al., J Neurochem. 64:2387-2395 (1995)), the honey bee (Blenau et al., J. Neurochem. 74:900-908 (2000)), the silk moth (Ohta et al., Insect Mol. Biol 12:217-223 (2003)).and C. elegans (Rex et al., J. Neurochem. 82:1352-1359 (2002)) that respond to tyramine suggested that tyramine may itself act as a neurotransmitter.
A better understanding of the physiological role of tyramine and tyraminergic signaling components in invertebrates can lead to new invertebrate-specific methods of controlling invertebrate pests.
A method of controlling an insect, nematode, or other invertebrate population is provided by the invention. The method involves contacting an invertebrate with an invertebrate tyrosine decarboxylase inhibitor in an amount sufficient to kill, incapacitate, or prevent reproduction by the invertebrate (e.g., insects or nematodes). The inhibitors may also be prophylactically applied to a site or organism to prevent infestation or infection by an invertebrate.
In a first aspect, the invention features a method of inhibiting proliferation of an insect at a site by contacting the site with a tyrosine decarboxylase inhibitor in an amount sufficient to inhibit the proliferation. The site can be, for example, on a plant, in or on an animal, or in a dwelling (e.g., a home).
In one embodiment of the above aspect, the insect is a beetle, grasshopper, locust, wasp, bee, mosquito, fly, midge, ant, cotton leaf perforator, flea, roach, termite, aphid, scale, mite, nematode, arachnid, or moth. Desirably, the invertebrate is a parasitic nematode.
In a related aspect, the invention features a method of inhibiting proliferation of an invertebrate at a site by contacting the site with a tyrosine decarboxylase inhibitor in an amount sufficient to inhibit the proliferation, wherein the inhibitor is a compound of formula I:
wherein n is 0 or 1; each of R1, R2, and R3 is, independently, selected from H, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, and C1-4 heteroalkyl; R4 is selected from H and acyl; and R5 is H, F, or OH.
In one embodiment of the above aspect, the invertebrate is a beetle, grasshopper, locust, wasp, bee, mosquito, fly, midge, ant, cotton leaf perforator, flea, roach, termite, aphid, scale, mite, nematode, arachnid, or moth. Desirably, the invertebrate is a parasitic nematode.
The invention also features a compound of formula I:
wherein n is 0 or 1; each of R1, R2, and R3 is, independently, selected from H, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, and C1-4 heteroalkyl; R4 is selected from H and acyl; and R5 is H, F, or OH.
The invention further features a composition for inhibiting the proliferation of invertebrates including a compound of formula I, or a suitable salt thereof, together with a diluent or dispersant. Suitable diluents and dispersants include, without limitation, Solvesso™, dipropylene glycol monomethyl ether, and N-methyl-pyrrolidone. The composition can be in the form of, for example, a spray, dust, granular material, a suspension, emulsion, pellet, or wettable powder.
The invention also features a kit including (i) a tyrosine decarboxylase inhibitor of the invention, or a salt thereof, and (ii) instructions for delivering the inhibitor to a site infested, or at risk of infestation, by an invertebrate population..
The invention further features a pharmaceutical composition including a tyrosine decarboxylase inhibitor of the invention, or a salt thereof, together with a pharmaceutically acceptable excipient.
In a related aspect, the invention features a method for identifying an inhibitor of invertebrate tyrosine decarboxylase. The method includes the steps of (i) contacting invertebrate tyrosine decarboxylase with tyrosine in the presence of a candidate compound; and (ii) monitoring the conversion of tyrosine to tyramine. In one embodiment, the tyrosine is labeled, e.g., radiolabeled. This method may be performed in vivo or in vitro, for example, in a high throughput cell-free assay.
In a certain embodiment of any of the above aspects, the compound of formula (I) is (2S)-2-(3-hydroxybenzyl)-2-hydrazinopropanoic acid, N-(DL-seryl)- N′-(2,4,-dihydroxybenzyl) hydrazine, or 4-hydrazinomethyl-benzene- 1,3-diol.
By “tyrosine decarboxylase” is meant an enzyme which is naturally occurring in an invertebrate and which catalyzes the in vivo conversion of L-tyrosine into tyramine. Tyrosine decarboxylases include, for example, Drosophila CG30446 protein and mosquito CP3581 protein.
By “tyrosine decarboxylase inhibitor” is meant a compound that can reduce the rate of conversion of tyrosine to tyramine by tyrosine decarboxylase by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 99% in comparison to the conversion rate in the absence of the inhibitor. Tyrosine decarboxylase inhibitors of the invention can bind to TDC- 1 with an affinity of less than 10 μM, 1 μM, or even 500 nM under physiological conditions.
As used herein, “biocide” refers to a compound which inhibits tyrosine decarboxylase and slows, delays, inhibits, or arrests the growth or reproduction of any invertebrate by at least 10%, 20%, 30%, 40%, 50%, 60%, or even by as much as 70%, 80%, 90%, 95%, or 99% in comparison to growth or reproduction in the absence of the biocide.
By “an amount sufficient” is meant the amount of a tyrosine decarboxylase inhibitor required to inhibit or arrest the growth of an invertebrate, inhibit reproduction in an invertebrate, prevent or deter infestation of a site by an invertebrate, or repel an invertebrate. A sufficient amount of tyrosine decarboxylase inhibitor used to practice the present invention for controlling invertebrate pests varies depending upon a number of factors, including, the invertebrate being controlled, the tyrosine decarboxylase being inhibitor used, the formulation used, and the site to which the inhibitor is applied. In preferred embodiments, the invertebrate pest is an insect. A dose-response curve, as described in the experimental results, can be used to determine a sufficient amount for any particular combination of factors.
As used herein, “inhibiting proliferation” refers to the application of tyrosine carboxylase inhibitor to either an invertebrate population or to a site at risk of infestation by an invertebrate population. Proliferation is inhibited when there is a reduction in invertebrate population growth rate in the presence of a tyrosine decarboxylase inhibitor in comparison to the growth rate observed for the same conditions in the absence of a tyrosine decarboxylase inhibitor.
By “parasitic nematode” is meant any nematode that lives on or within the cells, tissues, or organs of a genetically distinct host organism (e.g., plant or animal). For example, parasitic nematodes include, but are not limited to, any ascarid, filarid, or rhabditid (e.g., Onchocerca volvulus, Ancylostoma, Ascaris, Ascaris lumbricoides, Ascaris suum, Baylisascaris, Baylisascaris procyonis, Brugia malayi, Dirofilaria, Dirofilaria immitis, Dracunculus, Haemonchus contortus, Heterorhabditis bacteriophora, Loa loa, root-knot nematodes, such as Meloidogyne, M. arenaria,, M. chitwoodi, M. graminocola, M. graminis, M. hapla, M. incognita, Necator, M. microtyla, and M. naasi, cyst nematodes (for example, Heterodera sp. such as H. schachtii, H. glycines, H. sacchari, H. oryzae, H. avenae, H. cajani, H. elachista, H. goettingiana, H. graminis, H. mediterranea, H. mothi, H. sorghi, and H. zeae, or, for example, Globodera sp. such as G. rostochiensis and G. pallida) root-attacking nematodes (for example, Rotylenchulus reniformis, Tylenchuylus semipenetrans, Pratylenchus brachyurus, Radopholus citrophilus, Radopholus similis, Xiphinema americanum, Xiphinema rivesi, Paratrichodorus minor, Heterorhabditis heliothidis, and Bursaphelenchus xylophilus), and above-ground nematodes (for example, Anguina funesta, Anguina tritici, Ditylenchus dipsaci, Ditylenchus myceliphagus, and Aphenlenchoides besseyi), Parastrongyloides trichosuri, Pristionchus pacificus, Steinernema, Strongyloides stercoralis, Strongyloides ratti, Toxocara canis, Trichinella spiralis, Trichuris muris or Wuchereria bancrofti).
By “ortholog” is meant any polypeptide of an organism that is highly related to a reference protein or nucleic acid sequence from another organism. The degree of relatedness may be expressed as the probability that a reference protein would identify a sequence, for example, in a blast search. The probability that a reference sequence would identify a random sequence as an ortholog is ext4remely low, less than e−10,e−20, e−30, e−40, e−50, e−75, e−100. The skilled artisan understands that an ortholog is likely to be functionally related to the reference protein or nucleic acid sequence. In other words, the ortholog and its reference molecule would be expected to fulfill similar, if not equivalent, functional roles in their respective organisms.
By “substantially identical” is meant a polypeptide exhibiting at least 30% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein). Preferably, such a sequence is at least 40%, more preferably 50%, and most preferably 60% or even 75% identical to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e−3 and e−100 indicating a closely related sequence.
In the generic descriptions of compounds of this invention, the number of atoms of a particular type in a substituent group is generally given as a range, e.g., an alkyl group containing from 1 to 4 carbon atoms or C1-4 alkyl. Reference to such a range is intended to include specific references to groups having each of the integer number of atoms within the specified range. For example, an alkyl group from 1 to 4 carbon atoms includes each of C1, C2, C3, and C4. A C1-4 heteroalkyl, for example, includes from 1 to 3 carbon atoms in addition to one or more heteroatoms. Other numbers of atoms and other types of atoms may be indicated in a similar manner.
As used herein, the terms “alkyl” and the prefix “alk-” are inclusive of both straight chain and branched chain groups and of cyclic groups, i.e., cycloalkyl. Cyclic groups can be monocyclic or polycyclic and preferably have from 3 to 4 ring carbon atoms, inclusive. Exemplary cyclic groups include cyclopropyl and cyclobutyl groups. The C1-4 alkyl group may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, cyano, nitrilo, NH-acyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups. C1-4- alkyls include, without limitation, methyl; ethyl; n-propyl; isopropyl; cyclopropyl; cyclopropylmethyl; n-butyl; iso-butyl; sec-butyl; tert-butyl; and cyclobutyl.
By “C2-4 alkenyl” is meant a branched or unbranched hydrocarbon group containing one or more double bonds and having from 2 to 4 carbon atoms. A C2-4 alkenyl may optionally include monocyclic or polycyclic rings, in which each ring desirably has from three to four members. The C2-4 alkenyl group may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, cyano, nitrilo, NH-acyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups. C2-4 alkenyls include, without limitation, vinyl; allyl; 2-cyclopropyl-1-ethenyl; 1-propenyl; 1-butenyl; 2-butenyl; 3-butenyl; 2-methyl-1-propenyl; and 2-methyl-2-propenyl.
By “C2-4 alkynyl” is meant a branched or unbranched hydrocarbon group containing one or more triple bonds and having from 2 to 4 carbon atoms. The C2-4 alkynyl group may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxy, fluoroalkyl, perfluoralkyl, cyano, nitrilo, NH-acyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups. C2-4 alkynyls include, without limitation, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and 3-butynyl.
By “C1-4 heteroalkyl” is meant a branched or unbranched alkyl, alkenyl, or alkynyl group having from 1 to 4 carbon atoms in addition tol, 2, 3 or 4 heteroatoms independently selected from the group consisting of N, 0, S, and P. Heteroalkyls include, without limitation, tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiesters, phosphoramidates, sulfonamides, and disulfides. A heteroalkyl may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has three to six members. The heteroalkyl group may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, cyano, nitrilo, NH-acyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, hydroxyalkyl, carboxyalkyl, and carboxyl groups. Examples of C1-8 heteroalkyls include, without limitation, methoxymethyl and ethoxyethyl.
By “halide” is meant bromine, chlorine, iodine, or fluorine.
By “fluoroalkyl” is meant an alkyl group that is substituted with a fluorine.
By “perfluoroalkyl” is meant an alkyl group consisting of only carbon and fluorine atoms.
By “carboxyalkyl” is meant a chemical moiety with the formula -(R)- COOH, wherein R is selected from C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, or C1-4 heteroalkyl.
By “hydroxyalkyl” is meant a chemical moiety with the formula -(R)-OH, wherein R is selected from C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, or C1-4 heteroalkyl.
By “alkoxy” is meant a chemical substituent of the formula -OR, wherein R is selected from C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, or C1-4 heteroalkyl.
By “alkylthio” is meant a chemical substituent of the formula -SR, wherein R is selected from C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, or C1-4 heteroalkyl.
By “quaternary amino” is meant a chemical substituent of the formula -(R)- N(R′)(R″)(R′″)+, wherein R, R′, R″, and R′″ are each independently an alkyl, alkenyl, or alkynyl group. R may be an alkyl group linking the quaternary amino nitrogen atom, as a substituent, to another moiety. The nitrogen atom, N, is covalently attached to four carbon atoms of alkyl and/or aryl groups, resulting in a positive charge at the nitrogen atom.
By “acyl” is meant a chemical moiety with the formula R-C(O)-, wherein R is selected from C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C1-4 heteroalkyl, or amino acid acyl.
By “amino acid acyl” is meant a chemical moiety with the formula R-C(O)-, wherein R-C(O)- is selected from natural and unnatural α, β, and γ amino acids, including, for example, N-alkylated amino acids, and natural amino acids, such as alanine, serine, and glycine, among others.
Applicants have discovered genes encoding tyrosine decarboxylase which are present in invertebrates and necessary for the in vivo conversion of tyrosine to tyramine. The compounds of the invention, which can act as tyrosine decarboxylase inhibitors and block the biosynthesis of tyramine, are useful for controlling the proliferation of invertebrates.
FIGS. 3A-C show that tdc-1 encodes a tyrosine decarboxylase.
FIGS. 4A-D show TBH- 1 and TDC- 1 protein expression.
FIGS. 5A-SC are graphs showing that tdc-1 mutant nematodes are hyperactive in egg laying.
The invention features methods and compositions that disrupt tyrosine decarboxylase activity and are therefore useful biocides.
We identified and characterized a Caenorhabditis elegans tyrosine decarboxylase gene, tdc-1, as well as a tyramine beta-hydroxylase gene, tbh-1. Our findings demonstrate that C. elegans has distinct tyraminergic cells and that tyramine functions independently of octopamine in the Caenorhabditis elegans nervous system. Tyrosine decarboxylase proteins have been identified in the fruit fly, the honey bee and the mosquito. These proteins are closely related to the C. elegans TDC-1 protein. These proteins, inhibited by the methods of the invention, are the insect tyrosine decarboxylases required for tyramine and octopamine biosynthesis. Since tyramine and octopamine are restricted to the invertebrate nervous system, tyraminergic or octopaminergic signaling pathways provide highly specific biocide targets. Tyrosine decarboxylase inhibitors can be used to block the biosynthesis of tyramine in invertebrates and affect invertebrate viability.
Tyrosine Decarboxylase Inhibitors
Tyrosine decarboxylase inhibitors are biocidal agents that can be used to kill, or control the proliferation, of invertebrates. The methods and compositions of the invention are expected to be superior to available biocides, e.g., insecticides and nematicides, due to their very specific mode of action. Because the methods and compositions of the invention specifically target invertebrate proteins, they are less likely to produce adverse effects in mammals and plants to which they are administered.
The tyrosine decarboxylase inhibitor can be, for example, a compound of formula I:
In formula I, n is 0 or 1; each of R1, R2, and R3 is, independently, selected from H, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, and C1-4 heteroalkyl; R4 is selected from H and acyl; and R5 is H, F, or OH.
Compounds of formula I can be synthesized using the methods described herein for the synthesis of compounds 1-3, or using, for example, tyrosine and the synthetic protocols provided in U.S. Pat. Nos. 3,462,536 and 3,178,476, each of which is incorporated herein by reference.
Formulation and Use
Tyrosine decarboxylase inhibitors can be formulated for topical and/or systemic application to mammals, field crops, grasses, fruits and vegetables, lawns, trees, and/or ornamental plants. Alternatively, the inhibitors disclosed herein may be formulated as a spray, dust, powder, or other aqueous, atomized or aerosol for killing an invertebrate or controlling an invertebrate population.
Regardless of the method of application, the tyrosine decarboxylase inhibitor is applied in a biocidally-effective amount, which will vary depending on such factors as, for example, the specific target invertebrate to be controlled, the specific environment, location, plant, crop, or agricultural site to be treated, and the method, rate, concentration, stability, and quantity of the tyrosine decarboxylase inhibitor applied. The formulations may also vary with climatic conditions, environmental considerations, and/or frequency of application and/or severity of invertebrate infestation.
The biocidal formulation may be administered to a particular plant or target area in one or more applications, as needed, with a typical field application rate per hectare ranging on the order of about 50, 100, 200, 300, 400, or 500 g/hectare of active ingredient, or alternatively, 600, 700, 800, 900, or 1,000 g/hectare may be utilized. In certain instances, it may be desirable to apply the biocidal formulation to a target area at an application rate of about 1,000, 2,000, 3,000, 4,000, 5,000 g/hectare or even as much as 7,500, 10,000, or 15,000 g/hectare of active ingredient.
Tyrosine decarboxylase inhibitors of the invention can be formulated, for example, as a dust or granular material, a suspension in oil (vegetable or mineral), water, an oil/water emulsion, a pellet, or as a wettable powder, or in combination with any other carrier material suitable for application to a site of infestation. For example, suitable agricultural carriers can be solid or liquid and are well known in the art. Agriculturally acceptable carriers can include adjuvants, inert components, dispersants, surfactants, tackifiers, and binders, that are commonly used in insecticide or nematicide formulations. The formulations may be mixed with one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously mixing, blending and/or grinding the biocidal composition with suitable adjuvants using conventional formulation techniques.
The tyrosine decarboxylase inhibitors of the invention may also be used in consecutive or simultaneous application to an environmental site singly or in combination with one or more additional insecticides, pesticides, chemicals, fertilizers, or other compounds.
Other application techniques, including dusting, sprinkling, soil soaking, soil injection, seed coating, seedling coating, foliar spraying, aerating, misting, atomizing, fumigating, aerosolizing, and the like, are also feasible and may be required under certain circumstances, such as to eradicate insects that cause root or stalk infestation, or for application to delicate vegetation or ornamental plants. These application procedures are also well-known to those of skill in the art.
Therapeutic Formulations
The methods and compositions of the invention can be used for the treatment of an invertebrate infection in an animal, e.g., a hookworm infection.
The compositions of the invention may be administered with a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form. Administration may be transdermal, parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intracerebroventricular, intrathecal, intranasal, aerosol, by suppositories, or oral administration.
Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
Methods well known in the art for making formulations are found, for example, in “Remington: The Science and Practice of Pharmacy” (20th ed., ed. A. R. Gennaro, 2000, Lippincott Williams & Wilkins). Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Nanoparticulate formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) may be used to control the biodistribution of the compounds. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycolate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel. The concentration of the compound in the formulation will vary depending upon a number of factors, including the dosage of the drug to be administered, and the route of administration.
The compound may be optionally administered as a pharmaceutically acceptable salt, such as a non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like. Metal complexes include calcium, zinc, iron, and the like.
Administration of compounds in controlled release formulations is useful where the compound of formula I has (i) a narrow therapeutic index (e.g., the difference between the plasma concentration leading to harmful side effects or toxic reactions and the plasma concentration leading to a therapeutic effect is small; generally, the therapeutic index, TI, is defmed as the ratio of median lethal dose (LD50) to median effective dose (ED50)); (ii) a narrow absorption window in the gastro-intestinal tract; or (iii) a short biological half-life, so that frequent dosing during a day is required in order to sustain the plasma level at a therapeutic level.
Many strategies can be pursued to obtain controlled release in which the rate of release outweighs the rate of metabolism of the therapeutic compound. For example, controlled release can be obtained by the appropriate selection of formulation parameters and ingredients, including, e.g., appropriate controlled release compositions and coatings. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes.
Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc).
Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium.
Pharmaceutical formulations of compounds of formula I can include isomers such as diastereomers and enantiomers, mixtures of isomers, including racemic mixtures, salts, solvates, and polymorphs thereof.
The formulations can be administered to an animal in therapeutically effective amounts. For example, an amount is administered which prevents, reduces, or eliminates the invertebrate infection. Typical dose ranges are from about 0.001 μg/kg to about 2 mg/kg of body weight per day. Desirably, a dose of between 0.001 μg/kg and 1 mg/kg of body weight, or 0.005 μLg/kg and 0.5 mg/kg of body weight, is administered. The exemplary dosage of drug to be administered is likely to depend on such variables as the type and extent of the condition, the overall health status of the particular animal, the formulation of the compound, and its route of administration. Standard clinical trials may be used to optimize the dose and dosing frequency for any particular compound.
Invertebrate Pests
Virtually all field crops, plants, and commercial farming areas are susceptible to attack by one or more invertebrate pests. For example, crops can be susceptible to attack by insects and/or parasitic nematodes. All of the invertebrate pests described herein may be targeted with a tyrosine decarboxylase inhibitor to reduce the proliferation (e.g., slow, delay, inhibit, or arrest the growth, viability, or reproduction of) of the pest.
Tyrosine decarboxylase inhibitor of the invention are expected to demonstrate a very low acute toxicity towards non-targeted organisms, such as plants and animals. The compounds are expected to be useful in targeting a variety of invertebrate species, including, but not limited to, mosquitos, flies, midges, ants, cotton leaf perforator, fleas, roaches, termites, aphids, scales, mites, arachnids, spruce bud worm, and gypsy moths, among others. Particular species against which compounds of the invention may be expected to be effective include, without limitation, american cockroach (Periplaneta Americana), german cockroach (Blattella germanica), yellowfever mosquito (Aedes aeqypti), malaria mosquitos (Anopheles quadrimaculatus, Anopheles gambiae, and Anopheles arabiensis), northern house mosquito (Culex pipens), house fly (Musca domestica), house cricket (Acheta domesticus), corn earworm (Heliothis zea), differential grasshopper (Melanoplus differentialis), and yellow mealworm (Tenebrio molitor).
Insects that can be targeted using the methods and compositions of the invention include, without limitation, insects which attack vegetable, cole crops, and spices, such as alfalfa looper, armyworm, beet armyworm, artichoke plume moth, cabbage budworm, cabbage looper, cabbage webworm, corn earworm, celery leafeater, cross-striped cabbageworm, european corn borer, diamondback moth, green cloverworm, imported cabbageworm, melonworm, omnivorous leafroller, pickleworm, rindworm complex, saltmarsh caterpillar, soybean looper, tobacco budworm, tomato fruitworm, tomato hornworm, tomato pinworm, velvetbean caterpillar, and yellowstriped armyworm; insects which attack pasture and hay crops, such as armyworm, beef armyworm, alfalfa caterpillar, European skipper, a variety of loopers and webworms, as well as yellowstriped armyworms; insects which attack fruit and vine crops, such as achema sphinx moth, amorbia, armyworm, citrus cutworm, banana skipper, blackheaded fireworm, blueberry leafroller, cankerworm, cherry fruitworm, citrus cutworm, cranberry girdler, eastern tent caterpillar, fall webworm, fall webworm, filbert leafroller, filbert webworm, fruit tree leafroller, grape berry moth, grape leaffolder, grapeleaf skeletonizer, green fruitworm, gummosos-batrachedra commosae, gypsy moth, hickory shuckworm, hornworms, loopers, navel orangeworm, obliquebanded leafroller, omnivorous leafroller. omnivorous looper, orange tortrix, orangedog, oriental fruit moth, pandemis leafroller, peach twig borer, pecan nut casebearer, redbanded leafroller, redhumped caterpillar, rougliskinned cutworm, saltmarsh caterpillar, spanworm, tent caterpillar, thecla-thecla basillides, tobacco budworm, tortrix moth, tufted apple budmoth, variegated leafroller, walnut caterpillar, western tent caterpillar, and yellowstriped armyworm; insects which attack field crops, such armyworm, asian and other corn borers, banded sunflower moth, beet armyworm, bollworm, cabbage looper, corn rootworm (including southern and western varieties), cotton leaf perforator, diamondback moth, european corn borer, green cloverworm, headmoth, headworm, imported cabbageworm, loopers (including Anacamptodes spp.), obliquebanded leafroller, omnivorous leaftier, podworm, podworm, saltmarsh caterpillar, southwestern corn borer, soybean looper, spotted cutworm, sunflower moth, tobacco budworm, tobacco hornworm, and velvetbean caterpillar; insects which attack bedding plants, flowers, or ornamentals, such as armyworm, azalea moth, beet armyworm, diamondback moth, ello moth (hornworm), Florida fern caterpillar, lo moth, loopers, oleander moth, omnivorous leafroller, omnivorous looper, and tobacco budworm; insects which attack trees and shrubs, such as bagworm, blackheaded budworm, browntail moth, California oakworm, douglas fir tussock moth, elm spanworm, fall webworm, fuittree leafroller, greenstriped mapleworm, gypsy moth, jack pine budworm, mimosa webworm, pine butterfly, redhumped caterpillar, saddleback caterpillar, saddle prominent caterpillar, spring and fall cankerworm, spruce budworm, tent caterpillar, tortrix, and western tussock moth; and insects which attack grasses, such as armyworm, sod webworm, and tropical sod webworm.
Nematode that can be targeted using the methods and compositions of the invention include, without limitation, nematodes of the Family Longidoridae (e.g., Xiphinema spp. and Longidorus spp.) and Trichodoridae, (e.g., Trichodorus spp. and Paratrichodorus spp.), migratory ectoparasites belonging to the Families Anguinidae (e.g., Ditylenchus spp.), Dolichodoridae (Dolichodorus spp.) and Belenolaimidae (e.g., Belenolaimus spp. and Trophanus spp.).; obligate parasites belonging to the —Families Pratylenchidae (e.g., Pratylenchus spp., Radopholus spp., and Nacobbus spp.), Hoplolaimidae (e.g., Helicotylenchus spp., Scutellonema spp., and Rotylenchulus spp.), Heteroderidae (e.g., Heterodera spp., Globodera spp., Meloidogyne spp., and Meloinema spp.), Criconematidae (e.g., Croconema spp., Criconemella spp., Hemicycliophora spp.), and Tylenchulidae (e.g., Tylenchulus spp., Paratylenchulus spp., and Tylenchocriconema spp.); and parasites belonging to the Families Aphelenchoididae (e.g., Aphelenchoides spp., Bursaphelenchus spp., and Rhadinaphelenchus spp.) and Fergusobiidae (e.g., Fergusobia spp.).
The methods and compositions of the invention can be used to treat an infection in an animal (e.g., a human) by any parasitic nematode, including, without limitation, heartworm, hookworm, roundworm, whipworm, pinworm, and specifically, Strongyloides stercoralis, Onchocerca volvulus, Trichostrongylus colubriformis, Haemonchus contortus, Dictyocaulus viviparus, Ascaris suum, W. bancrofti, Necator americanus, Ancylostoma duodenale, Ascaris lumbricoides, and Trichinella spp.
The following experimental results are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compounds claimed herein are performed, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.
H13N06.6 Encodes Tyramine β-hydroxylase
The C. elegans genome sequence contains a single gene, H13N06.6, that encodes a protein with significant similarity to the Drosophila tyramine β-hydroxylase (TBH) (Monastirioti et al., J. Neurosci. 16:3900-3911 (1996)) and mammalian dopamine β-hydroxylase (DBH) (Lamouroux et al., EMBO J. 6:3931-3937 (1987)). We named this gene tbh-1 (tyramine β-hydroxylase) and obtained a 1.9 kb full-length complementary DNA (cDNA) clone for tbh-1 (
To determine whether TBH-1 is required for octopamine biosynthesis we measured the octopamine content of tbh-1 mutants using HPLC coupled to electrochemical detection. HPLC analysis of extracts of wild-type animals showed a peak with the same retention time as octopamine (
We also examined tyramine levels using thin-layer chromatography (TLC) of dansylated C. elegans extracts (
K01C8.3 Encodes Tyrosine Decarboxylase
We sought to identify a C. elegans L-aromatic amino acid decarboxylase (AADC) gene required for the decarboxylation of tyrosine, the first step in octopamine biosynthesis. AADCs are homodimeric pyridoxal 5′-phosphate (PLP) enzymes that can decarboxylate many naturally occurring L-aromatic amino acids. We identified five putative C. elegans AADC genes C05D2.3, C05D2.4, F12A10.3, K01C8.3 and ZK829 (
tdc-1 encodes two different splice variants, tdc-1a and tdc-1b, which differ at their 3′ ends (
We examined tyrosine decarboxylase activity in worm extracts by measuring the conversion of [3H]tyrosine to [3 H]tyramine. Tyrosine decarboxylase activity was present in wild-type extracts, but was almost undetectable in extracts from tdc-1 mutants (
TBH-1 is Expressed in a Subset of Cells that Express TDC-1
To analyze the expression patterns of tbh-1 and tdc-1, we generated polyclonal rabbit antibodies against the TBH-1 and TDC-1 proteins. TBH-1 antibodies recognized a single band of approximately 70 kDa in wild-type protein extracts, in accordance with the TBH-1 predicted size of 67 kDa. The 70 kDa band was absent in extracts from tbh-1 mutants (
C. elegans whole-mount staining with TBH-1 antibodies labeled a single pair of head interneurons in the lateral ganglion: we identified these neurons as the RICs (
TDC-1 was coexpressed with TBH-1 in the RICs and gonadal sheath cells (
Surprisingly, we found that TDC-1 was highly expressed in a few cells that did not express TBH-1. We observed bright staining of a pair of neurons in the lateral ganglion. We identified these cells as the RIM motor neurons (
tbc-1 Mutants are Hyperactive in Egg Laying
tbh-1 and tbc-1 deletion mutants were viable and healthy and had normal brood sizes (data not shown). tbh-1 and tdc-1 mutants both had a slightly reduced locomotion rate and defects in the inhibition of pharyngeal pumping and egg laying in the absence of food. Since tdc-1 and tbh-1 mutants both lack octopamine, the behavioral defects tbh-1 and tdc-1 mutants have in common suggests a role for octopamine in the modulation of locomotion, pharyngeal pumping and egg laying. tbc-1 mutants also had defects not shared with tbh-1 mutants: tdc-1 mutants were hyperactive in egg laying in the presence of food, failed to suppress head oscillations in response to touch and had defects in the coordination of forward and backward locomotion (see below). tbc-1 mutants had a reduced number of eggs in the uterus compared to wild-type animals and tbh-1 mutants (
tbc-1 mutants, unlike tbh-1 mutants, are hyperactive in egg laying behavior in the presence of food suggesting that tyramine plays a role independent of octopamine in the inhibition of egg laying in vivo. We therefore tested the effect of exogenous tyramine on egg laying behavior. Egg laying was inhibited on Petri plates containing 20 mM tyramine (
Anterior Touch Sensory Neurons Mediate the Suppression of Head Oscillations
We found that tbc-1 mutants failed to suppress head oscillations in response to anterior touch (see below). C. elegans locomotion is accompanied by oscillatory head movements during which the tip of the nose moves rapidly from side to side (
Light touch of an eyelash to the anterior half of the body induced a backing response. We found that during this backing response, head oscillations were suppressed in wild-type animals (
We noticed that head oscillations were usually not suppressed during spontaneous reversals. We asked whether other stimuli that, like gentle touch, induced backward locomotion, lead to the suppression of head oscillations. Nose touch, volatile repellents and high osmolarity induce an avoidance response and are mainly sensed by the ASH sensory neurons (Kaplan et al., Proc. Natl. Acad. Sci. U.S.A. 90:2227-2231 (1993)). Backward locomotion induced by these stimuli usually did not suppress head oscillations (
tbc-1 is Required to Suppress Head Oscillations in Response to Anterior Touch
Head oscillations were normal in tdc-1 mutants during forward locomotion and tbc-1 mutants normally induced backing in response to light touch. tbc-1 mutants, however, failed to suppress head oscillations during backward locomotion (
C. elegans head muscles are innervated by five classes of motor neurons (White et al., Philos. Trans. R. Soc. Lond. B. Biol. Sci. 314:1-340 (1986)): the IL1 sensory-motor neurons, the cholinergic RMD and SMD motor neurons, the GABAergic RME motor neurons, and the tyraminergic RIM motor neurons. Acetylcholine acts as an excitatory neurotransmitter at the C. elegans neuromuscularjunction. cha-1(p1152) mutants have a strongly reduced choline acetyl transferase activity and display uncoordinated locomotion (Rand et al., Genetics 106:227-248 (1984)) and uncoordinated head movement with few head oscillations. Ablation of RMD neurons results in the in the loss of head oscillations. GABA is the main inhibitory neuromuscular transmitter in C. elegans, and GABA-deficient unc-25 mutants and RME-ablated animals display loopy head oscillations. Ablation of the IL1 and OLQ sensory neurons also results loopy head oscillations suggesting an inhibitory role for the IL1 and OLQ neurons in head muscle contraction. To determine whether the suppression of head oscillations is GABA-dependent, we examined the anterior touch response in unc-25 mutants. unc-25 mutant animals had a reduced backing response but nonetheless suppressed head oscillations in response to anterior touch (
tbc-1 Mutants Have Defects in Reversal Behavior
We noticed that in response to anterior touch tbc-1 mutants backed less than wild-type animals and often displayed slightly jerky backward locomotion. Wild-type animals and tbh-1 mutants reversed on average 3.4 to 3.6 body bends, respectively in response to anterior touch (
C. elegans reversal frequency can be modulated by chemosensory cues (Pierce-Shimomura et al., J. Neurosci. 19:9557-9569 (1999), humidity (Zhao et al., J. Neurosci. 23:5319-5328 (2003)), temperature, and food (Tsalik et al., J. Neurobiol. 56:178-197 (2003)), which allows the animal to explore its environment in search of favorable conditions. On plates without food, wild-type animals made approximately 15 spontaneous reversals in 5 minutes (
The RIM Motor Neurons Modulate Reversal Frequency and are Required for the Suppression of Head Oscillations
The defects of tdc-1 mutants in egg laying, suppression of head oscillations and reversal behavior were not shared by the tbh-1 mutants suggesting a distinct role for tyramine in these behaviors. However, since tdc-1 mutants also lack octopamine we could not exclude a role for octopamine in these behaviors. We therefore analyzed the behavior of animals in which either the tyraminergic, or octopaminergic, neurons were killed by laser ablation. We found that animals in which the tyraminergic RIMs were ablated had an impaired backing response to anterior touch and failed to suppress head oscillations (
The AVA and AVD Backward-locomotion Command Neurons are Required for the Suppression of Head Oscillations in Response to Anterior Touch
The ALM/AVM and PLM mechanosensory neurons provide inputs to four pairs of locomotion command interneurons: the PVCs and AVBs, which are mainly required for forward locomotion, and the AVAs and AVDs, which generally drive backward locomotion (Chalfie et al., J. Neurosci 5, 956-964 (1985)). Laser ablation studies indicate that the locomotion command neurons cannot be strictly categorized in forward and backward (Hart et al., Nature 378:82-85 (1995); and Zheng et al., Neuron 24:347-361 (1999)), but rather form a bistable circuit that controls the direction of the animal's movement. Since the tyraminergic RIM motor neurons make gap junctions with the AVA and AVE backward command neurons, we tested the role of these neurons in the suppression of head oscillations in response to anterior touch (
Cell-ablation studies using laser microsurgery support a model in which the ALM and AVM touch neurons activate the AVD backward command interneuron via gap junctions and inhibit forward locomotion command neurons through synaptic connections with the PVC and AVB forward command neurons. To test the role of the AVD neurons in coupling the touch response to the suppression of head oscillations we ablated the AVD neurons. Five out seven AVD-ablated animals failed to suppress head oscillations, in response to anterior touch, consistent with the hypothesis that the AVD neurons transduce the touch response to the RIM neurons. We found that ablation of the AVD, AVA or AVE neurons did not affect the spontaneous reversal frequency (
Tyraminergic Cells are Distinct from Octopaminergic Cells
We identified two genes required for octopamine biosynthesis in C. elegans. tbc-1 a tyrosine decarboxylase gene required for the conversion of tyrosine into tyramine and tbh-1, a tyramine β-hydroxylase gene, is required for the conversion of tyramine to octopamine. Our characterization of tbc-1 provides the first description of an animal tyrosine decarboxylase gene. TBH-1 and TDC-1 are coexpressed in the RIC interneurons and gonadal sheath cells, indicating that these cells are octopaminergic. In contrast, TDC-1, but not TBH-1, is expressed in the RIM motor neurons and in the UV1 cells, indicating that these cells are tyraminergic. Thus, C elegans appears to have tyraminergic cells that are distinct from octopaminergic cells. Vertebrates use a similar strategy to generate dopaminergic and noradrenergic neurons: noradrenergic cells express DOPA decarboxylase and dopamine β-hydroxylase, and the dopaminergic neurons express DOPA decarboxylase but not dopamine β-hydroxylase (Muller et al., Neuroscience 11:733-740 (1984); Mercer et al., Neuron 7:703-716 (1991); and Chatelin et al., Brain Res. Mol. Brain Res. 97:149-160 (2001)). Until the mid-1950s dopamine was considered to be simply an intermediate in the biosynthesis of norepinephrine and epinephrine.
Tyramine was also initially thought to be simply a precursor octopamine. However, the identification of G-protein coupled receptors in Drosophila (Saudou et al., EMBO J. 9:3611-3617 (1990)), the locust (Vanden Broeck et al., J. Neurochem. 64:2387-2395 (1995)), the honey bee (Blenau et al., J. Neurochem. 74:900-908 (2000)), the silk moth (Ohta et al., Insect Mol. Biol. 12:217-223 (2003)). and C. elegans (Rex et al., J. Neurochem. 82:1352-1359 (2002)) that respond to tyramine suggested that tyramine may itself act as a neurotransmitter.
Tyramine Inhibits Egg Laying, Modulates Reversal Behavior and is Required for the Suppression of Head Oscillations in Response to Anterior Touch.
tbc-1 mutants have behavioral defects that are not shared by tbh-1 mutants, suggesting a specific role for tyramine in these behaviors. tdc-1 mutants are hyperactive in egg laying and exogenous tyramine inhibits egg laying, indicating that tyramine inhibits egg laying in vivo. The tyraminergic UV1 cells form adherens junctions with the utse and the vulF vulval cells and connect the uterus with the vulva. The close proximity of the UV1 cells to the vulval muscles suggests that they may be important for the tyramine-mediated inhibition of egg laying. The UV1 cells contain neurosecretory vesicles and express neuropeptides (Schinkmann et al., J. Comp. Neurol. 316, 251-260 (1992)) and several neurosecretory proteins, including SNT-1 synaptotagmin, (Nonet et al., Mol. Biol. Cell 10:2343-2360 (1999)), UNC-64 syntaxin (Saifee et al., Mol. Biol. Cell 9:1235-1252 (1998)), UNC-11 AP180 (Nonet et al., Mol. Biol. Cell 10:2343-2360 (1999)) and IDA-1 phogrin-IA-2 (Zahn et al., J. Comp. Neurol. 429:127-143 (2001), suggesting a paracrine role for the UV1 cells.
Light anterior touch, sensed by the mechanosensory ALM and AVM neurons, induces a backing response. We showed that during this backing response head oscillations are suppressed. tdc-1 mutants and RIM-ablated animals failed to suppress head oscillation in response to anterior touch. Our data suggested that the tyraminergic RIM motomeurons, which innervate the head muscles and make synaptic connections with the cholinergic RMD and SMD head motomeurons, are required for the inhibition of head muscle contractions.
The touch response is linked to the suppression of head oscillations. The RIM motomeurons also make synaptic contacts with the AVB forward locomotion command neurons and make gap junctions with the AVA backward locomotion command neurons (White et al., Philos. Trans. R. Soc. Lond. B. Biol. Sci. 314: 1-340 (1986)). Our results are consistent with the model of Chalfie et al., J. Neurosci. 5:956-964 (1985) in which tactile stimulation of the ALM/AVM anterior touch sensory neurons leads to the activation of the AVD command neurons, which in turn activate the AVA command neurons (
tbc-1 mutants and RIM-ablated animals also have defects in reversal behavior. Tyramine release from the RIMs may link the activation of the AVA neurons with the inhibition of the AVB neurons. Failure to properly inhibit the AVB forward command neurons during backing may lead to premature reinitiation of forward locomotion, as observed in tbc-1 mutants and RIM ablated animals. The connectivity of the RIM motor neurons with the locomotion command neurons may contribute to the coordination of the locomotion command neurons. RIM ablations or tyramine deficiency may change the steady state of the bistable circuit formed by the locomotion command neurons and lead to an increased reversal frequency.
C. elegans suppresses head oscillations in response to anterior touch but not in response to nose touch or posterior touch. The touch response of C. elegans could allow the animal to escape from nematophagous fungi, which use trapping devices along their hyphae to catch live nematodes. Fungi such as Arthrobotrys dactyloides and Dactylaria brochopaga use constricting rings to entrap nematodes. When a nematode moves into the ring, the contact triggers the swelling of the ring cells and can lead to the capture of the nematode. There is a lag time between the initial contact and the closure of the ring, allowing some nematodes to withdraw from the ring before being caught. The suppression of head oscillations in response to anterior touch may allow the nematode to smoothly retreat without the surrounding fungal ring and thereby and increase the chances of the nematode to escape from this death trap.
The experiments described above were carried out as follows.
Strains and Germline Transformation
All strains were cultured at 20° C. on NGM agar plates with the E. coli strain OP50 as a food source (Brenner, S., Genetics 77:71-94 (1974)). tbh-1 and tdc-1 deletion alleles were obtained by screening a chemical deletion library (Jansen et al., Nat. Genet. 17:119-121 (1997)). All deletions strains were outcrossed at least six times. Full-length tbh-1 cDNA sequence was obtained from expressed sequence tag (EST) clone yk722g9. Partial tdc-1 cDNA sequences were obtained from EST clones yk374c1 (tdc-1a) and yk303a5 (tdc-1b). The 5′ end sequence of the tbc-1 cDNA was determined by 5′ RACE. A tbh-1::gfp transcriptional fusion construct was made by cloning a 4.5 kb tbh-1 promoter fragment corresponding to nucleotide (nt)−4537 to +17 relative to the translational start site into the vector pPD95.67. A tdc-1::gfp reporter construct was obtained by cloning a PstI fragment corresponding to nt−4423 to +443 into the vector pPD95.69. GFP constructs were injected at 80 ng/μL into lin-15(n765ts) animals along with the lin-15 rescuing plasmid pL15EK at 50 ng/μL. A tdc-1 genomic NsiI fragment, corresponding to nt−917 to +2522, and a fragment corresponding to nt−4423 to +3042 relative to the translation start site, were subcloned in pBSK (Stratagene), resulting in pGTDC 1 and pGTDC2, respectively. pGTDC2-stop was derived from pGTDC2 by filling in and religating an AvrII site at nt 10541 of cosmid K01C8. pGTDC 1 (nEx1105) was injected at 50 ng/μL and pGTDC2 (nExll80) and pGTDC2-stop (nEx1181) were injected at 2.5 ng/μL into tdc-1(n3420); lin-15(n765ts) animals along with the lin-15 rescuing plasmid. The tbh-1::gfp extrachromosomal transgene was integrated by irradiating transgenic animals with gamma rays.
HPLC Analysis, Thin Layer Chromatography and Decarboxylase Activity Assays
Quantification of octopamine was performed using HPLC coupled with electrochemical detection. 30 μL of packed worms were homogenized with a pestle in 100 μL 0.3 M perchloric acid and centrifuged at 13,000 rpm at 4° C. to remove the insoluble residue. The supernatant was filtered through a 0.22 μm centrifugal filter and diluted five-fold with DEMO mobile phase (ESA, Bedford, Mass., USA), which contains 90 mM sodium phosphate, 50 mM citric acid, 1.7 mM 1-octanesulfonic acid, 10% acetonitrile adjusted to pH 3.0 with phosphoric acid. Samples were injected into an ESA MD 1 50/RP-C18 column at a flow rate of 0.5 mL/minute. Eluted compounds were detected electrochemically using an ESA model 5011 detection system. Detector potentials were set at −175 mV (channel 1), 175 mV (channel 2), 350 mV (channel 3), and 650 mV (channel 4). Under these conditions octopamine was oxidized at 650 mV. Octopamine levels were quantified using 2 pmol external octopamine standards (Sigma), and samples were spiked with octopamine to confirm the identity of the oxidizable substances.
Thin layer chromatography was performed as described (Eaton et al., Anal. Biochem. 172:484 (1988)) with slight modifications. 150 μL of packed worms were homogenized in 300 μL 0.1 M perchloric acid by sonication. Homogenate was centrifuged at 10,000 g for 30 minutes and the supernatant was used for dansylation. 750 μL of 1.2 M bicarbonate buffer pH 9.0 and 2.1 mL dansyl chloride in acetone (0.53 mg/mL) were added to the supernatant, mixed thoroughly and incubated for 20 minutes at 40° C. 300 μL 0.1 M proline was added, mixed thoroughly and incubated for an additional 20 minutes at 40° C. to remove excess dansyl chloride. A stream of dry nitrogen was directed over the reaction mixture to remove excess acetone. Dansylated amines were extracted with three 750 μL volumes of toluene and dried under a stream of dry nitrogen. Dansylated amines were resuspended in 10 μL of toluene, spotted on 10×10 cm Silica gel 60 plates (Sigma-Aldrich) and separated by chloroform: butyl acetate: ethyl acetate (3:3:1) in the first dimension and chloroform: butyl acetate: ethyl acetate: triethylamine (6:1:2:0.5) in the second dimension. Chromatographs were photographed under UV light. 50 pmol of dansylated tyramine, octopamine and dopamine were used as standards.
Immunohistochemistry and Microscopy
TBH-1 antibodies were raised in rabbits against a GST-TBH-1 (a.a. 244-586) fusion protein. TDC-1 antibodies were raised in rabbits against a GST-TDC-1A (a.a. 534-650) fusion protein. TBH-1 and TDC-1 antibodies were purified and used for western blot analysis and immunohistochemistry using standard methods. Identifications of TDC-1 and TBH-1 expressing cells were based on cell body positions and axon morphologies of strains that expressed tbc-1 and tbh-1 g-reporter genes and by immuno-staining. Cell ablations using laser microsurgery ablations were performed durig the second larval stage (L2) as previously described (Avery et al., Neuron 3:473-485 (1989)). GFP reporters were used to facilitate the cell identification and to confirm cell ablation at the L4 stage. The integrated tbh-1::gfp transgenic line, nIs107, was used for RIC ablations. An integrated nmr-1::gfp transgenic line, akIs3, (Brockie et al., Neuron 31, 617-630 (2001)) was used for RIM, AVA, AVD and AVE ablations.
Behavioral Assays
Behavioral assays were performed with young adults at room temperature (22-24° C.) and the different genotypes were scored in parallel on independent days. Egg-laying assays were performed as described by Koelle and Horvitz (Cell 84:115 (1996)). Suppression of head oscillations was tested by striking animals that were outside the bacterial lawn with a fine eyelash behind the posterior bulb of the pharynx; head oscillations were scored during backing response. Nose touch, osmotic avoidance touch were tested as previously described (see, for example, Way et al., Genes Dev. 3:1823-1833 (1989); or Bargmann et al., Cold Spring Harb. Symp. Quant. Biol. 55:529-538 (1990)). Response to harsh touch can only be assayed in animals that do not have functional light-touch sensory neurons and were scored in mec-7(e1527) mutants. Animals were tested twenty-four hours after they were picked as late L4. Laser-ablated animals were tested at least ten times. Animals that did not display any head oscillations during backward locomotion were scored as positive. Laser ablated animals were tested at least 20 times. Reversal assays were performed as described by Tsalik et al., J. Neurobiol. 56:178-197 (2003).
Synthesis of 4-hydrazinomethyl-benzene-1,3-diol (compound 1).
Hydrazine (115 μL, 3.62 mmol) and Pd/C (500 mg) were added to a dry 100 mL round bottom flask and suspended in 5 mL dry methanol. The flask was placed under hydrogen atmosphere using a balloon filled with hydrogen. 2,4-dihydroxybezaldehyde (Aldrich Cat. No. 168637) (500 mg, 3.62 mmol) was dissolved in dry methanol (2 mL) and added dropwise via syringe to the stirring hydrazine suspension. The reaction was allowed to proceed for 16 hours at room temperature. The reaction mixture was filtered through celite and concentrated to yield the crude product (586 mg). Reverse phase HPLC analysis (0-50% acetonitrile in 0.1% aqueous TFA, C18 column) revealed approximately 70% of the desired product, 4-hydrazinomethyl-benzene-1,3-diol, and 30% of the dimer (see Scheme 1) ([M+H]+: 155.07 exp.; 155.12 obsv.). The crude product was used without further purification.
Synthesis of N-(DL-seryl)-N′-(2,4,-dihydroxybenzyl) hydrazine (compound 2).
Commercially available N-Boc-Serine-(O-tBu)-OH (Novabiochem) was condensed with 4-hydrazinomethyl-benzene-1,3-diol under standard peptide conditions (see Scheme 2). The hydrazinomethylbenzene (226 mg, 1.5 mmol) and the protected serine (800 mg, 1.8 mmol) were dissolved in methylene chloride (2 mL). To this mixture were added standard peptide coupling reagenets PyBop, HOBt and triethylamine (1.8 mmol each). The reaction was stirred for 3 hours at room temperature, concentrated, redissolved in methanol, and purified by preparative HPLC (10-60% acetonitrile in 0.1% aqueous TFA, C18 column). ([M+H]+: 398.22 exp.; 398.30 obsv.).
The purified, protected product was dissolved in a 1:1 mixture of CH2Cl2:trifluoroacetic acid (1 mL) and allowed to stir at room temperature for 1 hour. The reaction was then concentrated, dissolved in water and purified by preparative HPLC.
Synthesis of (2S)-2-(3-hydroxybenzyl)-2-hydrazinopropanoic acid (compound 3).
Compound 3 can be synthesized, for example, from α-methyl tyrosine (Sigma Cat. No. M8131), hydrazine O-sulfonic acid, and hydroxylamine O-sulfonic acid using the methods described in Example 23 of U.S. Pat. No. 3,462,536.
Tyrosine Decarboxylase Inhibition Assay.
The screening of putative tyrosine decarboxylase inhibitors can be performed in vitro. For example, recombinant TDC-1, or worm protein extracts, can be combined with the test compound and the conversion rate of [3H]tyrosine to [3H]tyramine measured. A reduction of the conversion rate in the presence of the test compound is indicative of tyrosine decarboxylase inhibition.
Alternatively, putative tyrosine decarboxylase inhibitors can be screened in vivo by observing its effect on invertebrates. Test compounds which possess tyrosine decarboxylase inhibition activity are expected to alter invertebrate behavior, e.g., nematodes may fail to suppress head oscillations in response to anterior touch or may increase reversal frequency. Furthermore, a tyrosine decarboxylase inhibitor is expected to cause egg laying defects in nematodes, fruit flies, and other invertebrates.
TDC activity was assayed as described by McClung and Hirsh (Curr. Biol. 9:853 (1999)) with some modifications. 80 μL of packed worm were frozen in liquid nitrogen. Worms were thawed on ice after adding an equal volume of 50 mM Tris pH 7.5, 1 mM phenylthiourea, homogenized by sonication (10 seconds, 50% duty cycle) in a 1.5 ml tube, and centrifuged for 5 minutes at 13000 rpm. 8 μL of the supernatant was added to 32 μL of reaction buffer (0.1 M NaPO4 pH 6.8, 0.1 mM pyridoxal phosphate, 0.1 mM EDTA, 1 mM β-mercaptoethanol, 40 μCi/ml [3H]tyrosine) and incubated at 25° C. for 0, 30, 60, and 90 minutes. Heat-inactivated protein extract served as a negative control. Tyramine was extracted by adding 100 μL chloroform containing 0.1 M diethyl-hexylphosphoric acid and 300 μL 0.05 M NaPO4 pH 6.8. Samples were vortexed then centrifuged to separate the water phase, containing tyrosine, and the organic phase, containing tyramine. The organic phase was transferred to a new tube and washed once with 300 μL 0.05 M NaPO4 pH 6.8. The organic phase was transferred to a scintillation vial and counted in scintilation fluid.
When compound 2 was added to C. elegans grown on agar plates at a concentration of 0.3 mg/ml, the nematodes fail to suppress head oscillations in response to anterior touch. This change in behavior is consistent with strong inhibition of tyrosine decarboxylase by compound 2 (e.g., consistent with the behavior of C. elegans mutants which lack tyrosine decarboxylase).
The in vivo response test can also be used to determine the dose-response curve for a particular compound and particular invertebrate. From the dose-response curve, a minimum efficacious dose for inhibiting the proliferation of the invertebrate can be calculated.
Antibodies
TBH-1 antibodies were purified against a his-tagged TBH-1 (a.a. 183-586) fusion protein. TDC-1 antibodies were purified against a GST-TDC-1A (a.a. 534-650) fusion protein. TBH-1 and TDC-1 antibodies were used for western blot analyses and immunohistochemistry using standard methods. 1:2000 dilutions of TBH-1 and TDC-1 antibodies were used for western blot analysis. For whole-mount staining, animals were fixed in Bouin's fixative as described (Nonet et al., 1997). Fixed animals were incubated overnight with TBH-1, TDC-1 or GFP antibodies at 1:50 dilution, and incubated for 2 hrs with secondary FITC or Cy3 conjugated antibodies at 1:50 dilution from Jackson ImmunoResearch. GFP monoclonal antibodies were obtained from Chemicon International. Animals were mounted and analyzed with a Zeiss Axioplan microscope equipped with Nomarski optics and a fluorescent light source.
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
All publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication was specifically and individually indicated to be incorporated by reference.
This application claims the benefit of U.S. Provisional Application No. 60/673,829, filed Apr. 22, 2005, which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60673829 | Apr 2005 | US |
