Field of the Invention
The present invention relates generally to the field of anti-nematode compounds and methods of using the same.
State of the Art
Nematodes are elongated symmetrical roundworms that constitute one of the largest and most successful phyla in the animal kingdom. Many nematode species are free-living and feed on bacteria, whereas others have evolved into parasites of plants and animals, including humans. Human infections with parasitic nematodes are among the most prevalent infections worldwide. Over one billion people, predominantly in tropical and subtropical developing countries, are infected with soil and vector-borne nematodes that cause a variety of debilitating diseases. Liu et al., “Intestinal Nematodes” in 181 HARRISON'S PRINCIPLES OF INTERNAL MEDICINE 916-20 (McGraw-Hill, 1994).
Among these parasitic nematodes are Ancylostoma and Necator hookworms, which cause anemia and malnutrition, Ascaris roundworms, that can cause pulmonary and nutritional disorders, and Strongyloides stercoralis, that can effect a potentially life-threatening intestinal infection. Nematodes of the order Spirurida are responsible for onchocerciasis (river blindness) and lymphatic filariasis. Animal parasitic nematodes infect a wide variety of both domestic and wild animals. Major animal pathogens include Haemonchus contortus, which infects herbivorous vertebrates, Trichinella spiralis, the causative agent of trichinosis, and various members of the order Ascaridida, which infect pigs and dogs in addition to humans.
Plant parasitic nematodes also represent major agricultural problems and are responsible for many billions of dollars in economic losses annually. The most economically damaging plant parasitic nematode genera belong to the family Heterderidae of the order Tylenchida, and include the cyst nematodes (genera Heterodera and Globodera) and the root-knot nematodes (genus Meloidogyne). The soybean cyst nematode (H. glycines) and potato cyst nematodes (G. pallida and G. rostochiensis) are important examples. Root-knot nematodes infect thousands of different plant species including vegetables, fruits, and row crops. In contrast to many viral and bacterial pathogens, little is known about the molecular basis of nematode parasitism, limiting the available framework for rational anti-helminthic (anti-nematode) drug development. See David and Liu, “Molecular biology and immunology of parasitic infections,” in HARRISON'S PRINCIPLES OF INTERNAL MEDICINE 865-71 (McGraw-Hill, 1994).
Anti-nematode drug or pesticide discovery traditionally has relied on direct screening of compounds against whole target organisms or on chemical modification of existing compounds. These strategies have yielded relatively few classes of agents, acting against a limited number of known biological targets. For example, organophosphates and carbamates, the oldest extant class of nematicides, were developed many decades ago and target a single, biologically conserved enzyme, acetylcholinesterase. Imidazole derivatives such as benzimidazole exert their antiparasitic effects by binding tubulin. Levamisole acts as an agonist on the nicotinic acetylcholine receptor, and avermectins act as irreversible agonists at glutamate-gated chloride channels (Liu et al., 1996).
Unfortunately, there are certain debilitating nematode infections that are difficult if not impossible to cure with existing therapeutics. In onchocerciasis, for instance, the adult female Onchocerca volvulus worms are refractory to even newer generation drugs (Liu et al., 1996). In addition, drug resistance has emerged to all of these main classes of therapeutics, particularly in livestock animal applications in which their use is widespread (Sangster et al., 1999). To date it has not been possible to develop effective and practical vaccines. Even were such vaccines available, effective anti-nematode drugs still would be needed, for treating established infections and for offering the potential advantages of prophylaxis and treatment against a broad spectrum of nematode parasites.
The drawbacks of existing agents that are currently used to control plant parasitic nematodes are equally or more significant. Fumigant nematicides such as methyl bromide and 1,3-dichloropropene, which kill nematodes by slowly diffusing through the soil, are phytotoxic and must be applied well before planting. Environmental concerns, primarily groundwater contamination, ozone depletion, and pesticide residues in food (National Research Council, Pesticides in the Diet of Infants and Children (Washington, D.C.: National Academy of Sciences, 1993) have prompted the removal of Aldicarb, DGBCP, and other toxic nematicides from the market by the Environmental Protection Agency, with methyl bromide to be withdrawn in the U.S. by 2002. Johnson & Bailey, “Pesticide Risk Management and the United States Food Quality Protection Act of 1996,” in PESTICIDE CHEMISTRY AND BIOSCIENCE: THE FOOD-ENVIRONMENT CHALLENGE 411-20 (Royal Society of Chemistry, Cambridge, 1999). Physical control measures, such as solarization and hot water treatment, crop rotation and other biological control measures, and integrated approaches have been used to ameliorate the damage caused by plant parasitic nematodes. See, e.g., Whitehead, Plant Nematode Control, Wallingford: CAB International (1998). No single method or combination of measures is uniformly effective, however.
Molecular genetic methods, such as gene knockouts, can uncover the biological function of individual genes and proteins in an organism, information that can form the foundation for developing target-based compound discovery screens. At present, however, these techniques are difficult to perform in parasitic nematodes.
In contrast, such procedures can be performed in a straightforward manner in C. elegans. Furthermore, the complicated life cycle of many parasitic nematodes and their need for a suitable plant or animal host makes it inconvenient to propagate them in the laboratory.
The genome of C. elegans is predicted to contain 284 nuclear receptors (Gissendanner et al., 2004; Sluder and Maina, 2001). Forward and reverse genetic studies have uncovered roles for C. elegans receptors in diverse physiological processes, such as dauer formation, reproduction, and life span (DAF-12), larval molting (NHR-23), sex determination (SEX-1), xenobiotic metabolism (NHR-8), neuronal development (UNC-55, ODR-7, FAX-1) and lipid metabolism (NHR-49). Nevertheless, all nuclear receptors in worms remain “orphans,” since ligands regulating their function have not been identified (Lindblom et al., 2001; Sluder and Maina, 2001; Van Gilst et al., 2005a; Van Gilst et al., 2005b).
In contrast to other C. elegans nuclear receptors, a considerable amount of genetic evidence supports the existence of a steroid-like ligand for the orphan receptor, DAF-12. DAF-12 belongs to a group of over 30 genes, collectively called daf (dauer formation) genes, which transduce environmental signals that influence the choice between alternative developmental programs of dauer diapause or reproductive development (Antebi et al., 2000; Riddle and Albert, 1997).
Dauer diapause is a process in which animals at the second larval stage (L2) delay further reproductive development under conditions of diminishing food or overcrowding and instead form the non-feeding, non-reproductive, and long-lived dauer larva (Riddle and Albert, 1997). Upon entry into a more favorable environment, dauer larvae resume feeding and reproductive growth.
Mutations in Daf genes generally produce a dauer constitutive phenotype (Daf-c) or a dauer defective phenotype (Daf-d). Daf-c mutants always arrest as dauers, while Daf-d mutants bypass dauer, regardless of environmental signals. Loss of daf-12 results in Daf-d as well as L3 stage heterochronic phenotypes, indicating that daf-12 is required for dauer formation and for proper developmental timing in the reproductive state (Antebi et al., 1998; Antebi et al., 2000).
Detailed analysis of the dauer formation genes has revealed that favorable environments activate insulin/IGF-1 and TGFβ signaling pathways within the organism that converge on DAF-12 to inhibit its dauer promoting function and activate its reproductive function (Kimura et al., 1997; Ren et al., 1996; Schackwitz et al., 1996). Acting cell non-autonomously, these pathways are believed to activate, either directly or indirectly, the production of a DAF-12 ligand by the cytochrome P450, DAF-9 (Gerisch et al., 2001; Jia et al., 2002). Evidence for this model stems from the findings that insulin-like receptor (daf-2), TGFβ (daf-7), and cytochrome P450 (daf-9) signaling mutants are Daf-c. Furthermore, epistasis experiments have revealed that they act upstream of daf-12, since Daf-d alleles of daf-12 suppress the Daf-c phenotypes exhibited by these signaling mutants (Larsen et al., 1995). In addition to the Daf-d alleles, Daf-c mutants of daf-12 have been isolated that map to a single residue (R564) in the putative ligand binding domain of DAF-12 and are predicted to perturb ligand binding (Antebi et al., 2000). Phenotypically, these mutants arrest as partial dauers but recover and resemble weak daf-9 alleles that exhibit gonadal migration (Mig) defects (Gerisch et al., 2001; Jia et al., 2002). Thus, the predicted loss of hormone production in daf-9 null worms or loss of hormone binding by daf-12 Daf-c worms results in a failure to inhibit dauer-promoting functions and activate L3 stage reproductive functions of DAF-12.
Several lines of evidence suggest that DAF-12 ligands may be derived from cholesterol. First, C. elegans lacks the ability to synthesize cholesterol, which is required exogenously for normal growth and fertility (Chitwood, 1999). Second, cholesterol deprivation produces Mig and Daf-c phenotypes in wild-type worms and enhances the Mig and Daf-c phenotypes of weak daf-9 and daf-12 alleles (Gerisch et al., 2001; Jia et al., 2002; Matyash et al., 2004). Finally, worms lacking both homologs (ncr-1, ncr-2) of the human Niemann-Pick type C1 gene, a membrane glycoprotein implicated in lysosomal transport of cholesterol, arrest constitutively as dauers (Li et al., 2004).
These data indicate that a sterol-derived hormone promotes reproductive development in C. elegans. Evidence that lipid extracts from wild-type worms can rescue daf-9 phenotypes has strengthened the hormone hypothesis (Gill et al., 2004). Nevertheless, the identities of DAF-9-derived hormonal ligands that activate DAF-12 have remained elusive.
Accordingly, ligands must be identified that modulate the DAF-9/DAF-12 pathway, thereby to identify new agents that are active against pathogenic and parasitic nematode species, e.g., compounds active against animal or plant parasitic nematodes. A need also exists for new methodologies and screening technologies that allow for the identification of compounds active against nematodes. In particular, screening assays are needed that can be performed conveniently, in a high throughput format.