WO2000073493A2 - A nematode drug screen for modulators of mammalian disorders - Google Patents

Abstract

The invention provides rapid, versatile, and economic assay methods to screen for modulators of egg laying behavior in nematodes, by determining the activity of chitinase in the culture medium, thereby screening for candidate compounds that modulate a trait characteristic of mammalian disorders or diseases such as Alzheimer's disease, Ras-inducible cancer, and cardiopulmonary disease characterized by aberrant potassium channel function, among others. Also provided are modulators identified using the methods of the invention, pharmaceutical compositions including the modulators, and methods of using the modulators or pharmaceutical compositions to modulate a mammalian disorder or disease.

Description

A Nematode Drug Screen For Modulators Of Mammalian Disorders

FIELD OF THE INVENTION The invention relates to efficient screening methods for the identification of compounds capable of inhibiting egg laying in C. elegans and to methods for the identification of compounds capable of inhibiting APP processing in mammalian cells. The invention further relates to the identified compounds and methods for using those compounds.

BACKGROUND OF THE INVENTION The provision of modern health care at a reasonable cost presents a worldwide challenge that has not been fully met. Significant financial and emotional costs are associated with the health-related problems of humans and animals. As but one example, Alzheimer's disease (AD) is a degenerative neurological disorder that results in a progressive loss of mental faculties, ultimately leading to dementia and death. Several years ago, it was estimated that this disease afflicts 2-3 million people in the U.S. alone (WO 97/48983); today, the estimated figure has swollen to 4 million people, with an estimated annual treatment cost of 90 billion U.S. dollars. The development of rapid methods for screening candidate compounds for efficacy as therapeutics presents a promising avenue for lowering those costs. To the extent that such screening methods are useful against a broad spectrum of diseases or disorders, the rate at which health care costs will drop can be accelerated. One system that appears to provide a basis for developing broad- spectrum screening methods for a variety of therapeutics is the egg laying behavior of nematodes.

The progressive dementia associated with AD is associated with the formation of amyloid plaques and neurofibrillary tangles, accompanied by gliosis and neuronal loss. Three genes have been discovered to date which, when mutated, result in an autosomal dominant form of the disease (Lendon et al.,

JAMA 277:825-831 (1997)). These genes encode the amyloid protein precursor (APP) and a pair of related proteins in the presenilin pathway, Presenilin-1 (PS1) and Presenilin-2 (PS2). Mutations in any of the three genes enhance proteolytic processmg of APP \ ιa an intracellular pathw ay that produces amyloid-β (Aβ) peptide, a 40-42 amino-acid-long peptide that is the primary component of amyloid plaque in AD (Selkoe, Trends Neurosci. 16:403-409 (1993); Citron et al., Nat. Med. 3:67-72 (1997)). The proteases responsible for cleaving APP, or a processed form of APP, at the N- and C-termini to generate the Aβ peptide have been termed β-secretase and γ-secretase, respectively. The β-secretase recently was found by comparing the nematode genome with human expressed sequence tags (ESTs), see Yan et al., Nature 402:533-37 (1999); the γ-secretase may be associated with presenilins (DeStrooper et al., Nature 391 :387-90 (1998). Formation of the longer, 42-amino-acid form of the peptide is associated with accelerated AD pathogenesis. Some mutations in APP, PS1 or PS2 increase the ratio of Aβ 1-42 relative to total Aβ peptide production by subtly altering the site of γ-secretase processing (Citron et al., 1997). The cleavage site for γ-secretase processing is unusual in that it is located within the APP transmembrane domain. Insight into presenilin function has come from genetic studies of presenilin genes in model organisms such as the nematode, Caenorhabditis elegans, and from engineered deletions of the PS1 gene in mice. Three presenilin genes have been identified in C. elegans, sel-12, hop-] and spe-4 (Levitan et al., Nature 377:351 :354 (1995); Li et al., Proc. Natl. Acad. Sci. (USA) 94:12204-09 (1997); Westlund et al., Proc. Natl. Acad. Sci. (USA) 96:2497-2502.(1999); L'Hemault et al., J Cell. Biol. 119:55-68.(1992)). Loss-of-function mutations in sel-12 cause a defect in egg laying which can be rescued by human PS1 or PS2, demonstrating that features of the presenilin protein required for function have been conserved between nematode and man (Levitan et al., (1995); Levitan et al., Proc. Natl. Acad. Sci. (USA) 93:14940-44 (1996)). Sel-12 has also been shown to be required for Notch pathway signaling mediated by the C. elegans Notch receptor homologs, Lin-12 and Glp-1 (Levitan et al., (1995)). Absence of Sel-12 function suppresses Lin- 12 gain-of- function phenotypes, such as a multi vulva phenotype, and enhances Lin-12 and Glp-1 partial loss-of-function phenotypes. A link between PS1 function and Notch pathway signaling also is suggested by the phenotype of PS1 -- - null mouse embryos These embryos show altered spatiotemporal expression of Notch 1 w ith accompanying defects in somite segmentation and differentiation (Wong et al , Nature 387 288-292 (1997)) More interestingly from the perspective of AD pathophysiology, neurons cultured from PS 1 null embiyos have defects amyloidogemc processing of APP at the γ-secretase site (DeStrooper et al , Nature 391 387-390 (1998)) Thus, the presemhns are potential targets for therapeutic intervention in AD

Another major health threat to humans is cardiopulmonary disease, which perennially ranks at the top m terms of number of deaths and cost The control of the circulatory system, particularly the functioning of the heart, is known to involve the coordinated action of a vaπety of ion channel pumps, including sodium and potassium channel pumps The potassium channels, for example, control pacemaker function, resting potential and action potential duration Goodman & Gilman's The Pharmacological Basis of Therapeutics, 854 (Ninth Ed., McGraw- Hill, New York) A need continues to exist for compounds that selectively modulate the activity of these potassium channels

A third major health problem for human populations is cancer. At present, a great vaπety of cancers have been identified and, notwithstanding our incomplete understanding, there appear to be a number of causative agents. One significant contπbutor to the development of cancer is genetic predisposition, as evidenced by the research conducted on cellular oncogenes One well-studied example is the ras oncogene, which encodes a protein that interferes with the control of normal cell growth and development. Accordingly, a need continues to exist for modulating the activity of Ras Each of the above-enumerated examples of mammalian diseases or disorders illustrates the ongoing need for therapeutics useful in providing medically effective and economically feasible treatments As a first step towards the realization of therapeutics, those therapeutic compounds must be identified Accordingly, a need exists for methods of screening for potential therapeutics in an effective manner, for example using high-throughput formats Typical high-throughput screening formats are either inanimate in nature, or use cells or whole organisms having well-characterized, simple, and rapid life cycles not dependent on exotic or costly nutrients or conditions for maintenance and growth. One model organism that has been extensively studied is the free- living nematode, Caenorhabditis elegans. A large number of genes affect the ability of C. elegans to lay eggs (Trent et al., Genetics 104:619-47 (1983)). These genes include a large group of egl (egg laying defect) genes, as well as other genes. Collectively, mutations in apparent mammalian counterparts to members of this group of genes have been associated with a variety of mammalian disorders and diseases, including Alzheimer's disease, cardiopulmonary disease, and Ras- induced cancer.

In particular, the genome of C. elegans contains three orthologs of the Alzheimer's disease presenilin genes. When mutated in C. elegans, loss of presenilin function causes a defect in egg laying. In human cells, loss of presenilin- 1 (PS 1 ) function blocks amyloid beta peptide processing from the amyloid protein precursor (APP), as noted above. It is also known that gain-of- function mutants in a Shaw-type K⁺ channel cause an Egl phenotype in nematodes (Johnstone et al., Neuron 19:151-164 (1997)), suggesting an association between the Egl^" phenotype and cardiopulmonary diseases or disorders in mammals. Remarkably, egg laying defects in nematodes also appear to be associated with cancer causing conditions in mammals. Loss-of-function mutations in the C. elegans ras gene prevent the formation of the vulva. These mutants are egl (Horvitz et al., Nature 351 :535-41 (1991)). Overexpression of this ras gene, or the creation in it of activating mutations, leads to a multivulva phenotype; these mutants are not egl (Horvitz et al. (1991)). Further, activation of Ras requires farnesylation of the protein by an enzyme called farnesyltransferase. In C. elegans, inhibitors of Ras farnesyltransferase block the multivulva phenotype caused by an activating mutation in the ras gene (Hara et al., Proc. Natl. Acad. Sci. (USA) 92:3333-37 (1994)). The ras gene is known to be an oncogene involved in many human tumors, and inhibitois of human Ras farnesyltianserase are attractive as candidate anticancer drugs

Therefore, methods of screening for therapeutics that aie based on the egg laying behavior of the free-living nematode C elegans appear to provide a broad- spectrum set of assays designed to identify compounds that are risk factors for the development of a mammalian disorder or disease, as well as to identify potential therapeutics effective in treating a vaπety of mammalian diseases/disorders, such as Alzheimer's disease, cardiopulmonary disease and cancer, among others One way to discover such πsk factors is to measure the ability of test compounds to inhibit the egg laying of wild-type animals, one way to discover potential therapeutics, or drugs, is to revert the phenotype of egg laying deficient mutants to the wild-type In either case, the currently used method for measuπng egg laying is visual examination, under a microscope, of a large number of worms to determine if eggs are being shed into the environment or are hatching mside the hermaphroditic parent (mtra-parent hatching indicates that eggs are not being shed) Understandably, these assays are laboπous and slow Although nematodes have a relatively short hfespan, these assays also demand the time required for normal egg laying behavior to occur Thus, a need remains for a rapid, if indirect, measure of egg laying behavior of nematodes Analyses of the nematode eggshell have revealed that approximately 5% of the shell is chit , which contπbutes to the structural mtegnty of the egg Arnold et al , Mol Bio Parasitol 58 317-24 (1993), working with the parasitic nematode Hehgmosomoides polygyrus, showed that chitm is degraded by the combined action of chitinase and N-acetlyglucosamimdase Arnold et al further reported that chitmase plays a role in egg hatching, being a component of the mteπor of the egg that is released into the environment upon hatching Further, Arnold et al reported that the chitinase of H polygyrus was stable and accumulated in culture in a manner proportional to the increase in egg hatching in that culture Arnold et al , neither disclosed nor suggested that chitmase activity in culture bore any relation to egg laying behavior One enzvme assay tor chit ase uses substiates that can be discriminated from products through the use of fluorescence spectroscopy, as disclosed by McCreath et al N Microbiol Methods 14 229-237 (1992) This assay can be conducted on culture supematants obtained from wells of a culture plate, e g . a 96- well culture plate, or can be conducted in wells at the end of the incubation As fluorescence spectrometers capable of reading 96-well plates are commercially available, the assay can be easily adapted for high-throughput screening of candidate therapeutics or drugs Measurements of chitinase activity in cultures of non-parasitic nematodes such as C elegans, which rely on bacteπal food sources, have not been disclosed Analysis of the genome of this organism (Wormpep database, version 19) has not revealed the presence of a gene that resembles the chit ase genes of other organisms

Accordingly, a need continues to exist in the art for rapid and efficient methods of screening candidate compounds that modulate egg laying behavior m nematodes and/or modulate the progress of one or more mammalian disorders or diseases, such as Alzheimer's disease (e g , modulation of APP processing), cardiopulmonary disease (e g , modulation of potassium channel function), and cancer (e g , modulation of Ras function), among others A need also continues to exist for the candidate therapeutics themselves, as well as methods of using those compounds to treat mammalian disorders and diseases

SUMMARY OF THE INVENTION

The invention satisfies one or more of the aforementioned needs in the art by providing rapid and versatile methods of screening for candidate compounds effective in treating any one or more of a variety of mammalian disorders and diseases, such as Alzheimer's disease, cardiopulmonary disease and cancer. These methods, which are readily adaptable to high throughput formats, enjoy the advantage of rapidly, albeit indirectly, measuring the egg laying behavior of nematodes such as Caenorhabditis elegans by determining the level of chitinase released into the culture medium. As such, the methods of the invention combine the positive correlation of nematode egg laying behavior and mammalian disorders or disease states with the rapid and sensitive assay of chitinase activity released into worm culture media. Thus, the invention benefits from the discovery of a positive correlation between released chitinase activity and nematode egg laying behavior, not simply egg hatching behavior. The invention also provides the candidate therapeutics themselves, as well as methods of using those compounds. Accordingly, one aspect of the invention is drawn to a method of identifying an egg laying deficiency in Caenorhabditis elegans comprising monitoring the chitinase activity in the medium of a C. elegans culture, the egg laying deficiency being identified as a decreased chitinase activity in the medium as compared to the chitinase activity in the medium of a C. elegans culture free of any egg laying defect.

Another aspect of the invention is directed to a method of screening for a compound that mitigates an egg laying deficiency in C. elegans, comprising: (a) providing a culture of C. elegans comprising a non- wild-type allele of a gene associated with an egg laying deficiency; and (b) monitoring the chitinase activity in the medium of the culture in the absence and presence of a candidate compound, wherein a candidate compound that increases chitinase activity is identified as a compound that mitigates the egg laying defect. Suitable genes for use in this aspect of the invention include, but are not limited to, a gene selected from the group consisting of a gene in the presenilin pathway (e.g., sel-12, hop-1, and spe- 4), egl- , egl-5, egl-26/cog-4, egl- 38, egl-43. egl-44, egl-46. senι-1. sem-2, sem-4, rab-3, unc-31. egl-22. unc-51, lin-17, egl-20. egl-27, bar- 1, egl- 15. egl-1 7. unc-53. ιιnc-40, egl-8, egl-10. egl-30. ιnx-1, in.x-2. daf-4, daf-8. da/- 14. daf-7. egl-4. egl-32. lin-12(gf), let-60, lin-3, let-23, sem-5, lin-45, liπ-39, lin-l, lin-31, lin-2. lin-7, lin- 10, lin-l 1, lin-l 5, lin-l 9, lin-25, lin-26, lin-36, unc-86, sup-/ 7, sup- 10, sup-9, unc- 93, egl-2, egl-19, egl-23, egl-36, egl-1, PS-1, PS-2, and orthologs (e.g., transgenes) thereof, including human orthologs. A preferred concentration range for a candidate compound present in a nematode culture is 0.03 to 30 micromolar; another preferred concentration range for a candidate compound is 0.03 to 10 micromolar.

A related aspect of the invention provides a method of screening for a compound that modulates a mammalian disorder, comprising: (a) identifying a compound that mitigates an egg laying defect in C. elegans according to the method described above, (b) contacting a mammalian cell or cells exhibiting a trait characteristic of a disorder selected from the group consisting of Alzheimer's disease, Ras-inducible cancer, and a potassium channel disorder with the compound; and (c) monitoring the cell or cells in the presence and absence of the compound, wherein a compound that reduces at least one trait of the disorder is identified as a compound that modulates the disorder. For example, a trait characteristic of a disorder is APP processing, or production of an amyloid β peptide. The mammalian cell or cells of the invention may be found in culture, or may be found in an intact mammal; an exemplary mammalian cell is a human cell.

In a related aspect of the invention, the invention is drawn to the above- described method further comprising a step of manufacturing a composition comprising a compound identified according to step (c) of the method described immediately above as a compound that modulates the disorder, and a pharmaceutically acceptable carrier. The invention contemplates methods for modulating mammalian disorders comprising administering to a mammal an effective amount of a composition manufactured according to the method described in the pieceding sentence An exemplai) mammal to w hich these compositions ma\ be admimsteied is a human

In anothei aspect, the invention is directed to the compound identified as a modulator of a mammalian disorder descπbed

e Such a compound may modulate presenilin activity and may be found in a pharmaceutical composition comprising the compound m a dosage effective in treating a mammalian disorder selected from the group consisting of Alzheimer's disease, Ras-inducible cancer and potassium transport disorders

Another aspect of the invention is drawn to a method of identifying an inhibitor of C elegans egg laying, compπsing (a) providing a culture of C elegans, (b) measuring the chitinase activity in the medium of the culture in the absence and presence of a candidate compound, and (c) identifying as an inhibitor, a candidate compound that produces a decrease in the chitmase activity in the medium when present as compared to the chitmase activity in the medium in the absence of the candidate compound A related aspect of the invention is the above- descπbed method further compπsing determining whether the candidate compound modulates one or more of the following β-amyloid plaque formation in a mammal, APP processing in a mammalian cell that expresses APP, the activity of a component of the presenilin pathway, or the activity of a component of the C elegans Notch pathway, such as Glp-1 or Lm-12 In one embodiment, a gain-of- function lιn-12 strain of C elegans is used In a related aspect, the invention is drawn to the above-descnbed method further compπsing determining whether the candidate compound reverses the multivulva phenotype of the lm-12 gain-of- function strain of C elegans Still another aspect of the invention is directed to a method of screening for a candidate compound for the modulation of a disorder, compπsing (a) monitonng the chit ase activity of a C elegans culture in the presence and absence of a test compound, and (b) identifying a test compound that decreases the chitinase activity of the C elegans as a candidate compound for the treatment of a disorder selected from the group consisting of Alzheimer's disease, Ras-mducible cancer, and potassium tiansport disoiders A related aspect of the lm ention pro\ ιdes the above-described method, furthei compπsing (c) contacting a mammalian cell with the candidate compound for modulating a disorder, (d) measuring a trait associated with the disorder in the mammalian cell in the presence and absence of the candidate compound, and (e) identifying a candidate compound as one that affects the trait in a manner inconsistent with maintenance or progression of the disorder The invention also comprehends the above-described method further compπsing the step of manufacturing a composition comprising the candidate compound identified according to step (e) in a pharmaceutically acceptable earner Except where noted to the contrary, a preferred strain of C elegans for practice of the invention is a wild-type strain such as C elegans N2 Numerous other aspects and advantages of the present invention will be apparent upon consideration of the following drawings and detailed descπption

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 shows the compaπson of the wild-type (N2), DT6716 (eg -36) and SG1214(.ye/-12) strains of C elegans in asynchronous culture with regard to chitmase release The released chitmase activity in the eg -36 and se/-12 strains of C elegans is sιgnιficantly(p < 0 0001) lower than in the wild-type strain Figure 2 shows the percent variation (positive numbers-stimulation, negative numbers- inhibition) of chitinase release by vaπous test compounds

Figure 3 illustrates the frequency distribution for the percent vaπation (positive numbers- stimulation, negative numbers- inhibition) of chitmase release by vaπous test compounds Approximately 110 compounds cluster between -5 and -10% inhibition of chitmase activity m culture supematants

Figure 4 shows the percent vaπation (positive numbers- stimulation, negative numbers- inhibition) of chitmase enzyme activity by vaπous test compounds

Figure 5 illustrates the frequency distribution for the percent vaπation (positive numbers- stimulation, negative numbers- inhibition) of chitmase enzyme actι\ ity bv \ aπous test compounds Appioximately 225 compounds cluster around -20% inhibition of chitinase activity

Figure 6 demonstrates the concentration-related effect of test compound X on wild-type C elegans chitinase activity in synchronous culture m duplicate At 10 μM, test compound X inhibits chitinase activity by approximately 55%

Figure 7 illustrates the concentration-related effect of test compound Y on wild-type C elegans chitinase activity in synchronous culture in duplicate At 10 μM, test compound Y inhibits chitinase activity by approximately 60% Figure 8 shows the inhibition of chitinase enzyme activity by test compound X after one hour incubation At 10 μM, test compound X inhibits chitmase by 10-25% of the control

Figure 9 shows the inhibition of chitmase enzyme activity, in duplicate, by test compound Y after one hour incubation At 10 μM , test compound Y inhibits chitmase by 0-3% of the control Figure 10A shows an exemplary dose-response curve for a chitinase assay, and Figure 10B shows an exemplary dose response curve for the same compound acting on HEK125 3 cells ("X"- MTT cell viability assay, open triangle- soluble APP levels, closed square- Aβ42 levels, closed diamond- Aβ40 levels)

Figures 11, 12A and 12B show Western blot analyses of APP processing in HEK125 3 cells or muπne N2A-App cells (N2A-Appl in Figure 12B)

DETAILED DESCRIPTION OF THE INVENTION

The invention provides rapid, versatile and reliable methods of screening for candidate compounds such as therapeutics that modulate the egg laying behavior of nematodes such as C elegans The significance of the invention is partially found m the positive correlation that exists between egg laying defects in nematodes and mammalian disorders or diseases such as Alzheimer's disease, cardiopulmonary diseases and disorders, and Ras-inducible cancer, among others Accordingly, the invention also relates to methods for identifying candidate compounds for the treatment of these disorders and diseases The power and speed of the methods of the inv ention result from the discovery that chitmase, an enzyme found within nematode eggs, is released into nematode cultures in direct proportion to the capacity of the worms to lay eggs, not merely in direct proportion to the eggs that subsequently hatch. For example, nematodes that produce eggs with a normal or wild-type capacity to hatch, but which have an inability to lay such eggs, are eventually consumed by the internally hatched offspring. Contrary to expectations that cultures of such organisms would have elevated chitinase levels resulting from egg hatching and progressive destruction of the parents, chitinase levels were not elevated. Further, the methods of the invention can be performed using cultures of free-living nematodes such as C. elegans, whose food source is bacteria and whose cultures therefore contain these organisms, which often release a variety of destructive enzymes such as proteases.

In providing chitinase assays of nematode cultures, the invention makes available a rapid, easy and versatile approach to screening for candidate compounds such as therapeutic drugs effective in treating a variety of mammalian disorders and diseases. The methods of the invention are also suitable for identifying compounds that are potential risk factors for developing, prolonging or exacerbating one or more mammalian diseases or disorders. The methods of the invention avoid the need to use potentially demanding cell cultures in the initial screens and, in using nematodes. the methods avoid the time-consuming and costly need to visually inspect cultures for evidence of egg laying.

The significance of the invention is apparent in its many and varied embodiments. Because each of the specific members of the chitinase family of enzymes is found within nematode eggs, the methods can be performed by measuring general chitinase activities in culture, or by measuring any or all specific chitinase enzymes found in nematodes. Further, the screening methods of the invention are used to identify modulators of any mammalian disease and/or disorder shown to be related, genetically, biochemically, or otherwise, to nematode egg laying behavior. Examples of such mammalian disorders and/or diseases include, but are not limited to, Alzheimer's disease, cardiopulmonary diseases and disorders, and such forms of cancer as Ras-inducible cancer, among others. In preferred embodiments, the modulators or candidate compounds are present in a nematode culture at concentrations ranging from 0.03 to 30 micromolar, or from 0.03 to 10 micromolar. The methods of the invention secure the benefit of the thorough genetic investigation of the free-living nematode C. elegans, for which many distinct and useful genotypes and phenotypes are known and available. For example, in screening for compounds that may be risk factors for, or useful in the modulation or treatment of, any disease or disorder associated with nematode egg laying behavior, a wild-type strain of C elegans may be used to generally screen for compounds that lower chitinase levels in culture, due to interference or inhibition of egg laying. Alternatively, C. elegans mutants are used to target the screening methods to the identification of candidate compounds useful in the modulation or treatment of particular diseases or disorders, such as Alzheimer's disease. A sel-12 mutant strain of C. elegans exhibits defective egg laying, which may be effectively reverted, in part or full, by a candidate compound, with that effective reversion being detected as an increase in the chitinase levels in culture. In yet another embodiment, a lin-l 2(d) gain-of-function mutant is used, and compounds modulating chitinase levels by changing them to levels approaching or exceeding the wild-type level include compounds that inhibit Sel-12, which interacts with Lin-12. Beyond mutants in the presenilin pathway, the invention extends to screening methods using nematode strains containing mutations in a potassium channel gene, ras, and other genes associated with egg laying defects.

In addition to contemplating the many genotypes and phenotypes of nematodes that are known, the methods of the invention contemplate nematodes containing heterologous orthologs of any one or more of the nematode genes associated with egg laying defects. Further, the invention contemplates screens for toxicity to eliminate candidate compounds that inhibit egg laying by killing the adults. In prefeπed embodiments, the present invention descnbes a screening assay that may be used, foi example, as part of a drug discovery piogram to identify a putative therapeutic compound Specifically, the present invention contemplates the use of the screening assays of the present invention to screen for such compounds that modulate (increase or decrease activity) of chitmase activity In addition, the invention includes pharmaceutical compositions compπsing a candidate compound in an amount effective in modulating or treating one or more mammalian diseases or disorders associated with nematode egg laying The pharmaceutical compositions also include any pharmaceutically acceptable excipient, carrier, diluent, and/or adjuvant known m the art

The present invention provides methods of screening for stimulators of egg laying activity by monitoπng chitmase activity in the presence and absence of the candidate compound and compaπng such results It is contemplated that this screening technique will prove useful in the general identification of a compound that will serve the purpose of promoting, augmenting or increasing a therapeutic effect in at least one of a vaπety of disorders including, but not limited to, Alzheimer's disease, Ras-mediated cancer, non-Ras-mediated cancer, cardiovascular disorders, and the like. The screening methods of the invention can thus be employed to identify any compound whose therapeutic effect may be monitored by a conventional egg laying assay To this end, there are, as noted earlier, a very large number of genes and thus gene products which are associated with phenotypic alterations of egg laying in C elegans The use of this screen is suitable for use with any such nematode strain, provided that egg laying behavior is the nematode property being measured In specific embodiments, the present invention is directed to a method for determining the ability of a candidate compound to modulate a particular disease or disorder This screen takes advantage of the present discovery that the egg laying phenotype of a C elegans culture may be monitored using a chitmase assay As such, monitoπng the chitmase activity of such a culture in the presence and absence of a candidate compound will provide information regarding the beneficial or detnmental effects of a given compound on the egg laving properties of the cultuie, hich may then be correlated to a given disease state as discussed above The method generally includes the steps of

(a) monitoring the chitmase activity of a wild-type C elegans cell in culture m the presence and absence of a test compound, and

(b) identifying said test compound which alters the chitinase activity of said C elegans cell as a πsk factor for a disorder or as a candidate compound for the treatment of a disorder

To identify a candidate compound as being capable of modulating egg laying activity in the assay above, one would measure or determine the chitmase activity in the absence of the added candidate compound One would then add the candidate compound to the cell culture and determine the activity in the presence of the candidate compound A candidate compound which increases or decreases the activity relative to that observed in its absence is indicative of a candidate compound with desired modulatory capability Such assays may advantageously identify modulators that are either inhibitors of egg lay activity or, alternatively, are stimulators of egg laying activity, by identifying those compounds that decrease or increase chitmase activity, respectively

Another distinct aspect of the invention is a screening assay for candidate compounds that alter the egg hatching outside the body of C elegans, thereby increasing or decreasing the amount of chitmase activity present in a culture Such assays are performed using cultures of non-parasitic nematodes and are based on the positive correlation of egg hatching and chitmase enzyme activity in cultures of these nematodes

The candidate compound screening assays are straightforward to set up and perform A candidate compound contacts a C elegans culture under conditions suitable for chitinase expression, processing and/or trafficking, given that particular C elegans strain One measures the effect of the candidate compound on chit ase activity in cultuie and compaies that activity to the level ot activity in the absence of the candidate compound Likew ise, in assays for inhibitors after obtaining a similar culture, the candidate compound is admixed with the cell In this fashion the ability of the candidate inhibitory compound to reduce, abolish, or otherwise diminish a biological effect mediated that manifests as a defective egg laying phenotype in a C elegans culture

"Effective amounts" in certain circumstances are those amounts of a compound, substance, or agent that are effective in reproducibly altering a given nematode egg laying event relative to that event m the absence of the compound, substance, or agent Compounds that achieve significant desired changes m activity are used (e g , as measured by an generally accepted statistical parameter) Significant changes in chitinase activity are represented by an increase, or decrease, in chitmase activity of at least about 30%-40%, and most preferably, by changes of at least about 50%, with higher values of course being possible These changes in chitinase activity are identified relative to the chitinase activity in control (i e , drug-free) cultures

The invention also extends to the candidate compounds themselves, identified by one of the claimed screening methods As used herein the term "candidate compound" refers to any molecule that is suspected of being capable of modulating egg laying activity The invention does not restrict the sources for suitable candidate compounds Thus, the candidate compound may be a protein or fragment thereof, a small organic molecule, or even a nucleic acid molecule It may prove to be the case that the most useful pharmacological compounds identified through application of the screening assay will be compounds that are structurally related to other known compounds that have been identified through an egg laying assay and thus may be termed known modulators of egg laying activity The active compounds may include fragments or parts of naturally occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive However, pπor to testing of such compounds in humans or animal models, it ill be necessary to test a variety of candidates to determine which hav e potential

Accordingly, the active compounds may include fragments or parts of natuially occurring compounds or may be found as active combinations of known compounds which are otherwise inactive Thus, the present invention provides screening assays to identify compounds which stimulate or inhibit egg laying and ultimately have, or are developed through drug design to have, therapeutic efficacy in the treatment of a mammalian disease or disorder It is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and maπne samples may be assayed as candidates for the presence of potentially useful pharmaceutical compounds

It will be understood that the pharmaceutical compounds to be screened could also be deπved or synthesized from chemical compositions or man-made compounds Thus, it is understood that the candidate compound identified by the present invention may be a polynucleotide, a polypeptide, e g , antibodies

(including, but not limited to, monoclonal, humanized and single chain antibodies), small molecule inhibitors, or any other inorganic or organic chemical compound that may be designed through rational drug design starting from known stimulators or inhibitors of egg laying in C elegans, as determined using the screening aspects of the invention described herein

Advantageously, the screening assays of the invention are amenable to numerous high throughput screening (HTS) assays known m the art For a review of exemplary HTS assays, see Jayawickreme et al , Curr Opin Bwtechnol 8 629- 34 (1997) Automated and mmiatuπzed HTS assays are also contemplated, e g , as descπbed m Houston et al , Curr Opin Bwtechnol 8 734-40 (1997)

The compounds used in the screening assays, and particularly m the HTS assays, may be identified from hbraπes of chemical compounds There are a number of different braπes used for the identification of small molecule modulators including chemical libraries, natural product hbraπes, and combinatorial libraries comprised of random or designed peptides, ohgonucleotides, or organic molecules Chemical libraries consist of structuial analogs of known compounds or compounds that are identified as hits or leads via natural product screening or from screening against a potential therapeutic target Natural product libraries aie collections of products from microorganisms, animals, plants, insects, or marine organisms which are used to create mixtures for screening by, e g , feπnentation and extractions of broths from soil, plant or maπne organisms Natural product libraries include polypeptides, non-πbosomal peptides and non-naturally occurπng vaπants thereof For a review, see Science 282 63-68 (1998) Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture They are relatively simple to prepare by traditional automated synthesis methods, PCR cloning, or other synthetic methods Of particular interest will be libraries that include peptide, protein, peptidomimetic, multiparallel synthetics, recombmatoπal and polypeptide members A review of combinatoπal hbraπes and hbraπes created therefrom is available See Myers, Curr Opin Bwtechnol 8 701-07 (1997), incorporated herein by reference A candidate compound identified as a modulator by the use of vaπous hbraπes descπbed may then be optimized, e g , through rational drug design, and screened using the methods of the present invention

The goal of rational drug design is to produce structural analogs of biologically active compounds, substances or agents, e g , polypeptides or compounds with which they interact (agonists, antagonists, inhibitors, peptidomimetics, and binding partners, among others) By creating such analogs, it is possible to fashion drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of vaπous other molecules In one approach, one would generate a three-dimensional structure for a given peptide or a fragment thereof This could be accomplished by X-ray crystallography, computer modeling, or by a combination of both approaches An alternative "alanme scan" approach involves the random replacement of residues throughout the polypeptide molecule with alanine, followed by screening for modulation of relevant functions using the assays of the invention.

In addition, there has also been a rapid growth in the deliberate preparation and use of libraries and/or arrays of compounds. Each library contains a large number of compounds which are screened against a biological target such as an enzyme or a receptor. When a biological hit is found, the compound responsible for the hit is identified. Such a compound, or lead, generally exhibits relatively weak activity in the screen but forms the basis for a more traditional, targeted, medicinal chemistry program to enhance activity. The libraries may be prepared using the rapidly developing techniques of combinatorial chemistry or by parallel synthesis (DeWitt et al, Proc. Natl. Acad. Sci. (USA) 90:6909 (1993); Jung et al, Angew. Chem. Int. Ed. Engl., 31 :367-83 (1992); Pavia et al., Bioorg. Med. Chem. Lett. 3:387-96, (1993).

Alternatively, the compounds to be screened may be from a library based upon a common template or core structure [see for instance Ellman et al., J. Amer. Chem. Soc, 114:10997 (1992; benzodiazepine template), WO 95/32184 (oxazolone and aminidine template), WO 95/30642 (dihydrobenzopyran template) and WO 95/35278 (pyrrolidine template)]. The template has a number of functional sites, for instance three, each of which can be reacted, in a step-wise fashion, with a number of different reagents, for instance five, to introduce 5 x 5 x 5 different combinations of substituents, giving a library containing 125 components. The library will normally contain all or substantially all possible permutations of the substituents. The template may be a 'biased' template, for instance incorporating a known pharmacophore such as a benzodiazepine ring or an 'unbiased^' template, the choice of which is influenced more by chemical than biological considerations.

Thus, the present invention may be used to identify lead compounds for drug discovery. In addition to the library screening discussed above, such lead compounds may be generated by random cross-screening of single synthetic compounds made individually in the laboratory or by screening extracts obtained from natural product sources such as microbial metabolites, marine sponges, and plants.

In an alternative embodiment, the compounds may be generated through rational drug design based on the structure of known biologically active compounds and/or their sites of biological action. This has now been complemented by the powerful techniques of computer-assisted drug design. The goal of rational drug design is to produce structural analogs of biologically active molecules of interest. Such technologies have the potential to yield thousands of compounds for a particular indication, and each of the compounds may be advantageously screened for potentially beneficial effects using the screening assays of the present invention. The activities of the candidate compounds identified by the screens of the present invention may then be confirmed by screening against strains with suppressors for loss-of-function mutants of a given target. Additionally, it is contemplated that the activities of these candidate compounds may be further confirmed by performing additional screens to monitor the effects of the candidate compounds in mammalian cells exhibiting the particular measurable trait of a disorder to be treated by the compound.

Aspects of the invention disclosed in the preceding discussion may be better understood upon consideration of the following Examples, wherein Example 1 discloses a C. elegans chitinase assay, Example 2 describes a chitinase assay using a C. elegans strain having a defect in the presenilin pathway, Example 3 provides a chitinase assay using a C. elegans strain having a potassium channel defect, Example 4 discloses a chitinase assay using a C. elegans strain having a ras mutation, Example 5 describes an assay for chitinase inhibitors, Example 6 illustrates a chitinase assay using a lin-l 2(d) Notch pathway gain-of-function mutant nematode strain, Example 7 discloses a screening assay for candidate compounds that modulate a trait characteristic of Alzheimer's disease using human cells, and Example 8 provides chitinase assays to assess candidate compound toxicity. Example 1 C. elegans Chitinase Assav

C elegans cultuie medium was examined to deteimine if chit ase activity could be reliably detected in a medium containing a bactenal food source, such as E colt All strains of C elegans used in the examples descnbed herein, including the wild-type N2 strain, are available thiough the Caenorhabditis Genetics Center at the University of Minnesota

Sterile, basal medium for maintenance of C elegans was either an enriched peptone medium or Nematode Growth Agar (i e , NGA) The emiched peptone medium, based on the formulation of Schachat et al , Cell 15 405-411 (1978), contained 1 2 g NaCl, 20 g Bactopeptone, 1 ml cholesterol (5 mg/ml m ethanol), 1 ml 1 M MgSO₄»7H₂O, 25 ml 1 M KH₂PO₄ (pH 6), and 25 g Bactoagar per liter of glass-distilled water Twenty millihters were added to each 100 x 20 mm petπ dish, 100 ml were added to each 150 x 25 mm petπ dish The NGA medium contained 3 g NaCl, 2 5 g Bactopeptone, 1 ml cholesterol (5 mg/ml in ethanol), 1 ml 1 M CaCl₂, 1 ml 1 M MgSO₄'7H₂O, 25 ml 1 M KH₂PO₄ (pH 6) and 17 g Bactoagar per liter of glass-distilled water Twenty millihters were pipetted steπlely into each 100 x 20 mm petπ dish

Colonies of E coh used to inoculate broth cultures were developed on solid medium containing 8 g NaCl, 10 g Bactotryptone, 5 g yeast extract, 15 g Bactoagar and 5 mg thymidme per liter of glass-distilled water The medium was steπhzed by autoclavmg and poured into petπ dishes (e g , 20 ml per 100 x 20 mm dish) using standard techniques E coh broth medium contained 8 g NaCl, lOg Bactotryptone, 5 g yeast extract, 5 mg thymidme and 10 mg cysteme per liter of glass-distilled water Ten ml ahquots were dispensed into scintillation vials, autoclaved, and stored at 4°C

Each bottle of E coh broth was inoculated with a colony of E coh (strain OP50) The E coh broth culture was used to provide food for C elegans on solid media (e g , enπched peptone or NGA in petπ dishes or 96-well plates) One ml of a 4-day-old E coh broth culture was spread on each NGA or peptone agar dish (150 x 25 mm) while 200 μl ot broth as spiead on 100 \ 20 mm plates The agai containing E coh w as stored for one week at room tempeiatuie in a dra er beiore being inoculated with worms C elegans w ere transferred once a week onto new peptone or NGA agar plates coated containing E coh inoculated using standard techniques Strains of C elegans were maintained in a humid, low-temperature incubator set at 20°C

Chitinase assays of nematode cultures, such as C elegans cultures, are performed usmg either synchronous or asynchronous cultures To synchronize C elegans cultures, the worms were first harvested when gravid or full of eggs Harvesting was accomplished by washing each agar dish twice with 5 ml of steπle M-9 buffer for 100 x 20 mm dishes (7 5 ml for 150 x 25mm peptone dishes) and the washes were put into a 15 ml steπle centπfuge tube The worms were centπfuged in a Beckman centπfuge (TJ-6) at 1000 rpm (140 x g) for 2 minutes The supernatant was removed and the pellet was resuspended in 10 ml of M-9 buffer The resuspended pellet was centπfuged at 1000 rpm for 2 minutes

The supernatant was removed and the worm pellet was resuspended in 7 ml of a fresh, alkaline hypochloπte solution (6 ml sodium hypochloπte (4-6%) and 15 ml IN NaOH) The 15 ml tubes were gently hand-rotated for 7-8 minutes to prevent suffocation of the eggs The tubes were then centrifuged at 1000 rpm for 2 minutes and the supernatant containing the alkaline hypochloπte solution was removed The pellet was washed once with 10 ml of steπle water and twice with 10 ml of steπle M-9 buffer The pellet was centrifuged for 2 minutes at 1000 rpm between washes After the final wash with M9 buffer (12 g Na₂HPO₄, 6 g KH₂PO₄, 10 g NaCl, and 0 5g MgSO₄ ^«7H₂O per 2 liters of glass-distilled water, filtered into 1 liter polystyrene bottles using a 0 45 μm cellulose acetate membrane), the supernatant was removed and the pellet was concentrated by resuspension in 0 5 ml M9 buffer The egg pellet was transferred to a NGA dish containing 6-day-old E coh (Lewis et al , Basic Culture Methods In Methods in Cell Biology (48) Caenorhabditis elegans Mode Biology Analysis of an Organism 3-29 (Epsteom et al., eds. San Diego, Academic Press. Inc.) (1995)). The eggs hatched overnight in a 20°C incubator.

In another embodiment of the chitinase assays according to the invention, cultures w ere prepared in individual wells of 96-well plates. Medium for these wells contained 75-90 ml of sterile M-9 buffer, 10-25 ml of inoculated E. coh broth, 2 mg sulfamefhoxazole, 1 mg trimethoprim, and 100 μl of a methanol solution containing 5 mg cycloheximide and 10 mg chloramphenicol per 100ml volume. The exact amount of E. coli broth added to the medium was determined by the optical density reading at 570 nm to obtain 1.2 OD for 3.6 ml of inoculated E. coh broth. To determine the inoculum volume, the 1.2 OD was divided by the optical density reading of the E. coli broth and multiplied by 2 for synchronized cultures or 1 for unsynchronized cultures to determine the number of bottles of E. coli broth to be used in the test plates. The volume of M-9 buffer was adjusted by the amount of E. coli broth added. The primary assay volume in each well of the 96-well plate was 200 μl, which consisted of 161 μl of the test culture medium, 6 μl of M-9, 30%. DMSO control or a test compound at 300 μM, and 33 μl of the worm suspension. N2 worms from synchronized cultures were diluted in M-9 and 33 μl were placed on a glass slide for worm counting under a light microscope. The nematodes were diluted with M-9 until there were 50-90 worms per 33 μl. Worms were the final component added to the wells of the test plates. Six days after inoculation into the test wells, worms were fed 7 μl of a 3-day-old E. coli broth culture to ensure reproduction of the worms. The test culture medium volume was 147 μl The volume of nematodes was 33 μl, diluted where necessary as described above. Some of the initial characterizations of the DT6716, MT061 1, GS1214 and N2 strains of C. elegans were done with unsynchronized cultures. After the worms became gravid and had LI larvae in the test plates, as determined by visual inspection of control wells, the cultures were subjected to a fluorometric chitinase assay. Preferably, chitinase activity assays are conducted within 12 to 24 hours of egg laying, to minimize the time between test compound addition and completion of the assay. Typically, assays were conducted by transfeπing 10- 100 μl (a sufficient volume to contain detectable chitmase activity) of cultuie supernatant from indi idual w ells of a 96-well miciotiter plate Although 96-well plates were typically used, one of ordinary skill in the art will recognize that 24-well plates, 384-well plates, and a great variety of other containers would be suitable for use in practicing the methods of the invention To the aliquot of culture supernatant was added 10 μl of a DMSO solution containing 0 8 mM 4-methylumbelhferyl β-D-N,N',N"-tπacetylchιto-tπosιde (Sigma Chem Co , St Louis, MO) One of ordinary skill in the art understands that any labeled substrate known m the art may be used, e g , fluoroge c, radiometπc or coloπmetπc labeled chitmase substrates The mixture was then incubated at 37°C for 1 hour The assay was terminated with 100 μl of 1 M glycme/1 N NaOH (pH 10 6) and results were determined using a fluoπmeter (excitation 360/40, emission 460/40, and gam 75) according to McCreath et al , (1992)

Example 2

Chitinase Assays- Presenilin Pathway

The sel-\2 (GS1214) C elegans strain is a presenilin mutant, which carπes a loss-of- function mutation in the sel-12 gene encoding presenilin- 1 Because the

Lm-12 receptor requires interaction with Sel-12 for full activity, the defective Sel- 12 m this strain results in reduced Lm-12 activity This reduction of Lm-12 activity, in turn, results m an egg laying defect in C elegans (Sundaram et al , Genetics 135 755-763 (1993)) This strain was purchased from the Caenorhabditis Genetics Center at the University of Minnesota

C elegans was maintained, synchronized, cultured, and the cultures were fluorometπcally assayed, all as described in Example 1 The results of the chitmase assays on C elegans sel-12 cultures showed that reducing or eliminating Sel-12 activity caused a defect m egg laying Release of chitinase from the sel-12 cultures was reduced by 76% relative to the levels found with wild-type C elegans (strain N2) using the two-tailed Student's t-test, at p<0 001 (See Figure 1) Without wishing to be bound by theory, it appeared that eggs hatched inside the sel 12 adult hennaphrodite and eloped within the body cavity This development killed the adult and eventually led to the release of LI- or L2-stage larvae into the cultuie Surprisingly, the chitinase presumably released upon egg hatching did not appear in quantity in the culture medium Thus, chitmase release in the 96-well plate assay provides an efficient means to screen for compounds that interfere with the presenilin pathway and inhibit egg laying by wild-type C elegans

In addition to the preceding screens, 10,780 compounds were screened using the assays descπbed above The mean chitmase activities, and variances, vaπed between expeπments Cut-off values for selection of hits were based on the statistics of the distπbution with a threshold of z>3 0 (p<0 001) The pπmary screen yielded 37 hits of which 16 replicated on rescreening Visual examination was used to eliminate compounds, which decreased worm viability and allowed E coh overgrowth Dose-response curves for the active compounds were determined, and an exemplary dose-response curve is shown in Figure 10 Seven of the compounds had well-behaved dose-response curves with IC₅₀ values falling in the range 0 3-2 2 μM IC₅₀ values for the active compounds generally fell within the low micromolar range To test for direct effects of active compounds on chitmase enzymatic activity, compounds were incubated with worm-free supematants from N2 cultures containing hatched eggs No inhibition of chitmase activity was observed for two of the tested compounds

Example 3 Chitinase Assays- Potassium Channels To demonstrate that the chitinase assay could be used as a rapid and accurate measure of egg laying defects generally, rather than the subset of egg laying defects directly related to presenilin defects, chitmase assays were performed on cultures of C elegans strains exhibiting egg laying defects due to mutations outside the presenilin pathway proper In particular, C elegans strains n728 (DT6716) and n2332 (MT6011), each containing an egl-36 allele which confers a K 3 potassium channel defect, w eie subjected to the chitmase assay of the invention These two egl-36 strains are partially deficient in egg laying as a result of defects in the ability of the enteric and egg-laying muscles to contract (Johnstone et al , Neuron 19 151 -164 (1997)) The strains were obtained from the Caenorhabditis Genetics Center, University of Minnesota, and C elegans maintenance, synchronization, cultuπng and assaying of those cultures was performed in accordance with the descriptions provided in Example 1 Results showed a reduction in chitmase levels relative to the levels found in cultures of the wild-type N2 strain of C elegans However, these egl-36 strains of C elegans produced cultures containing more chitmase activity than was found in cultures of the sel-12 mutant strain of C elegans, which is completely defective in egg laying Thus, chitmase levels are positively correlated with the extent of egg laying, regardless of whether the defect in egg laying aπses from a mutation m the presenilin pathway

Example 4 Chitinase Assays- Ras-Inducible Cancer

Chitmase assays are also performed to identify modulators of Ras, an oncogene product implicated in a vaπety of mammalian cancers Compounds that cause an Egl phenotype in C elegans could act by inhibiting the function of the nematode Ras protein, either directly or through inhibition of farnesyltransferase C elegans maintenance, synchronization where desired, cultuπng for assay purposes, and culture assays for chitmase activities are performed as descπbed in Example 1 Re-testing positive compounds in cultures of C elegans strains that either over-express the wild-type nematode Ras protein or express an activated Ras mutem will distinguish compounds that act by directly inhibiting Ras function from those compounds that act at some other site that contπbutes to this phenotype Example 5 Assav foi Chitinase Inhibitoi

Chitmase assays using a w ild-type nematode stiain, such as C elegans N2, may be used to identify compounds that inhibit chitmase activity, thereby identifying chemicals that aie potential risk factors for symptoms associated with a variety of mammalian disorders or diseases, such as Alzheimer's disease Such assays were conducted, using the materials and methods described in Example 1 , along with the introduction of test compounds as described below

Test compounds were individually dissolved in DMSO to a concentration of 10 mM and 50 μl of each solution was added to separate wells of a 96-well plate Plates typically contained solutions of 88 distinct compounds, with 8 wells reserved for DMSO controls By seπal dilution with sterile M-9 buffer into fresh 96-well microtiter plate wells, 300 μM solutions of each test compound were prepared These test compound solutions were then used in the 96-well plate embodiment of the screening methods of the invention, as descπbed in Example 1 To maximize the sensitivity of the chitmase assays, test compounds , such as test compounds X and Y, were incubated in a low-temperature incubator at 20°C for 1 hour at 0 03, 0 1, 0 3, 1, 3, and 10 μM The test compounds were then subjected to the chitinase assay, in duplicate, to determine whether these compounds inhibited chitinase directly The activity of these compounds was compared to controls, ensuπng that the observed modulations of chitmase activity were real A summary of the results is presented m Figures 2-5 Four compounds out of 80 of the compounds tested showed a chitmase inhibition of greater than 50% relative to the wild- type (N2) C elegans activity Test compound X and test compound Y gave similar results when a dose-response study (using 0 03-10 μM of the test compounds) was conducted Re-screenmg of active test compounds confirmed the identities of those compounds which consistently reduced chitinase enzyme activity by at least 50%

Several control studies may also be performed to ensure that test compounds truly modulate chitinase enzyme activity by affecting egg laying behavior In order to detemiine whethei an active test compound w as toxic to the nematodes, thereby accounting foi the observed reduction in chitmase activ ity in the medium, plates fiom which supematants were removed for enzyme assays were stored at 20°C until the assay read-outs were completed Wells for which a reduction m chitmase activity was observed were examined under a dissecting microscope to determine if the abundance and stage-distribution of nematodes was similar to that observed in control wells Compounds that caused significant differences in terms of the number or stage of development of the worms, relative to the controls, were eliminated Active test compounds were also tested to determine if they directly inhibited the chitinase enzyme or interfered with detection of the fluorescence of the product of the enzyme assay Ahquots ( 10- 100 μl) of supematants from drug-free control wells were removed from individual wells of a 96-well plates and transferred to fresh 96-well plates Ahquots of vaπous concentrations (0-10 μM) of test compounds were added to the supematants and the plates were incubated for one hour at 37°C Chitmase assays were then performed as described above Any change in the apparent chitinase activity between drug-free and drug-containing wells identified test compounds that either directly inhibited the enzyme or interfered with detection of the fluorescent label Compounds producing such changes were then eliminated The chitmase drug screen was established to find compounds that inhibit egg laying in N2 C elegans Of 880 compounds tested, test compound X and test compound Y, when present at 10 μM, rendered the wild-type N2 strain of C elegans defective m egg laying Test compound X and test compound Y also produced a dose-dependent effect on chitinase activity (greater than 50% reduction in activity at 10 μM, see Figures 6-9), and showed no apparent toxic effects on the nematodes, demonstrating that chitmase activity can distinguish compounds that affect egg laying behavior to varying degrees

In the present invention, an assay for egg laying by measuπng chitinase was tested in wild-type C elegans (N2 strain), sel-12 presenilin mutants (see Example 2), and two egl-36

potassium-channel mutants (see Example 3) The sel-12 mutations cause a nearly complete cessation of egg laying, while the egl-36 strains were significantly (p<0.001 ). although incompletely, defective in egg laying, in comparison to the wild-type N2 strain of C. elegans. Approximately 1 1,000 compounds were screened at 10 μM for suppression of egg laying by wild-type womis, and several compounds showed some suppression of egg laying behavior. Active compounds in the N2 worm screen were defined as those that reproducibly yielded a 50% reduction in chitinase activity based on the statistics of the distribution. The N2 worm screen identified compounds inhibiting chitinase activity with fairly high specificity (18 potential inhibitors in 10,780 compounds tested, an identification rate of 0.2%). The most active compound in the N2 worm screen had an IC₅₀ of 100-300 nM and a maximal inhibition of 56% of the wild- type chitinase activity. A subset of these inhibitors is expected to block presenilin function. Thirty-seven potential hits were identified in the primary screen. All of these compounds were evaluated in a secondary assay looking to define the concentration (0.03-10 μM) range capable of inducing a response in terms of egg laying behavior. Sixteen compounds showed egg laying suppression with no general toxicity for C. elegans. These compounds were also evaluated for modulation of APP processing. Exemplary compounds for use in the screening methods of the invention include chloroquine (i.e., 7-chloro-4,4-diethylamino-2- methylbutylamino quinoline), primaquine (i.e., 8-(4-amino-l-methylbutylamino)- 6-methoxyquinoline), and the other compounds active in modulating APP processing as disclosed in U.S. Pat. No. 5,348,963, the mono- (e.g., 4- aminopyridine) and di-aminopyridines disclosed in U.S. Pat. No. 5,580,580, as well as the pyrimethamine, cromolyn sodium, and erythromycin disclosed in U.S. Pat. No. 5,567,720. U.S. Pat. Nos. 5,348,963, 5,580,580, and 5,567,720 are incorporated herein by reference in their entireties.

These compounds, and other candidate compounds, may be subjected to chitinase assays in the lin-l 2(d) C. elegans system to look for reversal of the egg laying defect. These secondary screens make use of knowledge regarding the genetic interaction of C. elegans Presenilin (sel-12) and Notch (lin-12), which is a Sel-12 receptoi To date, thirteen compounds ha e been identified that both inhibited egg laying in C elegans (percent inhibition ot chit ase activity in culture ranged from 37-87 with candidate compounds each at 10 μM) and also inhibited amyloid-beta peptide pioduction or APP processing in human cells (For candidate compounds each at 20-30 μM, percent inhibition of Aβ40 ranged from 48-77 and percent inhibition of Aβ42 ranged from 30-73 ) These compounds were identified using preferred, high throughput embodiments of the screening methods of the invention, rather than relying on the visual inspection of cultures to assess egg laying behavior

Example 6 Chitinase Assays- lin-12(d) Gain-of-Function Strains

Lin- 12(d) worms carry an activating mutation in the Lin- 12 receptor which causes such worms to be defective for egg laying (Sundaram et al , 1993) This strain was purchased from the Caenorhabditis Genetics Center at the University of Minnesota The hn- 12(d) mutation causes activation of signaling by Lm-12 through the Lm-12/Notch signal transduction pathway This activated signaling produces a defect in egg laying which can be partially rescued by sel-12 loss-of- function mutations (Levitan et al , Nature 377 351-354 (1995)) Although the Lin- 12(d) C elegans system provides another distinct aspect of the invention, the maintenance, synchronization, nematode cultuπng, and assaying of those cultures were performed as described m Example 1 To date, 1,700 compounds have been directly screened for modulation of chitinase activity in a primary screen using the hn- 12(d) system, additionally, the 16 compounds identified using the wild-type C elegans strain (see Example 2) were re-tested in a secondary screen using the hn- 12(d) system In the primary screen, release of chitinase activity from Lin- 12(d) worms averaged 5893 + 902 SD fluorescence units Assuming a Gaussian distribution, compounds which increase chitinase activity greater than 1 4 fold would have a p<0 01 significance level From the primary screen, 30 compounds were identified which exceeded this threshold, and 1 1 of the 30 compounds were also identified in the secondary assays. Three compounds were found to stimulate chitinase release more than 2-3 fold above background. By comparison, the activity of chitinase in wild-type N2 cultures averaged 16,000-25,000 fluorescence units under the conditions of the assay, a difference of 3-4 fold. Thus, the best compounds restored chitinase to levels approaching that seen in N2 worms. (See Figure 10 for an exemplary dose- response curve.) Notwithstanding the increase in chitinase activity, visual examination of the relevant cultures did not indicate that the compounds were restoring egg laying. The number of eggs per well in treated cultures did not appear to be different from that seen in DMSO-treated, control cultures oϊ lin-l 2(d) worms.

Apparent from the preceding discussion are embodiments of the methods of the invention that combine wild-type and/or mutant alleles of various genes, such as sel-12 and lin-l 2(d). The invention contemplates any combination of particular genes (i.e., any genotype) useful in screening for egg laying defects in nematodes, or the effective reversion thereof. Those of ordinary skill in the art, guided by the disclosure herein, will realize a number of promising genotypes that are suitable for use in practicing the methods of the invention.

Example 7

Alzheimer's Disease- Human Cells

Candidate compounds identified as modulators using a chitinase assay according to the invention are further tested in a mammalian cell line, such as the human embryonic kidney cell line HEK125.3, a derivative of HEK 293 cells engineered to express APP. Of course, any mammalian cell line known in the art to express APP may be used in the mammalian screening assay. Additionally, any

APP processing assay known in the art may be used., including, but not limited to, animal assays, such as assays involving transgenic mouse models.

In one embodiment of this aspect of the invention, 125.3 cells, derived from HEK293 cells, were transformed with a bicistonic vector derived from pIRES- EGFP(Clontech) containing the App695-Sw-KK coding sequence, an internal ribosome entry site, and an enhanced green fluorescent protein coding sequence in the second cistron. Yan et al. (1999).

The 125.3 cells were grown in DMEM (high glucose and without sodium pyruvate) medium supplemented with 10% fetal bovine serum, IX antibiotic- antimycotic, 1 mM sodium pyruvate, and 400 μg/ml G418 in a humidified incubator at 37°C with 5% CO,. The medium was aspirated from the T75 flask and the cells were rinsed with 10 ml lx PBS without calcium or magnesium. The PBS was then removed by aspiration and 5 ml of 0.05% Trypsin EDTA was added to the flask and allowed to remain in contact with the cells for 2-3 minutes at room temperature. The five millihters of trypsinized cells were added to 35 ml of medium and centrifuged at approximately 1000 rpm (140 x g) for 3-5 minutes. The medium was aspirated and cells were resuspended in 10-12 ml of medium. The cells were then either passaged into other T75 flasks (a 1 : 10- 1 : 12 dilution with a final volume of 10 ml) or counted on a hematocytometer with a final cell density of 5 x 10³ cells/ml. Cells (100 μl per well) were plated on Costar 96 Cell Culture Cluster plates. The next day (Day 2), an additional 100 μl of medium was added to each well of the 96-well plate. On Day 3, the test compounds were solubilized in DMSO at 10 mM. For initial screens, the compounds were diluted to 30 μM in medium. The two hundred microliter volume was removed by pipette from the cells and the volume was completely replaced with medium containing drug. The test plates were prepared in duplicate in which one plate was used for the 3 different enzyme immunoassays (i.e., EIAs). For the Aβ40 and Aβ42 EIAs, conditioned media were diluted 1 :20 and 1 :2, respectively, with 1% BSA in 0.05% of Tween20 in the soluble APP a fragment (sAPP , the APP fragment resulting from α-secretase cleavage between amino acids 17 and 18 of Aβ) EIA; the conditioned medium was diluted 1 :2 with 1%> BSA in 0.05% of Tween20 in Dulbecco's phosphate-buffered saline without calcium chloride or magnesium chloride. The duplicate plate was used in a MTT cell viability assay. After 48 hours, the supernatant from one of the duplicate test plates was aliquoted into -->->- another Costar 96 Cell Culture Cluster plate and stored at -80 C until the 3 different EIAs were perfonried.

The EIA for Aβ42 was performed as described (Pirttila et al., Neurobiol. of Aging 18: 121-27 (1997)). The sAPP assay was performed using the monoclonal antibody 22C1 1 (Boehringer-Mannheim Corp.) to capture sAPPα, with monoclonal antibody 6E10 (Senetek) used as the detecting antibody.

Fifteen of sixteen compounds identified as modulators of egg laying behavior in wild-type N2 C. elegans were screened for effects on APP processing in HEK125.3 cells. Compounds were evaluated at 10 μM and 30 μM. Six compounds decreased amyloid-β processing with very little cellular toxicity.

Toxicity of one other compound confounded the determination of its effects on APP processing. The six active compounds inhibited release of both Aβ40 and Aβ42 by about the same extent. Effects on sAPPcc release were variable. Two of the compounds had no effect on sAPPα release, one compound stimulated sAPPα release by 250%). The remaining four compounds decreased sAPPα release.

Dose-response curves were determined for each of the active compounds. Two of the compounds showed IC₅₀ values in the micromolar range; all began to show toxicity at doses greater than or equal to 30 μM, as assessed by reduction of MTT. For all of the compounds, the IC₅₀s for reduction of either Aβ40 or Aβ42 were similar. Processing of APP at the β-, α-, or γ-secretase sites should create fragments containing 99, 83, or 40 amino acids comprising the C-terminus of APP. In cultured cells, antibodies to the C-terminus of APP generally identify the 99- (C- Terminal Fragment-99 or CTF-99) and 83-amino-acid (CTF-83) fragments resulting from β- and α-secretase processing, respectively. For reasons unknown, the 49-amino-acid fragment that should result from γ-secretase processing is not detected. As shown by Western blot analysis (Figures 11 , 12A, and 12B), an antibody directed against the C-terminus of APP identified full-length APP, several unidentified fragments of 20-40 kDa, and a major fragment of about 9 kDa corresponding to CTF-99. In addition to APP piocessmg assays conducted using HEK125 3 cells, APP piocessmg assays were conducted using mouse N2A-App cells Several of the compounds alter APP processing intermediates in the mouse N2A-App cells as well as in the human HEK125 3 cells, as shown in Figures 1 1 , 12A and 12B Both cell lines have been transfomied with the same plasmid (descπbed above) which directs expression of APP695, having the Swedish NL-→KM mutation and a C- terminal di-lysme motif In both 125 3 cells and N2A-APP cells, one compound increased accumulation of an APP CTF corresponding in size to CTF-99 Two other compounds increased accumulation of an APP CTF corresponding in size to the α-secretase product, CTF-83, with concomitant stimulation of sAPPα release

In 125 3 cells treated with one of those two compounds, the increase in the quantity of the apparent CTF-83 product was so great that it overlapped the position of CTF-99 on the Western blot In N2A-App cells, treatment with yet another compound increased both CTF-99 and CTF83 The N2 worm screen yielded a surprisingly high percentage of compounds that suppressed Aβ40 and Aβ42 release by human HEK125 3 cells The majoπty of active compounds inhibited release of Aβ40, Aβ42, and sAPPα, one compound inhibited only Aβ peptide release, and yet another compound inhibited Aβ peptide release, but stimulated sAPPα release The effect of one candidate compound mimics that of protein kmase C

(PKC) activation Activation of PKC m cellular assays of APP processing stimulates entry of APP into the α-secretase processing pathway This causes an increase in the release of sAPPα from stimulated cells, and, in some cell lines, a concomitant reduction in the release of Aβ40 and Aβ42 (Gabuzda et al , Neurochem 61 2326-29 (1993)) Together with the increase in the release of sAPPα from treated cells, this candidate compound also increased the CTF-83 α- secretase processing product of APP in treated cells The compound is also active in assays of phosphohpase A2 inhibition, which suggests that it is acting through modulation of membrane hpid composition Loss of PS 1 activity in PS 1 null mouse neurons reduces Aβ processing but has little or no effect on the secretion of sAPPα (DeStrooper et al.. Nature 391 :387- 90 ( 1998). It also causes the accumulation of the CTF-99 β-secretase product of APP. A similar accumulation of the CTF-99 product is seen in HEK125.3 cells that have been treated with another candidate compound identified using the screening assays according to the invention. It is expected that this second compound acts as an inhibitor of the presenilin pathway.

APP expression in human cells was also assayed using Western blot analyses. A human embryonic kidney cell line, HEK125J cells, or the Neuro2A- APP1 cell line expressing APP695-Sw-KK, were grown in 100 mm plates in modified Eagle's medium (i.e., MEM) supplemented with 10% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, 1 mM non-essential amino acids, and 400 μg ml G418. After cells were grown to about 50-70% confluency, individual test compounds were added to the medium at a final concentration of 10 μM. The compounds were left in contact with the cells for 48 hours before preparation of the cell lysates. To prepare the lysates, cells were transferred to 15 ml conical tubes and centrifuged at 1 ,500 rpm (340 x g) for 5 minutes to remove the medium. The cell pellet was washed once with conventional phosphate-buffered saline (i.e., PBS). Cells were lysed in lysis buffer (10 mM HEPES, pH 7.9, 150 mM NaCl, 10% glycerol, 1 mM EGTA, 1 mM EDTA, 0.1 mM sodium vanadate, and 1%

Nonidet P-40). The lysed cell mixtures were centrifuged at 5000 rpm (2,040 x g) and the supematants were stored at -20°C. Protein concentrations were determined with the Bio-Rad Protein Assay (Bio-Rad Laboratory, Hercules, CA). Equal amounts of protein (50 μg) were used for electrophoretic analyses on 4-12% Tricine gels (Novex, San Diego, CA) followed by protein transfer to nitrocellulose membranes. The antibody C8, recognizing a C-terminal epitope of human APP, was purchased from the laboratory of Dennis Selkoe (Harvard Medical School and Brigham and Woman's Hospital, Boston MA) and used to detect the APP CTF-99 and CTF-83 fragments. Yan et al. (1999). Antibody 6E10 (Senetek) is also used for detection of fragments, such as CTF-99. For lmmunoblott g, cells were washed once with cold PBS, released horn the dish by scraping into PBS, and the cells were collected by centrifugation (2,000 rpm (593 x g) for 3 minutes) The pellets were collected from 6 w ell tissue culture plates and resuspended in 0 5 ml cold IPB lysis buffer (10 mM Tπs-HCl, pH 7 5, 5 mM EDTA. 1 % NP-40, 0 5% deoxycholate and 150 mM NaCl, plus 50X protease inhibitor cocktail, Boehπnger Mannheim) for 30 minutes on ice Lysates were centπfuged at 4°C (13,000 x g) for 15 minutes and the soluble proteins recovered in the supernatant The particulate fraction was resuspended in 0 1 ml lysis buffer and the protein content of each fraction was quantified using the Pierce method with BSA as the standard Equal amounts of particulate fractions (range 10-15 μg/lane were diluted in 4x NuPage (Novex) sample buffer containing dithiothreitol and heated to 70°C for 10 minutes Proteins were fractionated on NuPage 4-12% Bis-Tπs gels m MES-SDS running buffer at 200 volts for 40 minutes and then electro-transferred to polyv myhdene difluoπde (PVDF) membranes (Novex) in NuPage transfer buffer at 25 volts for 70 minutes

Membranes were blocked in TBS-T (25 mM Tπs-HCl (pH 7 5/ 0 15 M NaCl/0 1% Tween 20) containing 0 1% BSA (Sigma) and 5% nonfat milk (Bio- Rad) for 2 5 hours at room temperature Membranes used to detect CTF-99 were probed with a biotmylated 6E10 monoclonal antibody (Senetek) at 1 1000 dilution in TBS-T containing 0 1 % BSA and 5% nonfat milk overnight at 4°C Each membrane was washed 3 x 10 minutes with TBS-T and then probed with secondary antibody (cat anti-mouse IgG HRP (Santa Cruz), 1 5000 dilution containing 0 1% BSA and 5% nonfat milk) for 1 5 hours at room temperature Membranes used to identify aspartyl protease (i e , Asp2) were probed under the same conditions as descπbed above Pπmary antibody was a rabbit antisera (Rb 206, 1 200) and the secondary antibody was a goat anti-rabbit IgG-HRP (Dako), diluted 1 5000) Following 4 x 10 minutes washes with TBS-T, bands were detected with ECL (Amersham), using a one minute duration Film was exposed withm a range of 10 seconds to 3 minutes Using the Western blot assay to assess the amount of APP CTF-99 allows ready differentiation between potential inhibitors of β-secretase processing from potential inhibitors of γ-secretase processing. Compounds that directly or indirectly inhibit β-secretase cleavage have the effect of reducing the amount of CTF-99 produced by treated cells using Western blot as a detection assay because β-secretase cleavage is believed to be the first, and limiting, step in the formation of Aβ peptide. Correspondingly, if β-secretase cleavage occurs, but subsequent cleavage by γ-secretase is inhibited by a test compound directly or indirectly, there will be an increase in the amount of CTF-99 present in treated cells as CTF-99 is not processed further into Aβ peptide. Thus, beta secretase inhibitors are expected to decrease CTF-99, whereas increases in CTF-99 are expected to result from gamma secretase inhibitors.

Accordingly, Western blot analyses of treated lysates can be used to distinguish between potential beta and gamma secretase inhibitors. Western blot analysis also distinguishes between drug effects on cleavage events versus trafficking or maturation. Consequently, Western blot analysis provides a useful secondary screen for drugs that appear to be active in Aβ production.

To ensure that the candidate compounds were not toxic to human cells, standard MTT cell viability assays were performed. At hour 43, a 0.1% Triton X- 100 medium solution was added to 2-3 control wells on the plate and incubated for 40 minutes in a 37°C incubator with 5% CO₂. After 40 minutes, 25 μl of the MTT solution (5mg/ml MTT (3-(4,5-diethylthiazol-2-yl)-2,5-diphenyltetrazolium) in PBS, filter sterilized) was added to all of the wells and placed back in the incubator for 4-5 hours. After the 4-5 hour incubation, 100 μl of the cell lysis buffer (20% SDS, 50% dimethylformamide (i.e. , DMF), pH 4.7) was added to each well and returned to the incubator for overnight incubation. The next morning, the plate(s) were read on a SpectroMax250 plate reader at 562nm.

Enzyme-linked immunosorbent assays were also used to monitor the expression of APP, including the production of Aβ-40 and Aβ-42. The ELISA for Aβ-40 and Aβ-42 was performed as described (Mehta et al., Neurosci. Lett. 241 13-16 ( 1998)) The sAPP assay was perfonned using monoclonal antibody 22C 1 1 to capture sAPP, with antibody 6E10 used as the detecting antibody

Example 8 Chitinase Assays- Toxicity

The assays of the invention could be used in screens for general nematode toxicity as well, since compounds that kill orms will result in low chit ase levels in the culture Howev er, most general screens that use C elegans visually examine the worms foi effects on behavior and motility as well as viability and reproduction Because compounds that reduce egg-laying in cultures of wild-type animals could be simply toxic, positive wells are visually checked to determine if egg laying has been selectively affected For microscopic inspection, an aliquot of the culture supernatant is transferred to a fresh 96-well plate with an automatic or robotic pipettor, thereby preserving the original culture for quick visual inspection of positive wells

In one embodiment, a sel-12 mutant strain of C elegans was used to screen for compounds that would inhibit Hop-1, thereby creating a phenocopy of the lethal hop-1, sel-12 double mutant Thus, a screen was developed to identify compounds that would effectively render C elegans Hop-1 , which would be lethal only to sel-12 worms and not to wild-type N2 worms In conducting these assays, cultures were not synchronized Asynchronous cultures of sel 12 worms, producing a sufficient number of eggs that hatched, were used in the assay Cultures were grown on soft agar in 35 mm dishes One tested compound caused a malformation of eggs developing within the gonad of sel-12 adults Rather than seeing the normal linear array of well-formed eggs, these eggs were larger and lacked definition Two other tested compounds reduced the numbers of eggs within sel-12 adult worms and those eggs were larger than normal Three other tested compounds had no visible effect

While the present invention has been descπbed in terms of specific embodiments, it is understood that vaπations and modifications will occur to those skilled in the art Accordingly, only those limitations appeaπng in the appended claims should be placed upon the invention

Claims

What is claimed is

1 A method of identifying an egg laying deficiency in Caenoi habditis elegans comprising measuring the chitmase activity in the medium of a C elegans culture, said egg laying deficiency being identified as a decreased chitinase activity in said medium as compared to the chitmase activity in the medium of a culture of C elegans free of any egg laying deficiency

2 A method of screening for a compound that mitigates an egg laying deficiency in C elegans, compπsing a) providing a culture of C elegans comprising a non-wild- type allele of a gene associated with an egg laying deficiency, and b) monitoπng the chitmase activity m the medium of said culture in the absence and presence of a candidate compound, wherein a candidate compound that increases chitmase activity is identified as a compound that mitigates said egg laying defect

3. The method according to claim 2 wherein said gene is selected from the group consisting of genes in the presenilin pathway, egl-3, egl-5, egl- 26/cog-4, egl-38, egl-43, egl-44, egl-46, sem-1, sem-2, sem-4, rab-3, unc- 31, egl-22, unc-51, lιn-17, egl-20, egl-27, bar-1, egl-15, egl-17, unc-53, unc-40, egl-8, egl- 10, egl-30, ιnx-1, ιnx-2, da/-4, da/-8, daf-14, da/- 7, egl-4, egl-32, hn-12(gf), sel-12, let-60, hn-3, let-23, sem-5, hn-45, hn-39, lιn-1, lιn-31, lιn-2, lιn-7, lιn-10, lιn-11, lιn-15, lιn-19, lιn-25, lιn-26, lιn-36, unc- 86, sup-17, sup- 10, sup-9, unc-93, egl-2, egl-19, egl-23, egl-36, egl-1, and orthologs thereof

The method according to claim 2 wherein said gene is a presenilin gene

The method according to claim 4 wherein said gene is selected from the group consisting of sel-12, hop 1, and spe-4

The method according to claim 2, wherein said C elegans further comprises a non- wild-type hn- 12(d) allele

The method according to claim 2 wherein said gene is a human ortholog of a C elegans gene associated with an egg laying deficiency

The method according to claim 4 wherein said gene is selected from the group consisting of PS-1 and PS-2

A method of screening for a compound that modulates a mammalian disorder, compπsing

(a) identifying a compound that mitigates an egg laying defect m C elegans according to the method of claim 2,

(b) contacting a mammalian cell or cells exhibiting a trait characteπstic of a disorder selected from the group consisting of Alzheimer's disease, Ras-mducible cancer, and a potassium channel disorder with said compound, and

(c) monitoπng said cell or cells in the presence and absence of said compound, wherein a compound that reduces at least one trait of said disorder is identified as a compound that modulates said disorder

The method according to claim 9 wherein the cell of step (a) expresses APP, and wherein the monitoπng of step (b) comprises monitoπng APP processing in said cell

The method according to claim 9 wherein the cell ot step (a) is a human cell

The method according to claim 9 wherein said contacting step compπses adding said compound to a culture of a mammalian cell

The method according to claim 9 wherein said contacting step comprises administering said compound to a mammal, wherein said mammal compπses cells that exhibit a trait characteristic of a disorder selected from the group consisting of Alzheimer's disease, Ras-inducible cancer, and a potassium channel disorder

The method according to claim 9, further comprising a step of manufactuπng a composition comprising a compound identified according to step (c) as a compound that modulates said disorder, and a pharmaceutically acceptable earner

A method for modulating a mammalian disorder compπsing admmisteπng to a mammal an effective amount of a composition manufactured according to the method of claim 14

The method according to claim 15 wherein said mammal is a human

The compound identified as a modulator according to the method of claim 9

The compound according to claim 17 wherein said compound modulates presenilin activity

19 A pharmaceutical composition compπsing the compound according to claim 18 in a dosage effective in treating a mammalian disorder selected from the group consisting of Alzheimer^'s disease, Ras-inducible cancer and potassium transport disorders.

20. A method of identifying an inhibitor of C. elegans egg laying, comprising: a) providing a culture of C. elegans: b) measuring the chitinase activity in the medium of said culture in the absence and presence of a candidate compound; and c) identifying as an inhibitor, a candidate compound that causes a decrease in the chitinase activity in said medium when present as compared to the chitinase activity in said medium in the absence of said candidate compound.

21. The method according to claim 20, further comprising determining whether the inhibitor modulates β-amyloid plaque formation in a mammal.

22. The method according to claim 20, further comprising determining whether the inhibitor modulates APP processing in a mammalian cell that expresses APP.

23. The method according to claim 20, further comprising determining whether the inhibitor modulates the activity of a component of the presenilin pathway.

24. The method according to claim 20, further comprising determining whether the inhibitor modulates the activity of a component of the C. elegans Notch pathway. The method according to claim 24 herein said determining step comprises measuring the actn ity of a component selected from the group consisting of Glp-1 and Lin- 12

The method according to claim 20, wherein said providing step comprises a culture of a gain-of-function lιn-12 strain of C elegans

The method according to claim 26, further compπsing determining whether the inhibitor reverses the multivulva phenotype of said lιn-12 gam-of- function strain of C elegans

A method of screening for a candidate compound for the modulation of a disorder, compπsing

(a) monitoπng the chitmase activity of a C elegans culture m the presence and absence of a test compound, and

(b) identifying a test compound that decreases the chitmase activity of said C elegans as a candidate compound for the treatment of a disorder selected from the group consisting of Alzheimer's disease, Ras-inducible cancer, and potassium transport disorders

The method according to claim 28 further compπsing

(c) contacting a mammalian cell with said candidate compound for modulating a disorder,

(d) measuπng a trait associated with said disorder in said mammalian cell in the presence and absence of said candidate compound, and

(e) identifying a candidate compound as one that affects said trait in a manner inconsistent with maintenance or progression of said disorder

0. The method according to claim 29. further comprising the step of manufacturing a composition comprising the candidate compound identified according to step (e) in a pharmaceutically acceptable carrier.