WO2000034438A2

WO2000034438A2 - Method for constructing libraries of phenotypic profiles

Info

Publication number: WO2000034438A2
Application number: PCT/EP1999/009710
Authority: WO
Inventors: Titus Kaletta; Richard Feichtinger; Jonas Van Poucke; Anton Van Geel; Saskia Appelmans; Wim Van Criekinge; Thierry Bogaert
Original assignee: Devgen Nv
Priority date: 1998-12-07
Filing date: 1999-12-07
Publication date: 2000-06-15
Also published as: GB9826890D0; WO2000034438A3; JP2002531115A; EP1137754A2; AU1975000A

Abstract

Methods are provided for use in constructing libraries of phenotypic profiles in a nematode worm such as C. elegans. The methods require measurement of identifiable characteristics of the worm and systematic scoring of these characteristics. Also provided are methods of identifying compounds with potential pharmacological activity, for determining the mode of action of a given compound and for assigning genes to particular biochemical pathways.

Description

METHOD FOR CONSTRUCTING LIBRARIES OF PHENOTYPIC

PROFILES

The present invention is concerned with the field of ^λgenetic pharmacology' . Specifically, it relates to methods which can determine, among other things, whether a compound has potential pharmacological activity, whether a compound interacts with a particular gene or biochemical pathway in man or animals, what side effects are likely to be associated with a particular pharmaceutical compound and/or the mode or modes of action of any compound with biological activity. Additional uses for the methods of the invention include the assignment of function to particular genes or assignment of genes and their encoded proteins to particular biochemical pathways. In particular, the invention relates to the use of a microscopic nematode worm, for example Caenorhabdi tis elegans, and libraries of such worms in the aforementioned methods. These new methods are able to enhance and accelerate the drug discovery process. Prior to the early 1990 's the search for new compounds having the potential to combat human or animal disease was often begun by taking a compound known to have a particular pharmacological activity, synthesising structurally related variants and then testing those variants against the known target.

The test against the target might be carried out in vivo, for example by use of animal models of a human disease. Alternatively, if a particular molecule was known to be implicated in the progress of a disease, the compounds could be tested for interaction with the molecule in vi tro . The limitations of such methods are that in the event of a negative result no other information about the pharmaceutical potential of the compound tested is gained. For example, an in vi tro test might show a compound to have no inhibitory action against a particular target enzyme but that compound might have an inhibitory action against another enzyme in the same biochemical pathway as the target enzyme and therefore, in fact, have potential in treatment of the target disease. Animal tests, while providing a reasonable indication of both efficacy and toxicity, provide no information at all about the mode of action of the compound, and therefore the possible reasons for any toxicity. Furthermore, they are time- consuming and expensive and do not lend themselves to automation. Since the early nineties there have been two developments in particular which have revolutionized the drug discovery process, these being the new sciences of genomics' and ^combinatorial chemistry' . It has now been realised that a vast number of diseases have a genetic component and they are not purely the result of environmental influences. Indeed, it is possible that nearly all diseases are multifactorial and will have some degree of genetic basis, albeit very small in some cases. A huge amount of effort is being directed at the present time to the study of the organisation of the genomes of various unicellular and multicellular organisms, including humans. This involves the identification and sequencing of all the genes in a particular genome. Such activity does not only allow for hunting of genes which are directly associated with particular diseases but each of the genes found and the proteins they encode can become, directly or indirectly, a target against which compounds can be screened, whether or not that gene has yet been associated with a disease or indeed has any identified function at all. Furthermore, rather than starting from a compound of known ^activity' and relying on theoretical structure/function relationships to synthesise new candidate compounds, vast libraries of compounds, of uniform activity can be very rapidly synthesized in an automated manner by combinatorial chemistry. Thus, there is now potential to screen thousands of compounds against thousands of genes and the proteins they encode in very rapid high throughput screens (HTS) and to link compounds to genes and genes to disease .

The present inventors have discovered that these new technologies for drug discovery can conveniently be married with a particular multicellular organism, a nematode worm, C . elegans , which has been well characterised genetically and morphologically. They have thereby developed new methods, which are extremely powerful, rapid and convenient and can play an essential part in a drug discovery program. C. elegans is a microscopic nematode worm which occurs naturally in the soil but can be easily grown in the laboratory on nutrient agar inoculated with bacteria, preferably E . coli , on which it feeds. Each worm grows from an embryo to an adult worm of about 1 mm long in three days or so. As it is fully transparent at all stages of its life, cell divisions, migrations and differentiation can be seen in live animals. Furthermore, although its anatomy is simple its somatic cells represent most major differentiated tissue type including muscles, neurons, intestine and epidermis. Accordingly, differences in phenotype which represent a departure from that of a wild-type worm are relatively easily observed, either directly by microscopy or by using selective staining procedures, and many of these phenotypic differences submit to quantitative measurement. Many C. elegans mutants have been identified and their phenotypes described, for example, see C. elegans II Ed. Riddle, Blumenthal, Meyer and Priess, Cold Spring Harbor Laboratory Press, 1997. The C. elegans genome is now almost entirely sequenced as a result of the C. elegans genome project, carried out at the Sanger Center and Washington University School of Medicine. The sequence is available in a public database at http://www.sanger.ac.uk/projects/C_ elegans/. As a result of this it has emerged that C. elegans comprises genes which have equivalents that are widely distributed in most or all animals including humans.

Methods for creating mutant worms with mutations in selected C. elegans genes are known in the art, for example see J. Sutton and J. Hodgkin in ^ΛThe Nematode Caenorhabditis elegans' Ed. By William B. Wood and the Community of C. elegans Researchers CSHL, 1988 594- 595; Zwaal et al; Target-Selected Gene Inactivation in Caenorhabditis elegans by using a Frozen Transposon Insertion Mutant Bank' 1993, Proc. Natl. Acad. Sci. USA 90 pp 7431-7435; Fire et al, Potent and Specific Genetic Interference by Double-Stranded RNA in Caenorhabditis elegans 1998, Nature 391 860-811.

The possibility that C. elegans might be useful for establishing links between compounds and specific C. elegans genes by virtue of comparison of phenotypes generated by exposure to particular compounds and by selected mutations is considered by Rand and Johnson in Methods of Cell Biology, Chapter 8, vol 84, Caenorhabditis elegans: Modern Biological Analysis of an Organism Ed. Epstein and Shakes, Academic Press, 1995 and J. Ahringer in Curr. Op. in Gen. & Dev. 1_; 1997; 410-415.

However, these authors observe and attribute altered phenotypes on the basis of a single changed characteristic such as, for example, pharyngeal pumping rate or defecation frequency. Since that single characteristic may be determined by expression of a number of genes and the operation of several biochemical pathways such a crude assessment of phenotype is not sufficient to establish a link between any one gene or pathway and a compound to which the worm has been exposed. As such the procedure would not be sensitive enough for resolution of the properties of thousands of compounds in a high throughput compound screen. An additional problem with the proposals of the prior art is that known phenotypic characteristics have all been described differently by different workers in the C. elegans field. Phenotype descriptions in the literature largely omit aspects not directly related to or not recognised to be related to the principle interest of the individual researcher. There is no standard nomenclature to identify a specific change. Without this it is impossible to equate newly observed phenotypes with particular known phenotypes for comparison purposes.

The present inventors have developed methods which solve these problems and thereby have converted C. elegans into a really useful tool in the drug discovery field. Specifically, in respect of each worm a 'phenotype profile' or 'fingerprint' is established based on looking for plurality of changed characteristics in a particular mutant or worm which has been exposed to an environmental change or a compound. Furthermore, each profile is scored by following a strict standard protocol of measurement and a standard description is applied to each characteristic. The determination of a phenotypic profile in this way for a plurality of mutants or worms exposed to compounds illuminates differences between different mutants or otherwise treated worms which would not be apparent based on prior art methods. Furthermore, the standard scoring protocol and nomenclature allows the phenotypic profiles obtained to be collated into a library of reference profiles for direct comparison purposes. Thus, libraries of reference profiles can be established for mutant worms -and for worms exposed to particular environmental changes or different sorts of compounds. Such libraries allow complex patterns of linkage to be established between particular compounds and particular genes or biochemical pathways and between individual compounds of known or unknown biochemical or pharmacological activity.

In accordance with a first aspect of the present invention there is provided a method of constructing a library of phenotypic profiles of nematode worms which comprises the steps of:

(a) providing a worm having a defect in at last one gene.

(b) measuring any changes in identifiable characteristics of said worm compared to a worm without said defect,

(c) systematically scoring a plurality of any said changed characteristics to establish a characteristic phenotype profile associated with said defect,

(d) simultaneously or sequentially repeating steps (a) to (c) in respect of each of a plurality of worms each of which has a different defect, and

(e) collating the phenotypic profiles so obtained into a library of said profiles. Caenorhabdi tis elegans is the preferred nematode worm although the method could be carried out with other nematodes and in particular with other microscopic nematodes, preferably microscopic nematodes belonging to the genus Caenorhabditis . As used herein the term "microscopic" nematode encompasses nematodes of approximately the same size as C. elegans, being of the order 1mm long in the adult stage. Microscopic nematodes of this approximate size are extremely suited for use in mid- to high-throughput screening as they can easily be grown in the wells of a multi-well plate of the type generally used in the art to perform such screening. It is preferred to establish the phenotypic profile on the basis of the measurement and scoring of at least three different characteristics, preferably at least six characteristics and more preferably at least ten characteristics. It will be appreciated that the more differences which can be scored between a worm with a genetic defect and a worm without the defect the better the resolution between different mutants. Although not limited to such, at least one of the plurality of changed characteristics which can be measured and scored may be selected from the list shown in Table 1, and possibly each of all the changed characteristics scored is one of those shown in Table 1.

In a preferred embodiment, the method used to establish the phenotypic profile comprises measurement and scoring of two or more characteristics selected from the group consisting of: viability, life cycle, body shape, movement behaviour, mechanotransduction, pharynx pumping, defecation and fertility. This list provides a core set of measurable characteristics which can be used to establish an informative phenotypic profile for any type of worm. Furthermore, each of these characteristics is measurable using technical measuring apparatus, such as video image analysis, multiwell plate reader, and/or a technical assay procedure. In the most preferred embodiment, the method used to establish the phenotypic profile comprises measurement and scoring of all eight of the listed core characteristics. Measuring and scoring this set of core characteristics allows meaningful comparisons to be made between phenotypic profiles for worms subjected to diverse interventions. AS exemplified herein, comparisons can be drawn between profiles for two different mutant worms and between profiles for mutant worms and profiles for worms exposed to compound. It is to be understood the terms "measuring" or "measurement" as used in connection with any of the methods described and claimed herein are to be interpreted as including not just absolute quantitative measurement wherein a numerical value is assigned to the characteristic but also comparative measurement, wherein characteristics of a worm which has been subject to an intervention (i.e. mutation, exposure to compound, exposure to environmental change) are measured relative to the same characteristics of a wild-type worm and scored as being 'larger' , 'smaller' , 'longer' , 'shorter' , 'fatter', 'thinner', 'darker', 'paler' etc.

For comparison purposes it is essential that the scored characteristics are represented in the same order for each profile. For standardization of procedure between different workers or to facilitate automation, measurement and scoring of the characteristics could be carried out in a predetermined order according to a standard protocol. However, this is not essential to the operation of the method. In its simplest form and as shown in Example 5, the characteristics are recorded in a binary manner as 'present' or 'not present' based on deviations from wild-type worms.

It is desirable to establish a library which comprises a phenotypic profile in respect of a defect in each gene in the worm genome and/or different defects in the same gene (allelic variations). As aforesaid there are a considerable number of available mutants (see Riddle, Blumenthal, Meyer and Priess and Ahringer above) . In addition new ones can be generated by specific gene and site directed mutation and knockout methods known to those skilled in the art such as ethyl methanesulphonate (EMS) mutagenesis, transposon insertion or genetic interference using double stranded RNA (see Sutton and Hodgkin, Zwaal et al and Fire et al above) . The known or newly generated genetic defects may manifest themselves, for example, as the absence of expression of a gene, the reduction in expression of a gene, the over-expression of a gene, the expression of a functionally defective protein, the mis-expression of a protein, the ectopic mis-expression of a protein, the expression of a protein of altered stability or the alteration of gene expression as a function of time.

Generally, the manipulation of C. elegans to generate genetic defects can be carried out on wild- type worms or worms with existing single or multiple mutations. It may be desirable to genetically manipulate C. elegans carrying a reporter gene construct. The reporter molecule might be LacZ or green fluorescent protein but many other reporter molecules are known to those skilled in the art. Reporter gene constructs for C. elegans are described in Chalfie et al, 1994, Science 263 pp 802-805. It can also be desirable to genetically manipulate and then profile a transgenic worm, preferably a worm carrying a human gene, particularly where the gene is associated with, or is a candidate for association with a human disease and therefore a putative drug target. A list of human diseases for which a particular gene has been implicated is given in the paper by J. Ahringer (see above) and also provided by OMIM. Center for Medical Genetics, John Hopkins University and National Biotechnology Information, National Library of Medicine, 1996. http//www. ncbi .nlm.nih. gov/omim/, although these lists are not necessarily exhaustive.

It is easy to establish transgenic lines in C. elegans and the methodology is described in Craig Mello and Andrew Fire, Methods in Cell Biology, Vol 48 Ed. H.F. Epsein and D.C. Shakes, Academic Press, pages 452-480.

A form of the worm which may show a change in phenotype and may therefore be subject to profiling as described above is one in which the genetic defect and/or transgene and/or reporter gene is only present in a sub-set of the cells of the worm. It is possible for just the cells of a particular tissue to be the subject of a genetic manipulation.

The worm which is to be subject to determination of its phenotypic profile can be cultured by methods well-known in the art. C. elegans can grow on nutrient agar which has first been inoculated with bacteria on which the worms feed. Suitable culture methods are described in Rand and Johnson (see above) and in the examples given herein. Measurement of any changed characteristics which will determine the profile may be carried out using light microscopy, differential interference contrast optics or fluorescence microscopy. In addition immuno-chemical detection, colorimetric detection or detection of fluorescence, luminescence or radioactive labels may be used. In some cases the changed characteristics may be biochemical only and might be detected, for example by a pH change in the growth media or a change in electrical potential. Different characteristics may need to be determined at different points in the growth cycle of the worm. For example, some phenotypic characteristics may be manifested only in the larvae while others are only detectable in the adult worm. In some cases it may be necessary to make several measurements of the same characteristic at predetermined time intervals.

Phenotypic profiles generated by the methods described above can be collated into a library of profiles which are stored electronically on a database. However, it will be appreciated that the invention also provides a method of constructing a physical library or bank or worms each identifiable by their individual phenotypic profile. Such a worm library can be created using any or all of the methods described above and used for comparative purposes. The worms may be maintained by the culture methods described herein and/or frozen for long term storage by methods known to those skilled in the art. Libraries of phenotypic profiles or fingerprints of mutant worms or mutant worm libraries can be used to determine linkages between different genes and hence identify biochemical pathways. A particularly important use is the profiling of several mutations of the same gene and several genes of the same pathway. Different mutations in the same gene can have different phenotypes and often it is found that a careful analysis of the allelic series of a gene reveals important information that is hidden under a more severe phenotype of a null mutant (complete knock out, e.g. if it is lethal) . Phenotypic profiles of different mutations of the same gene allow characterisation of the gene by simply combining (logical OR) the profiles of all the mutations, whether they have been generated at the same time or not. It is possible, however, to handle the mutations separately and make more detailed connections, for example, concerning protein domains in case the similarity of phenotypes cluster with the sites of the mutations .

Described above are methods for constructing a library of phenotypic profiles for worms with a plurality of genetic defects or a library of mutant worms. However, in accordance with a second aspect the present invention provides a method of constructing a library of phenotypic profiles of nematode worms which comprises the steps of:

(a) exposing a worm to a compound,

(b) measuring any changes in identifiable characteristics of said worm as a result of exposure to said compound,

(c) systematically scoring a plurality of any said changed characteristics to establish a phenotypic profile associated with said compound,

(d) simultaneously or sequentially repeating steps (a) to (c) in respect of each of a plurality of different compounds, and

(e) collating the phenotypic profiles so obtained into a library of said profiles.

Methods for culturing C. elegans in the presence of a test compound are described by Rand and Johnson mentioned above and in the examples herein. In its simplest form a solution of the compound in a suitable solvent may be spread over a bacterial lawn on an agar plate before inoculation with the worm. Additional refinements include feeding the worm with bacteria, preferably E. coli, which have taken up the compound or attaching .the compound to a carrier compound which is particularly attractive to the worm.

The worms which are exposed to the compound may be wild-type worms, mutant worms, transgenic worms and/or worms carrying reporter gene constructs as already described herein. Further the measurement and scoring of a plurality of changed characteristics is carried out by exactly the same procedures as already described herein for the phenotypic profiling of mutant worms. This must be a standard format in order that direct comparisons can be made between profiles obtained on exposure to compounds and profiles exhibited by mutants. With compound screening it is possible to build up a series of different libraries depending on the compounds being tested. For example one library can comprise profiles generated in respect of each of the known compounds in a Pharmacopoeia, in other words compounds with known pharmacological activity.

Another library can comprise profiles generated by compounds known to interact with a particular biochemical pathway, which may or may overlap with those compounds from the Pharmacopoeia. Other libraries could include profiles for known compounds but with no known biological activity or compounds which are completely new molecules such as might be generated by combinatorial chemistry. As aforesaid the present invention is not limited to the production of phenotypic profile libraries but includes libraries or banks of worms whose phenotypic profile has been altered by exposure to compounds. In particular embodiments assays may be carried out with several concentrations of the same compound, and/or with mixtures of compounds . For example compounds from compound libraries may each be tested individually or with one or more other influencing compounds.

Furthermore, such compound testing protocols may be executed against identical worms or multiple mutant and/or transgenic backgrounds. In a particular example a panel of worm strains, covering a wide range of biochemical pathways and cellular activities by means of mutations in particular pathways, as well as reporter genes, is used for testing compounds. For each compound, potentially at several concentrations, a profile is recorded for the measurable phenotypes of each of the worm strains, either in parallel or sequentially.

In a third of its aspects the invention provides a method of constructing a library of phenotypic profiles of nematode worms which comprises the steps of:

(a) exposing a worm to an environmental change,

(b) measuring any changes in identifiable characteristics as a result of said environmental change,

(c) systematically scoring a plurality of any said changed characteristics to establish a characteristic phenotypic profile associated with said change,

(d) simultaneously or sequentially repeating steps (a) to (c) for each of a plurality of different environmental changes, and (e) collating the phenotype profiles so obtained into a library of said profiles.

The environmental change may be, for example, a change in pH, osmolarity, temperature, exposure to radiation or exposure to bacteria or viruses. Each of these external influences may result in the manifestation of a different phenotypic profile of characteristics so that libraries of said profiles and affected worms can be constructed. Again, measurements and scoring of the profile should follow a standard protocol in order that valid comparisons can be made between these profiles and those in mutant and compound libraries. The construction of worm and phenotypic profile libraries by the methods described above using the novel phenotypic profiling method described herein provides a very powerful tool for the discovery of new drugs. Profiles in each of the different libraries can be compared and links established between C. elegans genes and pathways, compounds and environmental effects. Preferably, the process of measuring and scoring the changed characteristics which go to make up the phenotypic profile is automated, making use of technical measuring apparatus. The profiles so generated may advantageously be stored electronically. Libraries of profiles can then be searched by computer which can identify identical or similar profiles, either within or between the different libraries. Quantitative data calculations, optionally in combination with boolean operations can be used. A comparison of the profile generated by a particular compound with the profiles of particular mutants may indicate the likely gene or biochemical pathway with which the compound interacts in the worm. Other databases can then be searched for a match of the worm gene with an equivalent human gene. The human gene might already be associated with a human disease as could be determined for example, from the OMIM database mentioned above. Thus, by use of the worm screen a potential candidate drug can be identified.

The discovery of the mode of action of a compound with known pharmacological or biochemical activity is facilitated by comparing its phenotypic profile in the worm with the mutant library or environmental change library of profiles to identify possible targets for the compound, other possibilities include finding a new potential medical indication of a known compound, a medical indication for a novel compound, an alternative method of treatment of a known disease or an indication of the reason for the side effect exhibited by some known pharmaceuticals. Testing worms with compounds, scoring the phenotypic profile in the novel manner described herein and then searching previously established libraries of profiles can potentially achieve all those goals. Once a compound has been identified as having the potential to be a therapeutic agent it can be processed through the more traditional drug discovery routes. The compound can be tested in more specific in vitro tests based on the new knowledge of the target for the compound and in animal models of the target disease. Structural variants then can be generated by medicinal chemistry with a view to improving activity.

The invention will now be described with reference to the following Examples, together with accompanying Figures, in which:

Figure 1 is a schematic diagram of the left lateral view of the body of C. elegans . The body of C. elegans is divided into a head, a body and a tail region. The head region stops at the end of the pharynx, the body stops at the rectum and the tail includes the tail whipe. C. elegans usually crawl on the right side. The ventral located vulva defines the ventral side of C. elegans . Figure 2 is a schematic diagram of C. elegans showing the characteristics "hypertrophy of the head and "extensions on head".

Example 1 General Profiling by Plate Drop Assay

4ml NGM agar (see 'The Nematode Caenorhabditis Elegans' Ed. by William B. Wood and the Community of C. elegans Researchers CSHL, 1988, pg 589) is poured into 3cm plate, and seeded with approximately 5μl of an E. coli overnight culture and grown preferably for one week at room temperature. If a compound is to be profiled lOμl of compound dissolved in DMSO or other appropriate solution is pipetted onto the bacterial lawn. The lawn should be covered completely. (This step can be omitted if a mutant, transgenic or other worm is being profiled without compound) . After overnight soaking in of compound one C. elegans (L4 stage) per plate is put in the bacterial lawn. Worms are checked after some hours, plates are incubated at 21°C and worms screened for phenotypes (control have LI progeny growing) . Plates are checked again after 4 days for phenotypes of FI progeny (control shows all stages up to gravid hermaphrodites) . Plates which have to be looked at again on subsequent days because of slow growth or for further checks are put aside. A plate protocol sheet such as that shown in Table 2 is completed deciding on one of the following routes: no effect/unspecific effect/needs to be applied at lower concentrations/needs to be profiled. If concentrations are appropriate and a decision can be made scoring of characteristics to produce a profile can be started using the profiling list in Table 1. Because the compound is pipetted onto a bacterial lawn rather than it being incorporated into the agar, as has been done in the prior art, this method is designated a 'plate drop assay' .

Table 1

1. Compound specific phenotypes

3. Life cycle

5. Movement

6. Mechanotransduction (Touch with a wire and with eyelash)

7. Sensory system

8. Environmental response

9. Pharynx

11. Rectum

Table 2

bacteria normal lawn happy grown as ring run away thin irregular movement crust slow movement died no movement

worm gone replaced by lost number and stage suicide in agar left progeny starved outside

died in compound movement body progeny normal normal gravid adult normal tracks more outside pumping defects reduced broodsize tracks not in center light brown messy gonad amplitude increased loopy pale with dark spots younger staged amplitude variable few eggs in gonad amplitude decreased pharynx stuffed oocytes enhanced movement foregut filled large coagulated eggs slow movement hindgut constipated dead eggs no movement protruding vulva dying hatchlings specific other crippled larvae

growth rate normal reduced broodsize younger staged

movement body brood viability normal normal gravid adult dead eggs population more outside pumping defects population not in center light brown messy gonad dead larvae amplitude increase, loopy pale with dark spots amplitude vaπable few eggs in gonad larval arrest amplitude decreased pharynx stuffed later sconng enhanced movement foregut filled large day of screen slow movement hindgut constipated no movement protruding vulva day of worm specific other compaπson of phenotypes progeny shows PC phenotype new worms show phenotype stage & age similar similar all stages worse worse young only a few only not all late larvae and adults weaker weaker adults only no effect not effect old adults compaπson to other plates compaπson to known drugs compaπson to known mutants Example 2

Profiling of a compound library (new compounds)

To profile new compounds from a library, the general profiling protocol is followed with the variations. Compounds are profiled once in undiluted concentration, the actual concentration being dependent on the compound library in question but will be between 0.01 mg and 1 mg of compound/lOμl D SO.

For compounds with a MW of 500 this calculates to 2- 200 mM stock. Dilution in 4ml agar would be at 5-500 μM. The high dose may create lots of unspecific effect problems e.g. bacterial death and worm starvation. Thus, if necessary the compounds are applied in a second round at lower concentrations which are dilutions in DMSO of 1/3, 1/10 and 1/30 of the undiluted concentration. A concentration is finally chosen for each compound which will allow a phenotype profile to be established according to the standard procedure.

Example 3

Profiling of known compounds (biotools , pharmacopoeia)

To profile known compounds from a library the general profiling protocol is followed with the following variations. The stock solution is preferred as lOOmM in DMSO and the experiment is started ab ini tio with a concentration series. The concentration series is used as described below. In one series of concentrations 15 or so worms (for a reasonable number of short term effects) are placed in the agar. In three series 1 worm each is placed on the agar to score a reasonable number of progeny. Lost worms of the latter three series of concentrations can be replaced from the large pool where worms have been exposed to the compound in the same way. The following concentrations can be used:

Example 4

Comparison of agar assay to drop assay

A set of compounds from the pharmacopoeia have been profiled using the general protocol (all compounds were of known activity and are described in Martindale: The Complete Drug Reference, 32nd edition, Pharmaceutical Press 1999) . The plate drop assay was compared against standard of pouring compounds into the agar as described in literature which method is designated agar assay. In the drop assay as well as in the agar assay, the compounds were added to the worm in a variety of concentrations, and the survival of the worm was scored as well as the phenotypic profile induced by the compound. The lowest concentration of a compound, still resulting in the death of the nematode was designated minimal lethal dose. The maximal concentration of a compound that did not result in the death of the nematode was designated maximal nonlethal dose. The minimal concentration of a compound that still resulted in a measurable phenotype was designated minimal effective dose. The concentrations of the compounds in the agar assay were compared to the concentrations in the drop assay. From this observation one may conclude that the newly described drop assay protocol turns out to be far more efficient for most compounds. The following table lists the calculated concentration ratio needed to get the same effect with the compound in the agar assay (in 2 ml agar) rather than the drop assay (in 4 ml agar) .

Table 3:

Minimal lethal dose: rate between the lowest concentration in which the compound is lethal to the worm in both assays Maximal non-lethal dose: rate between the highest concentration in which the compound is not lethal in both assays Minimal effective dose: rate between the lowest concentration in which the compounds results m a phenotype in both assays

Average: average of the rates Example 5

Preferred set of informative characteristics

Worms exposed to a compound, carrying a mutation or are transgenic are examined for the following 8 informative features/phenotypes :

1. Viability

Worms are examined for viability at all stages of the life cycle, being embryogenesis, larval stages 1 to 4 and adulthood. Dead embryos are defined by not hatching within 24h and dead worms are defined by not moving, by lack of pharynx pumping, by sick or pale appearance and by lack of response to mechanical stimulation.

Method:

Embryonic lethality is measured by counting the amount of unhatched worms after 24 hours (Elispot, Zeiss) . Counting of unhatched worms could also be automated using the FANS device, described below. Viability of larvae and adults is measured by dye uptake.

2. Life cycle Progeny are examined for the length of the generation cycle in comparison to control progeny (of a wild-type worm) . The stage of a synchronized progeny will be compared to the stage of a synchronized control progeny (N2, Bristol strain) after three days at 20°C. The developmental stages can be distinguished by vulva development, expression of stage-specific markers, such as collagen IV, body length and transparency.

Method: Measuring the body length of a population allows determination of the actual stage in the life cycle (For body shape measurement, see 3. Body shape). Expression of stage-specific markers can be examined using antibodies of the appropriate specificity, by way of example an antibody that recognizes an antigen on the surface of C. elegans LI larvae has been described by He mer et al . , (1991) J Cell Biol , 115(5) : 1237-47.

3. Body shape Worm size is determined by measuring worm length and worm diameter.

Method:

The body length of a synchronized progeny of adult worms is compared to the body length of a synchronized control progeny (N2, Bristol strain) . Measurement of body length can be achieved using a 'worm dispenser apparatus' which is commercially available from Union Biometrica, Inc, Somerville, MA, USA. This apparatus has properties analogous to flow cytometers, such as fluorescence activated cell scanning and sorting devices (FACS) . Accordingly, it may be commonly referred to as a "FANS" apparatus, for fluorescence activated nematode scanning and sorting device (FANS) . The FANS device enables the measurement of properties of microscopic nematodes, such as size, optical density, fluorescence, and luminescence.

Body size may also be measured via image analysis, in which case the measurements recorded may include worm diameter and deviation from the typical tube shape of a wild-type worm.

4. Movement behaviour The measurement of movement behaviour can include measurement of the speed of movement , or of the pattern of movement (e.g. direction) or both. A wild-type worm moves in a sinusoidal way forward and pauses or moves backward occasionally. Any deviation from this wild-type pattern of movement can be scored as a 'changed' characteristic.

Method:

An assay based on the following principles may be used to determine the speed of movement of a worm culture:

Nematode worms that are placed in liquid culture will move in such a way that they maintain a more or less even (or homogeneous) distribution throughout the culture. Nematode worms that are defective in movement will precipitate to the bottom in liquid culture. Due to this characteristic of nematode worms as result of their movement phenotype, it is possible to monitor and detect the difference between nematode worms that move and nematodes that do not move.

Advanced multi-well plate readers are able to detect sub-regions of the wells of multi-well plates. By using these plate readers it is possible to take measurements in selected areas of the surface of the wells of the multi-well plates. If the area of measurement is centralized, so that only the middle of the well is measured, a difference in nematode autofluorescence (fluorescence which occurs in the absence of any external marker molecule) can be observed in the wells containing nematodes that move normally as compared to wells containing nematodes that are defective for movement. For the wells containing the nematodes that move normally, a low level of autofluorescence will be observed, whilst a high level of autofluorescence can be observed in the wells that contain the nematodes that are defective in movement . In an adaptation of the movement assay, autofluorescence measurements can be taken in two areas of the surface of the well, one measurement in the centre of the well, and on measurement on the edge of the well. Comparing the two measurements gives analogous results as in the case if only the centre of the well is measured but the additional measurement of the edge of the well results in an extra control and somewhat more distinct results.

As an alternative to the above-described movement assay, specialist software such as SIMI Scout (designed for movement study of an athlete) may be used to determine speed of movement, deviation from sinusoidal movement and even the overall pattern of movement of the worm.

5. Mechanotransduction

Worms are examined for response to mechanical stimulation.

Method:

When the plate on which C. elegans are cultured is dropped wild-type worms react by enhanced movement and enhanced overall activity. The capability of a worm to respond to a mechanical stimulus is measured by the difference in speed of movement before and after stimulation.

6. Pharynx pumping

The phenotypes "Pumping frequency reduced, Pharynx pumping irregular" etc. describe the activity of the cyclic contraction of the pharynx muscles that occurs in a feeding adult about 3 times in a second. The contraction cycle can be descried as the nearly simultaneously contraction of the corpus, anterior isthmus, and terminal bulb, followed by relaxation.

Method:

The following pharynx pumping characteristics may be analyzed by image analysis: The frequency of pumping by counting the pharynx contraction. Pharynx contraction can be measured visibly by the opening and closing of the anterior corpus. The time of opened anterior corpus and the diameter of the opened corpus is used to measure hypercontraction, relaxation and strength of a contraction.

The following is an example of a pumping assay which allows measurement of the total efficiency of feeding of a worm, which is related to pumping:

The pumping rate of the pharynx is measured indirectly by adding a marker molecule precursor such as calcein- AM to the medium and measuring the formation of marker dye in the C. elegans gut. Calcein-AM is cleaved by esterases present in the C. elegans gut to release calcein, which is a fluorescent molecule. The pumping rate of the pharynx will determine how much medium will enter the gut of the worm, and hence how much calcein-AM will enter the gut of the worm. Therefore by measuring the accumulation of calcein in the nematode gut, detectable by fluorescence, it is possible to determine the pumping rate of the pharynx.

To perform the pharynx pumping screen with calcein-AM, a concentration of between 1 and lOOμM calcein-AM is added into the medium. Preferably 5 to lOμM calcein- AM is used. Fluorescence is measured using a multi- well plate reader (Victor2, Wallac Oy, Finland) with following settings: Ex/Em = 485/530. 7. Defeca ion

The defecation of C. elegans is a recurrent event comprising of the following steps: pBoc, aBoc and expulsion. Defecation in nematodes such as C. elegans is achieved by periodically activating a defined sequence of muscle contractions. These contractions are started in the anterior body wall muscles. At the zenith of the anterior body contractions the four anal muscles also contract. The four anal or enteric muscles are the two intestinal muscles, the anal depressor and the anal sphincter. In addition to this series of muscle contractions, specific neurons are also involved in the regulation of defecation, including the motor neurons, AVL and DVB.

Method:

In order to construct a phenotypic profile, well-fed adults are typically examined after one day for constipation. The time between two pBocs is also scored.

The rate of defecation of C. elegans can also be quantitatively measured using an assay based on the following principles:

The rate of defecation of nematodes such as C. elegans can be easily measured using a marker molecule which is sensitive to pH, for example the fluorescent marker BCECF. This marker molecule can be loaded into the C. elegans gut in the form of the precursor BCECF-AM which itself is not fluorescent. If BCECF-AM is added to nematode culture medium in the wells of a multi- well plate the worms will take up the compound which is then cleaved by the esterases present in the C. elegans gut to release BCECF. BCECF fluorescence is sensitive to pH and under the relatively low pH conditions in the gut of C. elegans (pH<6) the compound exhibits no or very low fluorescence. As a result of the defecation process the BCECF is expelled into the medium which has a higher pH than the C. elegans gut and the BCECF is therefore fluorescent. The level of BCECF fluorescence in the medium (measured using a multi-well plate reader on settings Ex/Em=485/550) is therefore an indicator of the rate of defecation of the nematodes.

8. Fertility

A wild-type adult hermaphrodite C. elegans lays about 8 eggs per hour.

Method:

The amount of eggs laid by 20 hermaphrodite C. elegans during at least 60 min is counted. The amount of eggs may be counted by simple visual inspection or using a FANS device, described above.

Example 6

Comparison of profiles within a library (daf-4 belongs to two pathways)

Mutant worms have been profiled according to the general profile protocol. Table 4 shows a summary of the profile, also called fingerprints, of one mutation of the indicated genes. Entries are binary with empty fields indicating a phenotype (deviation from negative control, here wild-type) not found assuming that it could have been measured. Any other entry including comments or quantitative data is read as measured phenotype in this binary scheme and indicated by *. The table lists only phenotypes that do have a positive entry, not necessarily complete, leaving pages of empty fields alongside and arranged according to a particular enquiry. The upper half consists of the hierarchical categories "dauer formation phenotypes" and "body shape phenotypes" as well as their relevant sub-phenotypes. The lower part consists of a set of hierarchically unrelated phenotypes subsumed under the enquiry categories, "increased activity" and "decreased activity". The complete list of characteristics is to be found in Table 1.

The point of including the lower part is to show the principle of recording all observed phenotypes, that they can be used to distinguish similar phenotypic profiles in detail and that they can be arranged in order to make comparisons. In this case it is seen that the dichotomy of long versus short body length does not correlate to the dichotomy of increased versus decreased activity.

The upper part shows 5 genes (i.e. a mutation in that gene) affecting dauer formation as well as 5 genes affecting body shape in a particular combination. A mutation in one gene, daf-4, is unique in sharing the characteristics of both phenotypic groups. The following picture illustrates the phenotypic overlap as found by comparing entries in the phenotypic profiles.

From this overlap a hypothesis of a mechanistic link can be put forward for daf-4. In this particular case the mechanistic link is confirmed by the molecular nature of the genes, which as far as known are all members of the TGFβ pathway by sequence similarity:

dbl-1 TGFβ like ligand daf-7 TGFβ like ligand sma-6 type I receptor daf-1 type I receptor daf-4 type II receptor daf-4 type II receptor sma-2 SMAD daf-3 SMAD sma-3 SMAD daf-14 SMAD sma-4 SMAD

The DAF-4 protein probably acts as a type II receptor in both pathways. The similarity of phenotypic profiles allows one to hypothesize mechanistic relationships in a manner analogous to sequence similarity of genes. For example a compound which induces the phenotypes: longer or shorter body length in combination with 2 or 3 of pale, thin and variable egg size, in worms exposed to it, is very likely to act on a protein of the TGFβ pathway.

Table 4:

Example 7 Comparison of phenotypes induced by acetylcholine esterase inhibitors

Wild type C. elegans adults have been exposed to acetylcholine esterase inhibitors at various concentrations. The worms have been profiled over two generations, meaning four profiles have been generated. All phenotypes from the phenotype list are displayed that have been measured in this experiment. Two phenotypes "loopy head movement" and "body dragged by head" are shared by most of the esterase inhibitors. This is called phenotype activity relationship (PAR, by analogy to structure activity relationship SAR) . The shared phenotypes are used to identify the action of a new compound. The unshared phenotypes are used to distinguish drugs or unravel side effects when these phenotypes are part of another PAR.

Table 5:

Example 8 Comparison of phenotypes of mutations in the acetylcholine neurotransmission pathway

C. elegans adults and larval stages that are homozygous for the mutations cha -1 , unc-1 7, snt-1 and cat-1 have been profiled, meaning fingerprints have been generated. All phenotypes from the phenotype list are displayed that have been scored in this experiment. The phenotypes "small", "resistance to CHA inhibitors (Ric)", "slow pumping" and "slow growth" are shared. This is called phenotype activity relationship (PAR, in analogy to structure activity relationship SAR) . The shared phenotypes are used to identify genes in a pathway. The unshared phenotypes are used to distinguish these genes or unravel further functions in parallel or new pathways when these phenotypes are part of another PAR. The fingerprint of ca t-1 is different because this gene is involved in the dopamine pathway.

Table 6:

Example 9

Method to profile an intervention (mutation, compound etc)

Profiling a mutation in the gene unc-1 7 that affects transportation of acetylcholine.

In the literature this phenotype is described, concerning movement, body size and feeding, as severe coiler, being rather small and thin and has only slow, irregular pumping of the pharynx (Riddle et al., "C. elegans II" Cold Spring Harbor Laboratory Press, 1997). By systematically describing unc-1 7 the resulting fingerprint unravels more details and new properties: Concerning movement, body size and feeding the phenotypes strong coiler, spiralling inwards posteriorly, curly jerky and moves better forward, being small have been profiled. In addition defects in the sensory system, defecation and reproductive system have been found, in detail: the touch response is gone, constipation, aberrant defecation cycle (aBoc) and egg laying defective (no egg retention) .

Example 10

Method to add biological information to a particular phenotype

One phenotype of the mutation unc-4 is "coiler" (looks like a snail) . The fingerprint of unc-4 adds for "coiler" the details "ventral side out" and "spiralling inwards posteriorly". This occurs when a set of neurons that control the forward movement of the ventral part of the worm (VA2 - VA10) gets the same input than another set of neurons that controls the backward movement of the ventral part (VB2 - VB10 ) .

In this case the ventral muscles get contradicting signals and only the dorsal muscles contract properly. The result is a coiler that has only the ventral side outwards. We explain most of the phenotypes as consequence of a mislead process, here synaptic input.

Example 11

Comparison of phenotypes induced by compounds acting on GABAnergic neurotransmission

Wild-type C. elegans adults have been exposed to GABA agonists (Muscimol) and GABA antagonists (Ivermectin and Fipronil) at various concentrations. Worms have been profiled and the scored phenotypes are displayed as fingerprints.

In addition, two mutations in the GABAnergic pathway have been profiled and compared with the compound induced phenotypes: unc-25 encodes for the decarboxylase and unc-49 encodes for a GABA receptor.

The phenotype "shrinker" is present in all fingerprints (see Table dark grey) . This phenotype is used as marker or diagnostic phenotype to identify activity of a compound or gene in the GABAnergic pathway. There are further phenotypes only shared by some compounds and mutants (see Table light grey) . These phenotypes are used to build a phenotype activity relationship (PAR) .

The shared phenotypes are used to identify the action of a new compound when "shrinker" cannot be used or to reveal more details on a compound action. For example, all compounds and unc-25 fingerprints contain constipation phenotypes but not the fingerprint of unc-49, although GABA is used for the defecation process. This is coincident with earlier findings that the UNC-49 gene product is not required for defecation.

These results may indicate the existence of another yet unknown GABA receptor in C. elegans . The unshared phenotypes are used to unravel toxic side effects or other mode of actions.

Table 7:

Example 12

Definition of body shape phenotypes

Aberrations of the body shape of C. elegans can be the result of mutations in a vast amount of genes. These genes may be required directly for the formation of the hypodermis, the hydroskeleton and the correct patterning of the worm body plan, e.g., collagen or even-skipped. They could be involved in the control of growth or metabolism like genes of the TGF β pathway or genes required for feeding. Eventually, mutations in certain genes that cause primary defects, e.g., absence of head muscle, cause secondary defects in the body shape like dystrophy in the head region. Body shape phenotypes are all visible or measurable deviations of the body shape, colour and content. Phenotypes are comparatively measured against wild- type (N2, Bristol strain) and scored as deviation of wild type in the corresponding developmental stage, sex and preparation. The scored phenotype comes with the percentage of worms positive for that phenotype within a population.

Table 8: Scientific definition of body shape phenotypes. The phenotypes listed in the left column are described and defined in the right column. Some phenotypes are derived from the classical worm jargon like "dumpy", which is still shorter than "short and thick worm".

Head defects

Body defects

It is possible to score body shape phenotypes by image acquisition followed by image analysis. The advantage in the automation of the profiling procedure is the quantification of the strength of a phenotype or the presence of the phenotype in a population. A disadvantage is that the procedure for analysing an image for every possible phenotype may be more elaborate than simply scoring by eye. Furthermore, certain details are difficult to access by video analysis e.g., blister versus protrusions.

Table 10: list of scientific body shape phenotypes, together with their corresponding technical definitions , in terms of characteristics which can be comparatively measured relative to wild-type characteristics using automated measuring apparatus.

Head defects

Example 13

Use of GFP in profiling C. elegans

A lot of features of C. elegans as described in Table 1 can be easily monitored, either automatically by image analysis, microtiter plate readers, or visual means, e.g. by normal microscopy or by Nomarski microscopy. Some features of C. elegans are more difficult to visualize. For these characteristics transgenic animals expressing a marker gene are very useful. Moreover, even for characteristics that are rather easily to score, the use of a nematode expressing a marker gene, such as GFP, LacZ, or luciferase, enhances the fingerprinting of C. elegans . The C. elegans can be a wild type, a mutant, or a strain subjected to a compound or environmental stress, or a combination of those.

C. elegans mutant unc-23 has a fingerprint, which comprises "jerky movement", "tend to coil", "bent head" and "egl". Expressing GFP in the muscle cells of the animal could result in identification and scoring of additional characteristics such as "improperly folded muscles", and/or "detached muscles in head region", and/or "no muscles in head region", and/or "defective muscle attachment", and/or "vulva muscle defects" (data not shown) .

Similarly, C. elegans mutant unc- 71 has a fingerprint which comprise "reduced movement", "weak amplitude", "strong kinker", and "slightly egl". When introducing GFP in the neurons of the animals no apparent extra fingerprint features where observed. A closer look at the neurons of this mutant worm revealed at least following extra phenotypes: "fasculation defects", "VD/DC connection defects" (data not shown) .

GFP-phenotypes are hence very important in allowing phenotypes which are not otherwise visible to be measurable with Nomarski or dissection microscopy. GFP-phenotypes are further important in the pinpointing of defects to certain tissues and cells, and moreover GFP-phenotypes are important in distinguishing between similar defects with different causes .

Claims

Claims :

1. A method of constructing a library of phenotypic profiles of nematode worms which comprises the steps of:

(a) providing a worm having a defect in at least one gene,

(c) systematically scoring a plurality of any said changed characteristics to establish a characteristic phenotypic profile associated with said defect,

(d) simultaneously or sequentially repeating steps (a) to (c) in respect of each of a plurality of worms each of which has a different: defect, and

2. A method as claimed in claim 1 wherein in step (c) at least three changed characteristics are scored.

3. A method as claimed in claim 1 or claim 2 wherein in step (c) at least six changed characteristics are scored.

. A method as claimed in any preceding claim wherein in step (c) at least ten characteristics are scored.

5. A method as claimed in any preceding claim wherein said worm is Caenorhabditis elegans.

6. A method as claimed in any preceding claim wherein steps (a) to (c) are carried out in respect of substantially every gene in the worm genome.

7. A method as claimed in any preceding claim which includes the step of manipulating said worm to generate said defect in said at least one gene.

8. A method as claimed in any preceding claim wherein said defect is selected from the absence of expression of said gene, the reduction in expression of said gene, the over-expression of said gene, the expression of a functionally defective protein, the expression of a truncated protein, the misexpression of a protein, the ectopic misexpression of a protein, the expression of a protein of altered stability or the alteration of gene expression as a function of time.

9. A method as claimed in claim 7 or 8 wherein said manipulation is carried out on wild-type C. elegans or a selected mutant thereof.

10. A method as claimed in claim 9 wherein said selected mutant harbours multiple mutations.

11. A method as claimed in claim 7 or 8 wherein said manipulation is carried out on C. elegans carrying a reporter gene.

12. A method as claimed in claim 11 wherein said reporter gene is LacZ or green fluorescent protein (GFP) .

13. A method as claimed in any one of claims 7 to 12 wherein said manipulation is carried out on a transgenic C. elegans .

14. A method as claimed in claim 13 wherein said transgenic C. elegans expresses a human gene.

15. A method as claimed in claim 14 wherein said human gene is a known drug target.

16. A method as claimed in claim 14 or claim 15 wherein said human gene is one associated with a human disease .

17. A method as claimed in claim 14 or 15 wherein said human gene is a candidate human disease gene .

18. A method as claimed in any of claims 7 to 17 wherein said manipulation is carried out on only a sub-set of C. elegans cells.

19. A method as claimed in any preceding claim wherein changed characteristics in said worm carrying said defect compared to a worm that does not carry said defect are identified by light microscopy, differential interference contrast optics, fluorescence microscopy, immunochemical detection or spectrophotometric detection, radiation detection, calorimetric detection, fluorescence detection or luminescence detection.

20. A method as claimed in any preceding claim wherein changed characteristics in said worm carrying said defect compared to a worm that does not carry said defect are identified by a pH change or a change in electrical potential.

21. A method as claimed in any preceding claim wherein said plurality of changed characteristics are scored in a predetermined order to generate said phenotypic profile.

22. A method as claimed in any preceding claim wherein the scoring of said plurality of changed characteristics is repeated at predetermined intervals of time.

23. A method as claimed in any preceding claim wherein said phenotypic profiles are stored electronically.

24. A method as claimed in any preceding claim wherein at least one of said plurality of characteristics is selected from the list shown in Table 1.

25. A method as claimed in any one of the preceding claims wherein step (b) comprises measuring changes in two or more characteristics selected from the group consisting of: viability, life cycle, body shape, movement behaviour, mechanotransduction, pharynx pumping, defecation and fertility.

26. A method of constructing a library of phenotypic profiles of nematode worms which comprises the steps of:

(a) exposing a worm to a compound,

(d) simultaneously or sequentially repeating steps (a) to (c) in respect of each of a plurality of different compounds and

27. A method as claimed in claim 26 wherein in step (c) at least three changed characteristics are scored.

28. A method as claimed in claim 27 wherein in step (c) at last six changed characteristics are scored.

29. A method as claimed in claim 28 wherein in step(c) at least ten changed characteristics are scored.

30. A method as claimed in any one of claims 26 to 29 wherein said nematode worm is C. el egans .

31. A method as claimed in any one of claims 26 to 30 wherein each of said plurality of different compounds has a known pharmacological activity.

32. A method as claimed in any one of claims 26 to 30 wherein each of said plurality of different compounds is one which is known to interact with a particular biochemical pathway.

33. A method as claimed in any one of claims 26 to 30 wherein each of said plurality of different compounds has no known pharmacological activity or biochemical interaction.

34. A method as claimed in any one of claims 26 to 30 wherein each of said plurality of different compounds is from a combinatorial library.

35. A method as claimed in any one of claims 26 to 34 wherein said worm to which said compound is exposed is wild-type C. elegans or a selected mutant thereof.

36. A method as claimed in claim 35 wherein said selected mutant harbours multiple mutations.

37. A method as claimed in any one of claims 26 to 34 wherein said worm to which said compound is exposed is C. elegans carrying a reporter gene.

38. A method as claimed in claim 37 wherein said reporter gene is LacZ or GFP.

39. A method as claimed in any one of claims 26 to 38 wherein said worm to which said compound is exposed is a transgenic C. elegans .

40. A method as claimed in claim 39 wherein said transgenic C. elegans expresses a human gene.

41. A method as claimed in claim 40 wherein said human gene is a known drug target.

42. A method as claimed in claim 40 wherein said human gene is one associated with a human disease.

43. A method as claimed in claim 40 wherein said human gene is a candidate disease gene.

44. A method as claimed in any one of claims 30 to 43 wherein said worm is exposed to said compound by feeding the worm on bacteria which have been exposed to said compound.

45. A method as claimed in claim 44 wherein said bacteria are E. coli .

46. A method as claimed in any one of claims 26 to 45 wherein said compound is linked to another compound or carrier substance.

47. A method as claimed in anyone of claims 26 to 46 wherein any changed characteristics in said worm resulting from exposure to said compound are identified by light microscopy, differential interference contrast optics, fluorescence microscopy, immunochemical detection, spectrophotometric detection, radiation detection, colorimetric detection, fluorescence detection or luminescence detection.

48. A method as claimed in any one of claims 26 to 47 wherein any changed characteristics in said worm resulting from said compound are identified by a pH change or a change in electrical potential.

49. A method as claimed in any one of claims 26 to 48 wherein said plurality of changed characteristics are scored in a predetermined order to generate said profile.

50. A method as claimed in any one of claims 26 to 49 wherein the scoring said plurality of changed characteristics is repeated at predetermined time intervals .

51. A method as claimed in any one of claims 26 to 50 wherein said scoring of changed characteristics is carried out using essentially the same scoring protocol as used in a method in accordance with any one of claims 1 to 25.

52. A method as claimed in any one of claims 26 to 51 which comprises the further step of storing the said phenotypic profiles electronically.

53. A method as claimed in any one of claims 26 to 52 wherein at least one of said plurality of characteristics is selected from the list shown in Table 1.

54. A method as claimed in any one of claims 26 to 53 wherein step (b) comprises measuring changes in two or more characteristics selected from the group consisting of: viability, life cycle, body shape, movement behaviour, mechanotransduction, pharynx pumping, defecation and fertility.

55. A method of constructing a library of phenotypic profiles of nematode worms which comprises the steps of:

(a) exposing a worm to an environmental change,

(c) systematically scoring a plurality of any said changed characteristics to establish a Characteristic phenotypic profile associated with said change, (d) simultaneously or sequentially repeating steps (a) to (c) for each of a plurality of different environmental changes and (e) collating the phenotypic profiles so obtained into a library of said profiles.

56. A method as claimed in claim 55 wherein in step (c) at least three changed characteristics are scored.

57. A method as claimed in claim 56 wherein in step (c) at least six changed characteristics are scored.

58. A method as claimed in claim 57 wherein in step (c) at least ten changed characteristics are scored.

59. A method as claimed in any of claims 55 to 58 wherein said environmental change is a change in the pH to which the worm is exposed and in step (d) each of the plurality of environmental changes comprises a different pH.

60. A method as claimed in any one of claims 55 to 58 wherein said environmental change is a change in the osmolarity to which the worm is exposed and in step (d) each of the plurality of environmental changes comprises a different osmolarity.

61. A method as claimed in any one of claims 55 to 58 wherein said environmental change is a change in the temperature to which the worm is exposed and in step (d) each of the plurality of environmental changes comprises a change in temperature.

62. A method as claimed in any one of claims 55 to 58 wherein said environmental change comprises exposure to radiation and in step (d) each of said plurality of environmental changes comprises a different level of radiation.

63. A method as claimed in any one of claims 55 to 58 wherein said environmental change comprises exposure to a virus and in step (d) each of said plurality of environmental changes comprises exposure to a different virus.

64. A method as claimed in any one of claims 55 to 58 wherein said environmental change comprises exposure to a bacterium and in step (d) each of said plurality of environmental changes comprises exposure to a different bacterium.

65. A method as claimed in any one of claims 55 to 64 wherein said worm is C. elegans .

66. A method as claimed in any one of claims 55 to 65 including a further feature as defined in any one of claims 5 to 54.

67. A method as claimed in any one of claims 55 to 66 wherein said scoring of changed characteristics is carried out using essentially the same scoring protocol as used in a method in accordance with claims 1 to 54.

68. A method as claimed in any one of claims 55 to 67 wherein step (b) comprises measuring changes in two or more characteristics selected from the group consisting of: viability, life cycle, body shape, movement behaviour, mechanotransduction, pharynx pumping, defecation and fertility.

69. A method of constructing a multiple library of phenotypic profiles of nematode worms which method comprises carrying out all of the methods of claims 1, 26 and 55.

70. A method as claimed in claim 69 wherein step (b) of the method of at least one of claims 1, 26 and 55 comprises measuring changes in two or more characteristics selected from the group consisting of: viability, life cycle, body shape, movement behaviour, mechanotransduction, pharynx pumping, defecation and fertility.

71. A method of determining the mode of action of a compound which method comprises the steps of;

(a) exposing a nematode worm to said compound

(b) measuring any changes in the identifiable characteristics of said worm as a result of exposure to said compound,

(c) systematically scoring a plurality of changed characteristics to establish a phenotypic profile associated with said compound and

(d) comparing said phenotypic profile with a library of reference phenotypic profiles wherein said library of reference profiles is obtainable by carrying a method in accordance with any of claims 1 to 70.

72. A method of determining whether a compound or combination of compounds interacts with a particular gene or biochemical pathway which method comprises the steps of;

(a) exposing a nematode worm to said compound or combination of compounds

(b) measuring any changes in identifiable characteristics of said worm as a result of said exposure,

(c) systematically scoring a plurality of any changed characteristics to establish a phenotypic profile associated with said compound or combination of compounds, and

(d) comparing said profile with a library of reference profiles said library of reference profiles being obtainable by carrying out the method of any one of claims 1 to 70.

73. A method of finding an alternative treatment for a human disease which method comprises the steps of:

(a) exposing a nematode worm to a candidate compound,

(c) systematically scoring a plurality of any changed characteristics to establish a phenotypic profile for said compound and

(d) comparing said profile with a library of reference profiles, said library of reference profiles being obtainable by carrying out a method in accordance with claim 31.

74. A method of finding a biochemical pathway in which a compound known to have pharmacological activity acts which method comprises the steps of:

(a) exposing a nematode worm to the known compound,

(c) systematically scoring a plurality of any changed characteristics to establish a phenotypic profile for said compound, and

(d) comparing said profile with a library of reference profiles, said library of reference profiles being obtainable by carrying out a method in accordance with claim 32.

75. A method of finding a potential new medicinal indication for a compound of known pharmaceutical activity which method comprises the steps of:

(a) exposing a nematode worm to the known compound,

(d) comparing said profile with a library of reference profiles, said library of reference profiles being obtainable by carrying out a method in accordance with any one of claims 1 to 70.

76. A method as claimed in claim 75 wherein said library of reference profiles is obtainable by carrying out a method in accordance with any one of claims 24 to 26.

77. A method of identifying the mechanism of action of any side effects associated with a compound of known pharmaceutical activity which method comprises the steps of;

(a) exposing a nematode worm to the known compound,

(d) comparing said profile with a library of reference profiles, said library of reference profiles being obtainable by carrying out a method in accordance with claim 32 and/or any of claims 1 to 25.

78. A method of attributing a particular gene to a particular biochemical pathway in C. elegans which method comprises the steps of:

(a) exposing a nematode worm to a compound known to operate in a particular biochemical pathway,

(b) measuring any changes in the identifiable characteristics of said worm as a result of exposure to said compound

(d) comparing said, profile with a library of reference phenotypic profiles said library of reference profiles being obtainable by carrying out a method in accordance with any one of claims 1 to 25.

79. A method as claimed in any of claims 71 to 78 wherein said nematode worm is selected from wild- type C. elegans, a mutant C. elegans comprising one or more mutations, a C. elegans carrying a reporter gene or a transgenic C. elegans .

80. A method as claimed in claim 79 wherein said transgenic C. elegans expresses a human gene.

81. A method as claimed in any one of claims 71 to 80 wherein step (b) comprises measuring changes in two or more characteristics selected from the group consisting of: viability, life cycle, body shape, movement behaviour, mechanotransduction, pharynx pumping, defecation and fertility.

82. A method for elucidating biochemical pathways in a nematode worm which method comprises the steps of:

(a) generating a defect in at least one gene in said worm,

(c) systematically scoring a plurality of any said changed characteristics to establish a phenotypic profile for said defect, and

(d) comparing said profile with a library of reference phenotypic profiles, said library of references profiles being obtainable by carrying out a method in accordance with any one of claims 1 to 25.

83. A method as claimed in claim 82 wherein said nematode worm is selected from wild-type C. elegans , a mutant C. elegans comprising one or more mutations, a C. elegans carrying a reporter gene or a transgenic C. elegans .

84. A method as claimed in claim 82 wherein said defect is selected from the absence of expression of said gene, the reduction in expression of said gene, the expression of a functionally defective protein, the expression of a truncated protein, the misexpression of a protein, the ectopic misexpression of a protein, the expression of a protein of altered stability or the alteration of gene expression as a function of time.

85. A method as claimed in any one of claims 82 to 84 wherein at least three, preferably at least six and more preferably at least ten changed characteristics are scored.

86. A method as claimed in any of claims 82 to 85 which includes the features described in any one of claims 19 to 25.

87. A method of constructing a library of nematode worms which method comprises the steps of:

(a) providing a worm having a defect in at least one gene.

(d) simultaneously or sequentially repeating steps (a) to (c) in respect of each of a plurality of worms, and

(e) producing a library of said worms each identifiable by their phenotypic profiles.

88. A method as claimed in claim 87 wherein said phenotypic profiles are collated into a library.

89. A method as claimed in claim 87 and 88 comprising any one of the features described in any one of claims 2 to 25.

90. A method of constructing a library of nematode worms which method comprises the steps of:

(a) exposing a worm to a compound,

(b) measuring any changes in identifiable characteristics of said worm as a result of exposure to said compound, (c) systemically scoring a plurality of any said changed characteristics to establish a phenotypic profile associated with said compound,

(d) simultaneously or sequentially repeating steps (a) to (c) in respect of each of a plurality of different compounds, and producing a library of said worms each identifiable by their phenotypic profiles.

91. A method as claimed in claim 90 wherein said phenotypic profiles are collated into a library.

92. A method as claimed in claim 90 or 91 comprising any one of the features disclosed in any one of claims 27 to 54.

93. A method of constructing a library of nematode worms which method comprises the steps of:

(a) exposing a worm to an environmental change,

(d) simultaneously or sequentially repeating steps (a) to (c) in respect of each of a plurality of different environmental changes, and

(e) producing a library of said worms each identifiable by their phenotypic profile.

94. A method as claimed in claim 93 wherein said phenotypic profiles are collated into a library.

95. A method as claimed in claim 93 or claim 94 comprising any one of the features disclosed in any one of claims 56 to 70.

96. A method of determining the mode of action of a compound which method comprises the step of:

(a) exposing a nematode worm to said compound,

(c) systematically scoring a plurality of any said changed characteristics to establish a phenotypic profile associated with said compounds, and

(d) comparing said phenotypic profile with the library of phenotypic profiles obtainable by the method of any one of claims 88, 91 or 94.

97. A method of determining whether a compound or a combination of compounds interacts with a particular gene or biochemical pathway which method comprises the steps of:

(a) exposing an nematode worm to said compound or combination of compounds,

(c) systematically scoring a plurality of any said changed characteristics to establish a phenotypic profile associated with said compounds or combination of compounds, and

(d) comparing said phenotypic profile with a library of reference profiles wherein said library of reference profiles is obtainable by the method of any one of claims 88, 91 or 94.

98. A method of finding an alternative treatment for a human disease which method comprises the steps of:

(a) exposing an nematode worm to a candidate compound,

(c) systematically scoring a plurality of any said changed characteristics to establish a phenotypic profile for said compound, and

(d) comparing said profile with a library of 35referenced profiles, wherein said library of referenced profiles is obtainable by carrying out the method in accordance with any one of claims 88, 91 or 94.

99. A method of finding a biochemical pathway in which a compound known to have pharmacological activity acts which method comprises the steps of:

(a) exposing a nematode worm to the known compound, measuring any changes in the identifiable characteristics of said worm as a result of exposure to said compound, (b) measuring any changes in the identifiable characteristics of said worm as a result of exposure to said compound,

(d) comparing said profile with a library of reference profiles, said library of reference profiles being obtainable by the method of any one of claims 88, 91 or 94.

100. A method of finding a potential new medicinal indication for a compound of known pharmaceutical activity which method comprises the steps of:

(a) exposing an nematode worm to the known compound,

101. A method of identifying the mechanism of action of any side effects associated with a compound of known pharmaceutical activity which method comprises the steps of:

(a) exposing a nematode worm to the known compound,

102. A method of attributing a particular gene to a particular biochemical pathway in C. elegans which method comprises the steps of:

(c) systemically scoring a plurality of any said changed characteristics to establish a phenotypic profile for said compound, and

(d) comparing said profile with a library of reference phenotypic profiles, said library of reference profiles being obtainable by carrying out the method in accordance with any one of claims 88, 91 or 94 .

103. A method as claimed in any one of claims 96 to 102 wherein said nematode worm is selected from wild-type C. elegans, a mutant C. elegans comprising one or more mutations, a C. elegans carrying a reporter gene or a transgenic C. elegans .

104. A method as claimed in claim 103 wherein said transgenic C. elegans expresses a human gene.

105. A method of establishing a phenotypic profile for a nematode worm which method comprises measuring and scoring at least three, preferably at least six and more preferably at least ten characteristics of said worm which are not exhibited by wild-type worms.

106. A method as claimed in claim 105 wherein said characteristics not exhibited by wild-type worms are selected from the list shown in Table 1.

107. A method as claimed in claim 105 or claim 106 which comprises measuring and scoring changes in two or more characteristics selected from the group consisting of: viability, life cycle, body shape, movement behaviour, mechanotransduction, pharynx pumping, defecation and fertility.

108. A method as claimed in any one of claims 105 to 107 wherein said phenotypic profile is established for a nematode worm which is selected from a worm having one or more mutations, a worm which has been exposed to a compound or combination of compounds, a transgenic worm, a worm carrying a reporter gene or a worm which has been exposed to an environmental change .

109. A method as claimed in claim 108 wherein said transgenic worm comprises a human gene.

110. A method as claimed in claim 108 wherein said compound has known pharmacological activity.

111. A method as claimed in claim 108 wherein said compound is known to be active in a particular biochemical pathway.

112. A method as claimed in claim 108 wherein said compound or combination of compounds is from a combinatorial library of compounds.

113. A compound which has potential therapeutic activity in a mammal which has been identified in a method as claimed in any one of claims 71 to 81 or 96 to 104.

114. A library of nematode worms obtainable by a method as claimed in any one of claims 87 to 95.

115. A library as claimed in claim 114 wherein said nematode worm is C. elegans .