WO2000063427A2 - Compound screening method - Google Patents

Compound screening method Download PDF

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WO2000063427A2
WO2000063427A2 PCT/IB2000/000575 IB0000575W WO0063427A2 WO 2000063427 A2 WO2000063427 A2 WO 2000063427A2 IB 0000575 W IB0000575 W IB 0000575W WO 0063427 A2 WO0063427 A2 WO 0063427A2
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nematode worms
worms
nematode
elegans
compound
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PCT/IB2000/000575
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English (en)
French (fr)
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WO2000063427A3 (en
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Philippe Verwaerde
Christ Platteeuw
Gwladys Cuvillier
Thierry Bogaert
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Devgen Nv
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Priority claimed from GBGB9908670.4A external-priority patent/GB9908670D0/en
Application filed by Devgen Nv filed Critical Devgen Nv
Priority to JP2000612504A priority Critical patent/JP2002542466A/ja
Priority to AU41376/00A priority patent/AU780574B2/en
Priority to CA002365707A priority patent/CA2365707A1/en
Priority to MXPA01010175A priority patent/MXPA01010175A/es
Priority to EP00920972A priority patent/EP1175506A2/en
Publication of WO2000063427A2 publication Critical patent/WO2000063427A2/en
Publication of WO2000063427A3 publication Critical patent/WO2000063427A3/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082Supracellular entities, e.g. tissue, organisms
    • G01N33/5085Supracellular entities, e.g. tissue, organisms of invertebrates
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406Neurotransmitters
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00Genetically modified animals
    • A01K2217/05Animals comprising random inserted nucleic acids (transgenic)
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43526Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms
    • G01N2333/4353Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms from nematodes
    • G01N2333/43534Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms from nematodes from Caenorhabditis
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701Details
    • G01N2333/4709Amyloid plaque core protein
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701Details
    • G01N2333/4712Muscle proteins, e.g. myosin, actin, protein

Definitions

  • the invention relates to the field of pharmacology and in particular to the screening of chemical substances with potential pharmacological activity using nematode worms such as Caenorhabditis elegans .
  • the invention relates to methods adapted for high-throughput screening which are performed in a multi-well plate format.
  • Caenorhabditis elegans is a nematode worm which occurs naturally in the soil but can be grown easily in the laboratory on nutrient agar or in liquid nutrient broth inoculated with bacteria, preferably E. coli , on which it feeds. Each worm grows from an embryo to an adult worm of about 1mm long in three days or so.
  • C. elegans make it an extremely useful tool in the drug discovery process.
  • C. elegans may be used in the development of compound screens, useful in the identification of potential candidate drugs, in which worms are exposed to the compound under test and any resultant phenotypic and/or behavioural changes are recorded.
  • the possibility that C. elegans might be useful for establishing interactions between external molecules and specific genes by comparison of C. elegans phenotypes which are generated by exposure to particular compounds and by selected mutations is considered by Rand and Johnson in Methods of Cell
  • This method is in effect a standard compound screen in which worms are exposed to candidate compounds and changes in the phenotype, behaviour, biochemistry or physiology of the worms as a result of exposure to the compound are recorded.
  • Such assays may be performed using wild-type nematodes, in which case the changes' detected in step (c) will generally be changes away from wild type behaviour etc.
  • compound screens can also be carried out using non wild-type worms, for example mutant or transgenic worms which may display non wild-type characteristics. In this case the ⁇ change' detected in part (c) may be a reversion towards wild-type.
  • compound screening assays involve running a plurality of assay mixtures in parallel with different concentrations of the chemical substance under test. Typically, one of these concentrations serves as a negative control, i.e. zero concentration of test substance. Changes in behaviour, phenotype, biochemistry or physiology etc resulting from exposure to the compound may then be evaluated in comparison to the negative control.
  • the invention provides a method of determining the mode of action of a chemical substance using nematode worms, which method comprises the " steps of:
  • basic compound screening methodology can be extended to determine the mode of action of a chemical substance. This may be done, for example, by detecting/measuring properties or characteristics of worms exposed to the compound and comparing the result with properties or characteristics of mutant worms carrying mutations in known ⁇ proteins.
  • Example 4 of the accompanying examples provides an illustration of this in the CNS field.
  • the invention provides a method of identifying further components of the biochemical pathway on which a compound having a defined effect on nematode worms acts, which method comprises the steps of:
  • This method of the invention is, in effect, a classic a genetic suppressor screen performed in a multi-well format.
  • a suppressor screen the aim is to identify a mutation which suppresses the phenotype generated by exposure of the worm to a chemical.
  • Worms carrying suppressor mutations are usually identified on the basis that they exhibit a more ⁇ wild type' phenotype in the presence of the compound, as compared to the phenotype generated by exposure of wild type worms to the same compound.
  • the invention provides a method of identifying chemical substances which modulate the effect of a first compound, which compound has a defined effect on nematode worms, which method comprises the steps of:
  • This method may be used to screen for antagonists of a given compound. This principle is illustrated in the accompanying Example 8.
  • elegans being of the order 1mm long in the adult stage. Microscopic nematodes of this approximate size are extremely suited for use in id- to high-throughput screening as they can easily be grown in the wells of a multi-well plate.
  • non- visual detection means refers to any means of detecting a signal which does not require visual inspection by the human eye.
  • non-visual detection system represents a major advantage over previously known screening methods using which require visual inspection of the nematodes by eye in order to detect gross " ⁇ phenotypic or behavioural changes.
  • the worms can take- up the marker molecule.
  • worms may take up the marker as a result of the action of a chemical substance under test.
  • the worms can be pre-loaded with the marker molecule prior to the addition of chemical substance or the marker molecule can be delivered via the media in which the worms are cultured or via bacteria or other food particles on which the worms feed.
  • the marker molecule can also be added to the nematodes as a 'precursor' molecule which can undergo chemical changes in the nematodes as a result of the biochemical activity of the nematode. This biochemical activity on the precursor changes its properties resulting in the generation of a signal which can be measured.
  • a typical example of this system is the use of a precursor marker molecule which can be cleaved by enzymes present in the gut of the nematode worms to generate a marker molecule with a detectable property such as, for example, . fluorescence.
  • FANS fluorescence activated nematode scanning and sorting device
  • the FANS device enables the measurement of properties of microscopic nematodes, such as size, optical density, fluorescence, and luminescence.
  • a FANS is a preferred detection instrument.
  • the use of a FANS is not limited to these experimental conditions, FANS could be generally used for all the screening methods described herein.
  • a screening method using a FANS device is quite analogous to the screening method described for the multi-well plate reader.
  • worms are contacted with the chemical substances with or without the addition of a marker molecule.
  • the multi-well plates are submitted to trie FANS apparatus and in a fully automated procedure the worms are analysed well-by-well for features such as overall size, fluorescence, luminescence or optical density. The desired features are then scored. With the use of the FANS device screens can also be performed quantitatively.
  • worms added to the wells may vary depending upon the type of screen being performed and the required sensitivity. In all plate formats, including 96 well plates, it is preferred to use 1 to 100 worms per well, more preferably 10 to 80 worms per well and most preferably 80 worms per well.
  • Various methods can be used to ensure that substantially equal numbers of worms are added to each of the wells.
  • One way in which this can be achieved is by taking worms cultured according to the standard procedures known to those skilled in the art in solid or liquid media and re-suspending the worms in a viscous solution to form a homogeneous suspension.
  • the viscosity of the solution maintains an even distribution of worms in the suspension, thus substantially equal numbers of worms can be dispensed by adding equal volumes of the homogeneous worm suspension to each of the wells.
  • Suitable viscous solutions include a solution containing a low concentration of a polymer material (e.g. 0.25% low melting point agarose) , glycerol etc.
  • an equal distribution of worms over the wells of the multi-well plate can be achieved using a worm dispensing device, such as that developed by Union Biometrica, Inc.
  • the worm dispenser can be programmed to add a set number of worms to each of the wells of the plate.
  • it can be used to select worms in such a way that only hermaphrodites or males or dauers are dispensed and it can also select on the basis of size so that specifically eggs, LI, L2, L3, L4 or adult worms are dispensed.
  • the inventors have observed that use of a viscous medium in the methods of the invention can have advantages over and above ensuring that equal numbers of worms are added to the wells of the multi-well plate.
  • the multi-well screens described by the inventors are performed in liquid medium.
  • nematodes such as C. elegans
  • solid (e.g. soil) growth in liquid medium results in less healthy worms.
  • Worms grown in liquid medium " are longer and thinner, the pharynx pumps at a reduced rate, the worms show less movement and lay less eggs.
  • the inventors have found a solution to this problem by adding a water soluble polymer to the medium in order to increase its viscosity (i.e.
  • the screening assays described herein may also be improved by the addition of a water soluble polymer, possibly at a lower concentration than is required to increase the viscosity of the medium, at a concentration sufficient to prevent the nematodes from sticking to the wells of the microtiter plate.
  • a water soluble polymer to the assay medium in order to prevent the nematodes from sticking to solid surfaces such as the walls of a microtiter plate may result in a significant improvement in any of the specific types of assays described herein, including the pharynx pumping assays, movement assays, mating assays, egg laying assays and defecation assays, and indeed any other type of assay using nematodes such as C. elegans which is performed in a microtiter plate.
  • Standard methods for culturing nematodes are described in Methods in Cell biology Vol. 48, 1995, ed. by Epstein and Shakes, Academic press. Standard methods are known for creating mutant worms with mutations in selected C. elegans genes, for example see J. Sutton and J. Hodgkin in "The Nematode Cae ⁇ or ajditis 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.
  • the 'chemical substances' or 'compounds' to be tested in the methods of the invention may be is any foreign molecules not usually present in the worm or to which the worm would not normally be exposed during its life cycle. These terms may be used interchangeably.
  • the worm may be exposed to a chemical substance/compound listed in a pharmacopoeia with known pharmacological activity.
  • the chemical substance/compound may be one known to interact with a particular biochemical pathway or gene.
  • a further alternative is to test known molecules with no known biological activity or completely new molecules or libraries of molecules such as might be generated by combinatorial chemistry. Compounds which are DNA, RNA, PNA, polypeptides or proteins are not excluded.
  • the methods of the invention are performed using transgenic C. elegans expressing a transgene which comprises a 'toxic gene' .
  • the term 'toxic gene' encompasses any nucleic acid sequence which encodes a protein which is toxic to the cell. Suitable examples include nucleic acid encoding ataxin, alpha-synuclein, ubiquitin, the tau gene product, the Huntington' s gene product (huritingtin) , the best macular dystrophy gene product, the age-related macular dystrophy product or the unc- 53 gene product.
  • 'Toxic genes' encoding proteins involved in apoptosis or necrosis could also be used with equivalent effect.
  • transgenic C. elegans can be constructed which express one or more toxic genes in a single tissue, in a subset of cell types, in a single cell type or even in a single cell, for example a single neuron. Expression of the toxic gene will generally result in abnormality/malfunction of the cells and tissues expressing the toxic gene.
  • tissue-, cell type- or developmentally- specific promoters are known for use in C. elegans .
  • Synchronized worms are worms that are in the same growth stage.
  • the various growth stages of nematode worms such as C. elegans are eggs, the LI stage, L2 stage, L3 stage, L4 stage and adult stage.
  • the synchronized nematode worms are of a specific sex.
  • the synchronized cultures can be hermaphrodites or males or nematodes in special larval stage, designated dauers. Techniques suitable for use in generating the various synchronized cultures are known in the art, see for example Methods in Cell Biology, vol 48, ibid. The main population of a standard C.
  • elegans culture consists of hermaphrodite worms, so it does not require special techniques to generate synchronized hermaphrodite nematodes in different growth stages.
  • To generate male worms several techniques have been described in the literature. C. elegans cultures that are enriched or consist exclusively of male worms, have been described in C. elegans II, ed, By Fiddle, Blumenthal, Meyer and Pries, 1997, CSHL press. Strains for making enriched or pure male samples have been described by Johnathan Hodgkin, Worm breeder's gazette 15(5), 1999).
  • C. elegans dauers several techniques have been described (Elegans II, ibid) .
  • the step of detecting a signal indicating phenotypic, physiological, behavioural or biochemical changes in the nematode worms using non-visual detection means comprises detecting changes in the pharynx pumping rate of the nematode worms.
  • C. elegans feeds by taking in liquid containing its food (e.g. bacteria). It then spits out the liquid, crushes the food particles and internalises them into the gut lumen. This process is performed by the muscles of the pharynx.
  • the process of taking up liquid and subsequently spitting it out is called pharyngeal pumping or pharynx pumping. Because the process of pharynx pumping involves both the muscles of the pharynx and also pharyngeal neurons, measurement of the rate of pharynx pumping can be exploited to provide a useful screen to identify chemical substances which have an effect on muscle and/or nerve activity.
  • the rate of pharynx pumping can be readily measured by detecting the accumulation of a marker molecules in the worm gut. If this is done using a multi-well plate reader then the assay can be performed rapidly and quantitatively.
  • the pharynx pumping rate may be measured by using a marker molecule precursor which is cleavable by enzymes present in the gut of the nematodes, as described above.
  • Calcein-AM is particularly preferred for this purpose.
  • Calcein-AM is an esterase substrate, and upon cleavage of calcein-AM by esterases, calcein (a fluorescent molecule) is released.
  • esterases are present in the gut of nematodes such as C. elegans, the pharynx pumping rate can be measured indirectly by measuring calcein fluorescence.
  • calcein-AM has been used to measure the rate of pharynx pumping in C. elegans in the presence or absence of several chemical substances. These measurements can be performed in a quantitative high throughput way, allowing selection for chemical substances that alter the pumping rate of the C. elegans pharynx.
  • This method is not restricted to the use of calcein-AM and other precursor substrates could be used, such as: With a fluorescent read out:
  • marker molecules which are fluorescent at low pH have the additional advantage that they can be used together with a nematode food source, e.g. bacteria, which then should not interfere with the read-out as it is dependent only on a pH change and is not enzyme related.
  • ion channel proteins and ion pump proteins are also potential targets for such chemical substances. Examples of such ion channels are sodium/calcium channels, calcium channels, sodium channels and chloride channels. In general, drugs or chemical substances that affect the pumping rate of the C.
  • elegans pharynx and which are identified using the pharynx pumping screen will most probably be compounds that show the following activities : -molecules that have influence on neurotransmitter molecules or that are precursors for the synthesis of a neurotransmitter,
  • the pharynx pumping assays can be used to screen for a broad range of chemical substances with potential pharmacological activity that may have a therapeutic use as anti-psychotic, anti-depressant, anxiolytic, tranquillizer, anti-epileptic, muscle relaxant, sedative or hypnotic agents.
  • the assays may also be used to identify chemical substances that may effect Parkinson's disease and Alzheimer's disease.
  • anti-pruritic, anti-histaminic, and anti- convulsant drugs may also be isolated using the pharynx pumping assay.
  • the pumping assay may also be used to identify nematocides and insecticides.
  • the pharynx pumping assay may also be used to identify chemical substances which modulate the neurotransmitter pathways involving acetylcholine, dopamine, serotonin, glutamate, GABA and octopamine. This can be achieved by using selected mutant C. elegans which exhibit altered levels of one or more of the above-listed neurotransmitters .
  • the pharynx pumping assay methodology can, in addition to the screens described above, be adapted for use in determining the mode of action of a chemical substance, or to select for chemical substances which act on a specific target
  • transgenic or humanized nematodes are dispensed into the wells of a multi-well assay plate.
  • a sample of the chemical substance under test is then added to each of the wells and changes in the rate of pharynx pumping are detected as described above.
  • the rate of pharynx pumping in the absence of any chemical substances is also scored.
  • the pharynx pumping assay can thus be used to identify chemicals which enhance or suppress the rate of pharynx pumping in a defined mutant, transgenic or humanized strain.
  • mutant and transgenic C. elegans strains which are useful in this aspect of the invention. Mainly these mutants and transgenics relate to neurotransmitter synthesis, neurotransmitter signal transduction and ion channels. More specifically, examples of mutant, transgenic and humanized worms are given which relate to neurotransmitter receptors such as muscarinic receptors, glutamate receptors, hormone receptors, 5- HT receptors, cannabinoid receptors, adrenergic receptors, dopaminergic receptors, opioid receptors, GABA receptors, adenosine receptors, VIP receptors and nicotinic receptors, proteins involved in neurotransmitter synthesis, neurotransmitter release pathways and G-protein coupled receptor proteins, G- protein coupled second messenger pathways such as adenylate cyclase, protein kinase A, cAMP responsive element binding proteins, IP3, diacylglycerol, protein kinase C, phospholipase Q and proteins
  • mutants which are suitable for use in this aspect of the invention are provided in the examples given herein.
  • transgenic or humanized strains it is possible to screen for chemical substances which act on a specified target and thus identify a broad range of chemical substances that may have a therapeutic use as anti-psychotics, anti-depressants, anxi ⁇ lytics, tranquillizers, anti-epileptics, muscle relaxants, sedatives or hypnotics, but the screen will also result in chemical substances that may have an effect on Parkinson's disease and Alzheimer's disease.
  • anti-pruritic, anti-histaminic, and anti- convulsant drugs may be isolated.
  • the transmitter pathway that maybe effected by chemical substances and hence may be detected by the assay are the pathways for acetylcholine, dopamine, serotonin, glutamate, GABA and octopamine.
  • acetylcholine dopamine, serotonin, glutamate, GABA and octopamine.
  • mutant, transgenic and humanized worms also allow the development of screens for chemical substances that have an activity in well-defined biochemical pathways. For example, it is possible to screen for compounds that rescue the phenotype of selected mutant C. elegans which carry a defined mutation in a known gene or compounds which enhance the phenotype of the selected mutant C. elegans .
  • the pharynx pumping screen may used to screen for compounds having potential insecticidal activity. The inventors have observed that exposure of C. elegans to compounds having pesticidal activity, such as herbicides, insecticides, nematocides or fungicides, has an effect on the pharynx pumping rate.
  • the pharynx pumping assay methodology can be used to identify further components of the biochemical pathway on which a compound having a defined effect on nematode worms acts. Using this screen it is possible to identify genes that enhance, suppress or modulate the activity of a selected compound. The screen can be done directly and rapidly as using multi-well plates thousands of worms can be screened at once.
  • the assay is performed using C. elegans which are transgenic, or mutant or humanized for the Sarco/endoplasmic reticulum calcium ATPase gene (SERCA) and/or for its regulators Phospholamban (PLB) and Sarcolipin (SLN) . These genes are important for the regulation of the internal storage of calcium in the cell .
  • SERCA Sarco/endoplasmic reticulum calcium ATPase gene
  • PHB Phospholamban
  • SPN Sarcolipin
  • Chemical substances that alter the pumping rate of the pharynx in these mutant, transgenic or humanized worms are substances that modulate the activity of SERCA or PLB or SLN or that alter the interaction of SERCA-PLB or that alter the interaction of SERCA-SLN or that alter the activity of the SERCA pathway.
  • Such chemical substances may be useful as therapeutics or may be hit compounds useful for further drug development in the area of cardiovascular diseases including hypertension, cardiac hypertrophy and cardiac failure, but also in the area of diabetes mellitus and in the area of skeletal muscle diseases including Brody disease.
  • the assay may be performed using nematodes which exhibit aberrant pharynx morphology and/or function.
  • the pharynx of the nematode consists of several cell types and all of these are required for the pharynx to function properly.
  • pharynx pumping is regulated by several neurons.
  • the cells essential for pharyngeal morphology and pharyngeal function are the pharyngeal muscles, the pharyngeal epithelial cells, the pharyngeal glands and the pharyngeal neurons. If one of these cell types is altered, degenerated or dysfunctional, the pharynx will have an aberrant morphology or an aberrant function which results in an altered pumping of the pharynx.
  • C. elegans mutants that exhibit an altered pumping rate as a result of an altered pharyngeal morphology.
  • C. elegans worms which exhibit a defect in one or more of the cell types required to maintain the morphology and/or function of the C. elegans pharynx. This can be achieved by expressing 'toxic genes' in cells of the pharynx.
  • the term 'toxic genes' encompasses any nucleic acid sequence which encodes a protein which is toxic to the cell.
  • Suitable examples include nucleic acid encoding ataxin, alpha-synuclein, ubiquitin, the tau gene product, the Huntington' s gene product (huntingtin) , the best macular dystrophy gene product, the age-related macular dystrophy• product or the unc-53 gene product.
  • 'Toxic genes' encoding proteins involved in apoptosis or necrosis could also be used with equivalent effect. Expression of the toxic genes in the pharynx or in particular cell types within the pharynx can be achieved using tissue- specific or cell type-specific promoters which are capable of directing the appropriate expression pattern.
  • the myo-2 promoter can be used to direct expression in the pharynx and the unc-129 promoter can be used to direct expression in the pharyngeal neurons.
  • Other suitable promoters included the tropomyosin promoter tmy-1 and the daf-7 promoter. Expression of a toxic gene in one or more cell types of the pharynx or in the pharyngeal neurons will result in a changed morphology and/or function of the pharynx, and hence an alteration of the pharynx pumping rate. Interestingly, disruption of the ASI neuron by expression of a toxic gene under the control of the daf-7 promoter results in dauer formation. This is directly the result of a lack of insulin hence C.
  • elegans in which the ASI neuron is disrupted can be used to perform screens which may be useful in relation to diabetes. These screens could be performed using the pharynx pumping assay read-out or alternatively the movement assay read-out described below (see example 12) .
  • Mutant or transgenic worms which exhibit an altered pharynx pumping rate can be used to screen for chemical substances which further alter the pharynx pumping rate e.g. which rescue the mutant/transgenic phenotype or which enhance the mutant/transgenic phenotype.
  • the chemical substances thus isolated may be useful as therapeutic agents or as hit compounds for further drug development in disease areas such as anti-depressants, anti-psychotics, anxiolytics, tranquillizers, anti-epileptics, muscle relaxants, sedatives, anti-migraine drugs, analgesics and hypnotics.
  • Mutant or transgenic worms which exhibit an altered pharynx pumping rate may further be used to screen for pesticides such as herbicides, nematocides, insecticides and fungicides.
  • the performance of the pharynx screens may also be improved by the addition of a water soluble polymer to the assay medium at a concentration sufficient to prevent the nematode worms from sticking to the wells of the multi-well plate.
  • a water soluble polymer are polyethylene glycol, particularly PEG8000, PVA and PVP, with PEG8000 being most preferred.
  • the optimum concentration of polymer added to the medium may be determined by routine experiment. For PEG8000, a concentration of 0.1% is particularly preferred.
  • the inventors have observed that addition of PEG to the assay medium in the pharynx pumping assay results in an increased quality, mainly due to a reduction in the numbers of dead or harmed individuals.
  • the nematodes need to be divided over the different wells and plates, either manually or using automated systems. During these manipulations of the nematodes, there ⁇ is a risk that the worm will stick to the wall of the pipette or other tool. The flow of the medium in which the nematode is suspended may then result in the death of the worm.
  • addition of PEG8000 to the medium results in more pumping and less variation in the pharynx pumping assay (see Figure 10) .
  • the step of detecting a signal indicating a phenotypic, physiological, behavioural or biochemical changes in the nematode worms using non-visual detection means comprises detecting changes in the intracellular levels of ions, metabolites or secondary messengers in cells of the nematode worms.
  • the activity of a chemical substance is not detected indirectly be measuring a signal from a marker molecule, but by measuring the activity of genetically encoded sensor whose properties are altered in the presence of specific ions, metabolites or secondary messengers.
  • changes in intracellular levels of Ca 2+ can be detected using the genetically encoded calcium sensor molecules GFP-calmodulin or aequorin.
  • GFP-calmodulin is known to be fluorescent in the presence of calcium ions.
  • intracellular calcium levels are low, no fluorescence can be detected but if the calcium levels increase calcium binds to the GFP-calmodulin causing a conformational change which results in a fluorescent molecule which can be detected, for example using a multi-well plate reader.
  • transgenic C. elegans which express the genetically encoded sensor in all cells, or in specific tissues, or in selected cells. This can be achieved with the use of tissue-specific or cell type- specific promoters with suitable activity.
  • the method cen be performed using transgenic worms which express GFP-calmodulin in any cells/tissues of the nematode which are sensitive to calcium signalling, including cells of the pharynx, the vulva muscles, the body wall muscles and neurons.
  • the transgenic worm can be of wild-type genetic background, a mutant transgenic or a humanized strain.
  • intracellular calcium levels in the cells of the pharynx are correlated with the pharynx pumping rate
  • the fluorescence detected in transgenic nematodes expressing GFP-calmodulin in these cells is an indication of the pharynx pumping rate and these transgenic worms can also be used to screen for chemical substances that influence the pumping rate of the pharynx.
  • the step of detecting a signal indicating a phenotypic, physiological, behavioural or biochemical changes in the nematode worms using non-visual detection means comprises detecting changes in the movement behaviour of the nematode worms.
  • 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.
  • nematode autofluorescence fluorescence which occurs in the absence of any external marker molecule
  • optical density can be observed in the wells containing nematodes that move normally as compared to wells containing nematodes that are defective for movement.
  • a low level of autofluorescence or optical density will be observed, whilst a high level of autofluorescence or optical density can be observed in the wells that contain the nematodes that are defective in movement.
  • Optical density is measured using a variation of the platelet aggregation assay, which is well known in the art. Using the MRX revelation device from Dynex (USA) , optical density can be measured at multiple points per well, showing the precipitation pattern of the nematodes.
  • autofluorescence or optical density measurements can be taken in two areas of the surface of the well, one measurement in the centre of the well, and on measur ment 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 .
  • the movement assay can be used for the same purposes as the pharynx pumping assay described above i.e. the movement assay can be used to identify chemical substances that alter the movement behaviour of the nematode and hence may have an effect on muscle and/or neuronal activity, for the identification of genetic enhancers, suppressors and modulators of a selected compound having a known effect on nematode worms or for the identification of chemical substances that are enhancers, suppressors or modulators of a selected compound.
  • Chemical substances which are identified using the movement assay as having an effect on the movement behaviour of nematode worms are generally found to belong to the class of CNS-related drugs but also include GABA antagonists, NMDA antagonists, m-Glu antagonists and adrenergic antagonists.
  • the movement assay is based on the principle that moving nematodes will stay suspended in the medium, whilst nematodes which do not move anymore will sink to the bottom of the well. This difference in the location of the nematodes results in a difference in OD when measured centrally in the well as previously described. Although moving worms stay diluted in the medium they tend to sink down over time as a result of gravity pull.
  • concentration of polymer used in any given assay is, of course, dependent on the specific type of assay one wishes to perform; if the polymer concentration is too low the viscosity of the medium will be insufficient, too high a concentration of polymer will result in formation of a gel, preventing non-moving worms from sinking during the assay.
  • concentration of polymer required for optimum performance of the assay can readily be determined by routine experiment.
  • the performance of the movement assays described herein may further be improved by the addition of a water-soluble polymer to the assay medium at a concentration sufficient to prevent the nematode worms from sticking to the wells of the multi-well plate.
  • a water-soluble polymer are polyethylene glycol, particularly PEG8000, PVA and PVP, with PEG8000 being most preferred.
  • the optimum concentration of polymer added to the medium may be determined by routine experiment. For PEG8000, a concentration of 0.1% is particularly preferred.
  • the movement assay methods are preferably carried out using microscopic nematode worms, particularly those of the genus Caenorhabdi tis and most preferably C.
  • the movement assay can be performed using synchronised worm cultures at different growth stages, using male, hermaphrodite or dauer worms or using mutant, transgenic or humanized worms.
  • One mutant C. elegans strain, the ace-1; ace-2 double mutant is particularly suitable for use in movement assays. This strain does not show any movement and has a spasm-like phenotype. It can therefore be used to screen for chemical substances which rescue the defective movement phenotype. These chemical substances may have a pharmacological effect on muscle and/or neuronal activity.
  • the step of detecting a signal indicating a phenotypic, physiological, behavioural or biochemical changes in the nematode worms using non-visual detection means comprises detecting changes in the mating behaviour of nematode worms .
  • the mating behaviour of nematodes is very complex, involving at least following steps: recognition, backing, tail curling, vulva locat-ion and copulation.
  • the male nematode has at least 41 specialized additional muscles, 79 additional neurons, 36 extra neuronal support cells, 23 proctodeal cells, and 16 hypodermal cells associated with mating structures. The function of some of the neurons has been described. Also several mutants have been described that show defects in mating behaviour (C. elegans II, ibid; J. Sulton et al., W13G 7(2)22; Loer and Kenyon WBG 12 (2).80, 1992; Hadju et al., International worm meeting abstract 15 1, 199 1) .
  • either the male worms or the hermaphrodite worms can be transgenic worms which stably express a marker molecule such as an autonomous fluorescent protein (GFP or BFP) or a luminescent marker in some or all cell types.
  • a marker molecule such as an autonomous fluorescent protein (GFP or BFP) or a luminescent marker in some or all cell types.
  • the offspring generated from mating of these transgenic worms will also express the marker molecule and hence can be easily measured using a multi-well plate reader or a FANS device.
  • the hermaphrodites do not need to be 'non-selfers' since only offspring resulting from the mating of males and hermaphrodites will express the marker whilst offspring generated from hermaphrodite self-fertilization will not harbor the marker molecule.
  • the offspring resulting from mating and self-fertilization can thus be distinguished.
  • the hermaphrodite worm is the transgenic strain expressing the marker molecule the hermaphrodite strain is preferably also a 'non-selfer' strain .
  • the mating assay can also be performed using C. elegans in which the function of a male-specific neuron involved in mating behaviour is disrupted.
  • the examples included herein provide a list of male- specific neurons involved in mating behaviour. The function of one or more of these neurons can be disrupted for example by expression of one of the toxic genes listed above in connection with the pharynx pumping assays.
  • C. elegans which have defects in one or more specific neurons it is possible to perform screens to identify chemical substances which act on a specific neuronal signalling pathway.
  • the chemical substances identified using such screens may have CNS-related pharmacological activity.
  • the mating assay can also be performed using transgenic C. elegans which exhibit altered mating behaviour as a result of the expression of a toxic gene in a specific tissue or cell type.
  • Suitable transgenic C. elegans can be constructed according standard techniques known in the art using one of the toxic genes listed above under the control of an appropriate tissue- or cell type-specific promoter. Promoters which may be useful for this purpose include the her-1 P2 promoter which directs gene expression in CP9, the mab-18 (alternative splice of pax-6 homologue vab-3) promoter which directs gene expression in ray 6 and the spe-Tl promoter which directs gene expression in 60 cells of the spermatheca.
  • the step of detecting a signal indicating a phenotypic, physiological, behavioural or biochemical changes in the nematode worms using non-visual detection means comprises detecting changes in the egg laying behaviour of the nematode worms.
  • assays based on detection of egg laying can be used for the same purposes as the pharynx pumping and movement assays described herein.
  • the number of eggs layed is detected by counting the numbers of resultant offspring using the techniques described above for the mating assay.
  • the egg laying assays and the mating assays are based on the measurement of the eggs and the offspring.
  • the quantity of eggs can be measured by applying specific antibodies to the eggs, and counter staining with dyes specific to the antibodies which recognize the eggs as known in the art.
  • the methods may comprise detecting the numbers of eggs produced with the use of specific dyes which recognize the eggshell.
  • detection of the number of eggs in a well is carried out using a dye which recognizes a substance released on hatching of the eggs.
  • the enzyme chitinase is released into the medium.
  • the enzyme recognizes the substrate 4-methylumbelliferyl 2 -D-N, N, N, -triacetylchitotrioside (or 4-Methylumbelliferyl ⁇ - D -N,N' -diacetylchitobioside) which is a fluorescent precursor molecule (provided by Sigma, St. Louis, MO).
  • N-acetyl- 2 -glucosaminide or CM-DCF-NAG provided by Molecular Probes, Eugene, OR, or provide by Sigma.
  • Egg laying assays using chitinase substrates may be carried out using the following general methodology: Place 30 nematodes in a microtiter plate in 80 ⁇ l of M9 medium. Add the compound to be tested at appropriate concentrations in lO ⁇ l. Add the chitinase substrate at an appropriate concentration in lO ⁇ l. Measure fluorescence, luminescence, or colour formation at various time intervals. Results of typical experiments are shown in Figures 16 and 17, clearly indicating that increasing the time interval results in better readouts.
  • the step of detecting a signal indicating a phenotypic, physiological, behavioural or biochemical change in the nematode worms using non-visual detection means comprises detecting a change in the defecation behaviour of the nematode worms.
  • Defecation in nematodes such as C. elegans is achieved by periodically activating a stereotyped 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.
  • the defecation assay is preferably performed using C. elegans mutants which have a defective defecation behaviour and particularly with C. elegans mutants which are constipated.
  • C. elegans mutants which have a defective defecation behaviour and particularly with C. elegans mutants which are constipated.
  • Several mutants with all kinds of defects in the defecation cycle have been reported (Thomas, Genetics 124: 855-872, 1990; Iwasaki et al., PNAS 92: 10317-10321, 1995; Reiner et al., Genetics 141: 961-976, 1995).
  • the defecation assay can also be performed using wild-type worms or worms with no defecation defects which allow screening for compounds which are inhibitors of defecation.
  • defecation in C. elegans requires the activity of muscles and neurons, compounds which alter the rate of defecation may potentially have C
  • 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 the medium in the wells of the 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.
  • 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 is therefore an indicator of the rate of defecation of the nematodes.
  • Defecation can also be measured using a method based on the luminescent features of the chelation of lanthanides such as terbium in the presence of an aromatic group, such as aspirin.
  • the method requires two pre-loading steps, first the wells of a multi-well plate are pre-loaded with aspirin conjugated to a chelator such as DTPA (prior to the addition of the nematode worms) and second, bacteria or other nematode food source particles are pre-loaded with terbium using standard techniques known in the art. C. elegans are then placed in the wells pre-loaded with aspirin conjugated to a chelator such as DTPA and are fed with the bacteria pre-loaded with terbium.
  • a chelator such as DTPA
  • the terbium present in the pre-loaded bacteria added to the wells will result in a low level of background luminescence.
  • the bacteria When the bacteria are eaten by the nematodes the bacterial contents will be digested but the terbium will be defecated back into the medium.
  • the free terbium will then be chelated by the aspirin which was pre-loaded into the wells resulting in measurable luminescence.
  • the luminescence thus observed is therefore an indicator of nematode defecation.
  • a further method to detect defecation is based on esterified chelators of lanthanides. This method is essentially the similar to the method described above to detect defecation by aspirin chelation of Terbium.
  • the main advantage of the lanthanide chelation method is that the chelator does not to be coated on the wells, but can be added to the liquid medium in which the jiematode is placed.
  • Lanthanides are rare earth metals that are known to exhibit a long lifetime fluorescence when chelated in presence of an aromatic group.
  • Well known lanthanides are Europium and Terbium; a typical chelator is diethylenetriaminepentaacetic acid (DTPA) .
  • DTPA diethylenetriaminepentaacetic acid
  • the assay is based on the principle that an esterified DTPA cannot chelate terbium. After ingestion by C. elegans such esterified chelator will be processed by gut esterases. Upon release by defecation it will readily chelate terbium, thus allowing detection using time-resolved fluorescence, as known in the art.
  • This method allows the detection of very small amounts of material.
  • the method may allow monitoring of defects in the defecation process.
  • Figure 1 is an overview of the neurons and transmitters that are known to have a direct influence on the pumping rate of the C. elegans pharynx.
  • Figure 2 shows an example of the detection of enhancers of the pumping rate of the C. elegans pharynx, using a fluorescent read-out.
  • Figure 3 shows an example of the detection of inhibitors of the pumping rate of the C. elegans pharynx, using a fluorescent read-out.
  • Figure 4 shows dose-response curves for the inhibitors tamoxifen, BP554 and pimazide.
  • Figure 5 shows a dose-response curve for the enhancer clomipramine, showing the toxic effect of DMSO.
  • Figure 6 shows a dose-response curve for thapsigargin showing the enhancer effect at high concentrations and the inhibitor effect at high concentrations.
  • Figure 7 illustrates the principle of the movement assay.
  • Figure 8 illustrates the principles of chemical substrate selection and antagonist selection using the movement screen.
  • Figure 9 shows the results of a representative movement assay illustrating the change in nematode autofluorescence (y-axis) with time (x-axis) .
  • Figure 10 illustrates the result of an experiment to show the effect of PEG8000 on performance of the pharynx pumping assay.
  • 100 worms (strain HD8) were incubated for 3 hours in the presence of 0.5 ⁇ M calcein-AM. They were handled with or without the addition of 0.1% PEG.
  • Figure 11 illustrates the results of experiment to show the effect of viscosity of the medium on performance of the movement assay.
  • Figure 12 and Figure 13 illustrate the effect of viscosity of the medium on performance of the movement assay for various C. elegans mutants in a comparative study.
  • 100 worms were incubated in a round bottom shaped microtiter plate.
  • OD was measured at 340nm in various viscous media (M9, medium viscosity carboxymethylcellulose and high viscosity carboxymethylcellulose) . Measurements were done in triplicate .
  • Figures 14 and 15 illustrate the effect of viscosity of the medium on the pharynx pumping screen.
  • N2 + MC denotes wild-type worms in medium containing carboxymethylcellulose .
  • Figures 16 and 17 illustrate the kinetics of egg laying assays using N2 worms based on detection of chitinase activity using a fluorescent substrate.
  • the assays were carried out in the presence of varying concentrations of clomipramine and fluoxetine, respectively.
  • Figures 18 to 21 illustrate the effect of compounds of known insecticidal activity on the pharynx pumping rate of C. elegans .
  • a reduction in the pharynx pumping rate on exposure to insecticide is clearly seen.
  • Example 1 Distribution of nematodes, and dilution of compounds The basic protocol for performing a screen using the method of the invention is described for multi- well plates with 96 wells, but other multi-well plates with 6, 12, 24, 3 84 or 1536 wells could be used.
  • synchronized worms are used.
  • the production of large amounts of synchronized worms has been described in (Methods in cell biology, Vol. 48, ibid) .
  • an assay buffer 40mM NaCl, 6mM Kcl, ImM CaCl 2 , ImM MgCl 2
  • 10 X M9 buffer 30g KH 2 P0 4 , 60 g Na 2 HP0 4 , 50 g NaCl, 10 ml MgS04 1M, made up to 1 litre with H 2 0
  • Other buffers than M9 buffer can be suitable for this purpose.
  • the worms are then diluted and resuspended in semi-soft agar (final concentration of 0.25% low melting agarose in M9 buffer) .
  • This procedure results in an equal, homogenous and stabilised suspension of the nematodes.
  • Other polymers than low melting agarose can be used in this procedure.
  • the presence of a homogenous worm suspension facilitates the equal distribution of the worms in the multi-well plates, but is not essential for the described screening assay. Any other method that results in a homogenous distribution of the nematodes worms over the wells will he useful. More specifically, the use of a worm dispenser will result in even a better, and hence a more equal distribution of the worms over the wells of the multi-well plate.
  • the worms are distributed in the multi-well plates using electronic 8 channel pipettes.
  • 40 +/- 5 worms are added to every well of the microtiter plate.
  • the chemical substances are made soluble in DMSO. Any other solvent can be used for this purpose, but most selected chemical substances appear to be soluble in DMSO.
  • the chemical substance is added in the wells at various concentrations, but preferentially a concentration between 3 to 30 ⁇ M is chosen as this gives the clearest results. It possible to screen for dosage effects by varying the concentration of the chemical substance from less than 1 ⁇ M up to lOO ⁇ M.
  • the concentration of the DMSO should not be too high and preferentially should not exceed 1%, more preferentially the concentration of the DMSO should not exceed 0.5% and even more preferentially, the concentration of the DMSO is lower than 0.3%.
  • C. elegans strains can be used. Screens to select for chemical substances inhibiting the pumping rate of the C. elegans pharynx are generally performed with mutant C. elegans strains which have a constitutively pumping pharynx. Wild-type worms can also be used in this screen, but the mutants worms are preferred. Other C. elegans mutants can be used in this screen to select for inhibitors of pumping. The selected mutant C. elegans with the constitutively pumping pharynx pumps medium into the gut at a constant rate and reduction/rescue of this phenotype can easily be scored, which facilitates the detection and selection of chemical substances.
  • the screen is generally performed using wild-type C. elegans worms but other mutants could be used in this screen.
  • a wild type worms will not pump or show a reduced pumping rate in liquid medium that doesn't contain any food source as the food source is one of the signals to induce pharynx 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.
  • a marker molecule precursor such as calcein-AM
  • 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.
  • Chemical substances that alter the pumping rate of the pharynx will result in more or less uptake of the calcein-AM and hence in more or less fluorescent signal.
  • the fluorescence can be measured rapidly and quantitatively, resulting in a fast, quantitative high throughput screening method for the identification of chemical substances with potential pharmacological activity.
  • a concentration of between 1 and lOO ⁇ M calcein-AM is added into the medium.
  • Preferably 5 to lO ⁇ M calcein-AM is used.
  • This measurement of the pharynx pumping rate by detecting the accumulation of a marker molecule is not limited to calcein-AM.
  • Other precursors can be used and thus the assay as described here can be changed to be__suitable for other precursors.
  • the precursor can be cleaved by esterases, but could also be a substrate for other enzymes in the nematode gut.
  • the marker molecule should not necessary be a fluorescent molecule, but can be a molecule detectable by other methods. Most of these precursor substances are commercially available or could be synthesized according to methods known in the art. Some examples are: With a fluorescent read out:
  • FDP Fluorescein diphospate
  • DNAses Other target enzymes present in the gut for which substrates can be found or developed are DNAses,
  • the drugs were randomly distributed over the wells of two 96-well multi-well plates.
  • the pumping rate of the C. elegans pharynx was measured using calcein-AM as described in Example 2.
  • C. elegans wild-type strain N2 was used to select for enhancers of the pumping phenotype, and a mutant C. elegans strain with a constitutively pumping pharynx was used to detect inhibitors of pumping.
  • the substrate calcein-AM was added to the medium at the same time as the worms and the compounds.
  • the fluorescence was measured after approximately one hour.
  • compounds and worms are added to the medium first and incubated for approximately 1 hour. After this incubation period that allowed for the chemical substances to activate or to inhibit the pumping rate of the pharynx, calcein-AM was added. The plates where then further incubated for one hour prior to fluorescence measurement in the microtiter plate reader.
  • pharyngeal neurons At least the neurons, 11, 12, 13, M3, MC, NSM, Ml, RIP and M4 have been shown to be important for pharynx pumping.
  • the neurons MC, M3, M4 and NSM are known to regulate the contraction/pumping rate of the pharynx. They control respectively the rate of pumping, timing of muscle relaxation, isthmus peristalsis and the perception of food.
  • elegans are acetylcholine and serotonin, glutamate, octopamine, dopamine and GABA (The nematode Caenorhabditis elegans ed. by W. B. Wood et al . , CSHL press 1988, page 337-392) .
  • the pharynx pumping rate is influenced by inhibitors and agonists of neurotransmitters, and by compounds that inhibit or enhance neurotransmitter pathway calcium channels, sodium/calcium channels, chloride channels.
  • These chemical substances are used in a very wide range of prescribed drugs, such as anti-depressants, anti- psychotics, anxiolytics, tranquillizers, antiepileptics, muscle relaxants, sedatives, anti- migraine drugs, analgesics and hypnotics.
  • Some of these Central Nervous System (CNS) related drugs have applications in disease areas such as CNS related genetic diseases as Parkinson' s disease and Alzheimer's disease.
  • CNS related drugs it is best to classify them according to their biochemical function in the neurotransmitter pathway cascade.
  • CNS related drugs can at least have influence on the following features of the pathway:
  • a CNS drug can have influence on the precursor compounds, or can be precursor molecule for the synthesis of a neurotransmitter.
  • a CNS drug can enhance, inhibit or modulate the synthesis of a neurotransmitter
  • a CNS drug can have a function in the depletion _-of the transmitter.
  • a CNS drug can prevent or stimulate the release of the transmitter from the synaptic vesicles in the synaptic cleft.
  • a CNS drug can function as a receptor inhibitor or stimulator.
  • a CNS drug can mimic the transmitter.
  • a CNS drug can function as conduction inhibitor or activator.
  • a CNS drug can function as an activator or inhibitor of the conduction blockade. - A CNS drug prevent or stimulate the re-uptake of transmitter after firing of the neuron.
  • a CNS drug can functions as a false transmitter
  • CNS related drugs can be found in the classes of chloride channel blockers, sodium/calcium channel blockers, calcium blockers, and other ion channel blockers.
  • screening methods designated as “in vitro binding assays” or “cloned transporter assay systems” are well known to persons skilled in the art.
  • cell membranes harboring a specific type of receptor are isolated from mammalian tissue or specific tissue cultures. In most cases these membranes are isolated from cells that over-express the desired receptor.
  • neurotransmitters such as acetylcholine, dopamine, serotonin, glutamate, GABA and octopamine, but also hormonal substances such as norepinephrine, adrenaline and . others are the subject of the screening assay.
  • the receptor ligand being the neurotransmitter in most cases
  • Experimental conditions can then be set-up that compares the binding rate of the radioactive ligand to the receptor.
  • Putative CNS drugs and other chemical substances can then be isolated that alter the binding of the ligand to the receptor.
  • these screening assays can be used to isolate antagonist of neurotransmitters, these "in vivo" assays do not reflect the in vivo effect of the isolated compound, as only the association with the desired receptor is monitored. Moreover for every potential target in the neurotransmitter pathway cascade, an "in vitro binding assay” needs to be developed. Furthermore, for some of the putative targets for CNS related drugs as described above, no assays have been developed or these assays are difficult to develop, or no high throughput screening is possible. All known assays with tissue and animal models also suffer from the latter problem. Moreover the assays using animal tissues or organs involve the killing of large amounts of animals, and screening methods based on the use of higher animals, especially mammals, are increasingly to be avoided due to issues of animal welfare.
  • the pharynx pumping assay methodology can be used to determine in which neurotransmitter pathway a compound shows activity (acetylcholine, dopamine, serotonin, glutamate, octopamine, GABA, etc.).
  • C elegans nematode mutants have been constructed which are defective in one or more genes.
  • the defect can be introduced stably by standard technology (i.e. gene knock-outs) but can also be transiently introduced by RNAi technology. Both techniques are well known in the field of C. elegans genetics.
  • the genes that are affected in the nematodes of this collection are genes that are those involved in one or more neurotransmitter pathways.
  • affected genes are genes that code for neurotransmitter receptors such as muscarinic receptors, glutamate receptors, hormone receptors, 5- HT receptors, cannabinoid receptors, adrenergic receptors, dopaminergic receptors, opioid receptors, GABA receptors, adenosine receptors, VIP receptors, nicotinic receptors, proteins involved in neurotransmitter synthesis or neurotransmitter release pathways and G-protein coupled receptors, genes encoding proteins for G-protein coupled second messenger pathways such as adenylate cyclase, protein kinase A, cAMP responsive element binding proteins, phospholipase C, genes encoding for functions in gap junctions and genes encoding for ion channels and ion pumps .
  • neurotransmitter receptors such as muscarinic receptors, glutamate receptors, hormone receptors, 5- HT receptors, cannabinoid receptors, adrenergic receptors, dopaminergic receptors, opioid
  • mutants are tested in a pharynx pumping screen as described in the previous Examples and the results are stored for reference. Compounds having an unknown mode of action are then tested in the pharynx pumping screen and the results obtained compared with the reference results obtained from the mutants in order to determine the mode of action or pathway of the compound.
  • transgenic worms have also been constructed.
  • C elegans can be engineered to express human genes using standard technology (described in Methods in Cell Biology, vol. 48).
  • transient and stable transgenic nematodes can be constructed, and the methods for engineering the expression of heterologous and homologous transgenes in the nematode C. elegans are well known within the field.
  • These transgenes can be expressed solely in cells of the pharynx with the use of pharynx-specific promoters, but could also be expressed solely in the neurons affecting the pumping rate of the pharynx.
  • the transgenes expressed in the transgenic C. elegans can encode neurotransmitter receptors such as muscarinic receptors, glutamate receptors, hormone receptors, 5 - HT receptors, neurotransmitter synthesis, neurotransmitter release pathways and G-protein coupled receptors.
  • neurotransmitter receptors such as muscarinic receptors, glutamate receptors, hormone receptors, 5 - HT receptors, neurotransmitter synthesis, neurotransmitter release pathways and G-protein coupled receptors.
  • These transgenes can be protein encoding sequences of human origin. At least 400 G- protein coupled receptors have been sequenced so far.
  • genes encoding proteins for G-protein coupled second messenger pathways such as adenylate cyclases, protein kinase A, cAMP responsive element binding proteins, phospholipase C and genes encoding for functions in gap junctions and genes encoding for ion " -channels and ion pumps could be expressed in the pharynx or in the neurons of the nematode.
  • the transgenic C. elegans described above can have a wild-type genetic background or can be mutant C. elegans strains.
  • the worms are humanized, which means that a transgene which is a protein-encoding nucleic acid sequence of human origin is expressed in a worm made mutant for the C. elegans gene encoding the corresponding protein.
  • C. elegans mutants with mutations in genes encoding components of neuronal signalling pathways are listed below.
  • the expression of transgenes encoding the corresponding C. elegans and human proteins can be engineered in C. elegans wild- type or C. elegans mutant strains resulting in transgenic and humanized worms respectively: Table 1
  • a range of C. elegans mutants may be obtained from the C. elegans mutant collection at the C elegans Genetic Center, University of Minnesota, St Paul, Minnesota.
  • specific mutants may be generated by standard methods. Such methods are described by Anderson in Methods in Cell Biology, Vol 48, "C. elegans : Modern biological analysis of an organism" Pages 31 to 58.
  • Several selection rounds of the PCR technique can be performed to select a mutant worm with a deletion in a desired gene.
  • Other methods of generating mutants with targeted defective gene expression are described by Sutton and Hodgkin, Zwaal et al and Fire et al as described above.
  • dilution series were made for some chemical substances. These include the chemicals clomipramine, tamoxifen, BP554, pimazide and thapsigargin. A concentration range was made from less than 1 ⁇ M up to 100 ⁇ M, and the pumping assay was repeated as described in previous examples. From these results distinct dose-response curves could be drafted.
  • GFP-calmodulin was expressed in the pharynx of C. elegans and fluorescence was observed using two-photon microscopy. It has been shown that inhibitors of pumping such as ivermectin and enhancers of pumping such as serotonin influence the observed fluorescence of the GFP-calmodulin in a predicted way.
  • the human SERCA-2 protein is known to be negatively regulated by at least one protein, known as phospholamban (PLB) . Both are expressed in the heart of vertebrates, and an extensive list of literature exists on the features of this interaction.
  • PLB phospholamban
  • An increase of the internal storage of calcium is general considered to be important for the strength of muscle contraction, and consequently an improvement or increase of this muscle contraction can be realized by enhancing the SERCA activity.
  • Chemical substances that enhance the SERCA activity or inhibit the SERCA-PLB interaction are considered as potential therapeutics, or as hits for the further development of therapeutics in the disease areas which are the cause of a malfunction of the calcium biology of the cell or organism. Examples of disease areas where an increase of SERCA activity may be beneficial are cardiac hypertrophy, cardiac failure, arterial hypertension, Type 11 diabetes and Brody disease.
  • pig and/or human SERCA under the SERCA and/or myo-2 promoter.
  • pig and/or human SERCA under the SERCA and/or myo-2 promoter in a C. elegans mutated for the C. elegans SERCA (Knock-outs and selected mutants) .
  • pig and/or human PLB under the SERCA and/or the myo-2 promoter.
  • pig and/or human PLB under the SERCA and/or the myo-2 promoter in a C. elegans mutated for the C. elegans SERCA (Knock-out and selected mutants) .
  • transgenic and mutant animals show a clear change in pharynx pumping rate as could be measured by the fluorescence of calcein in the gut using the calcein-AM pharynx pumping assay. Some of these strains were considered to be useful for further screen development.
  • the transgenic and mutant animals were placed in the wells of multi-well plates. Calcein-AM and chemical substances under test were then added. The fluorescence of the calcein formed in the gut was measured in a multi-well plate reader set to measure fluorescence. Chemical substances that altered the properties of the pharynx pumping rate, and hence altered the function and activity of the SERCA pathway were selected for further analysis, and can be considered as potential compounds for the-ra-peutic use, or as hits for the further development of therapeutics.
  • the anatomy of the pharynx of the nematode consists of several parts, containing several cells and cell types. These include the pharyngeal muscles, the pharyngeal epithelial cells, the pharyngeal glands, and .the pharyngeal neurons. At least 14 neurons are involved in the function of the neuron from--which the most important are II, 12, 13, M3, MC, NSM, Ml, RIP and M4 (reviewed in "The nematode C. elegans ed. by W.B. Wood, 1988, CSHL Press) .
  • Mutations or dysfunctions in any part of the pharynx will result in an altered pumping rate of the pharynx.
  • Several mutations are known in the literature to give rise to an altered pumping rate, or to have an altered pharynx morphology.
  • Another way to alter the cells involved in pharynx function, pharynx pumping and pharynx morphology is by applying using transgenic techniques to the nematode.
  • Transgenic C. elegans can be constructed which express these genes in a tissue specific way.
  • the myo-2 promoter will induce expression in the pharynx muscles
  • the unc-129 promoter will induce expression in the neuronal cells.
  • a cell type-specific or tissue-specific promoter can be selected so that degeneration of the tissues can be precisely controlled. Promoters can be selected in such a way that the expression of the toxic gene is only induced in one specific cell.
  • the ASI neurons of C. elegans are chemical-sensory neurons and are essential for food perception and pharynx pumping. It has previously been reported that the disruption of the ASI or ADF or ASG or ASJ neuron results in dauer formation. These experiments that kilt " one or more of these neurons were performed with laser ablation. (Schackwitz WS et al., Neuron 17:719- 728, 1996) . Furthermore it was reported that the Daf- 7 (a member of the TGF-beta family) is expressed specifically in the ASI neuron.
  • Example 13 Specific example of the assay, with dauers.
  • Daf-2 ts is a nematode mutant, which grows normally at 15°C but generated 100 % dauer formation at 25°C, these mutants can also be used in screens to isolate chemical substances that cause worms to bypass the dauer phenotype.
  • Hexamethonium and mecamylamine are well known acetylcholine receptor antagonists and hence should repress the overload of acetylcholine in the synapses of the acel; ace2 mutant, resulting in restoration or rescue, of the movement.
  • receptor antagonist hexamethonium will also result in a decrease of movement, as it prevents proper signalling.
  • hexamethonium represses the movement of wild-type worms, but significant less than neostigmine (100% represents normal movement of wild-type worms).
  • Example 15 Example of a mating assay using hermaphrodite non- selfers .
  • the hermaphrodite chosen in this experiment has a reduced self-offspring or the offspring is non-viable or preferentially the hermaphrodite is self-sterile, such as the hermaphrodites mutant in the fer or spe genes. Furthermore, to enhance mating the self- sterile hermaphrodite has preferentially a reduced movement or no movement phenotype.
  • the males in this experiments can be wild-type males, or mutant males, or transgenic males, or humanized males.
  • Mating behaviour is assessed by measuring the total number of offspring produced, as described above .
  • a mating assay has also be performed with a specific self-sterile transgenic hermaphrodite that has a reduced movement phenotype and expresses stably GFP. All offspring of this mating assay express GFP and hence the number of offspring can easily be detected by measuring the GFP fluorescence using a multi-well plate reader or a FANS. Hermaphrodites expressing other makers such as luminescent markers can be used in an analogous experiment.
  • hermaphrodites were chosen in following combinations: a) The hermaphrodites were wild-type hermaphrodites, or hermaphrodites showing a reduced movement phenotype b) The male nematodes were wild-type, transgenic, mutant or humanized nematodes, expressing GFP.
  • the offspring of the self- fertilization of the hermaphrodite, and the offspring resulting from the genuine mating could be distinguished by following the fluorescence of the GFP as only the offspring resulting from a mating showed GFP expression.
  • Table 3 lists C. elegans male-specific neurons and their role in mating behaviour. Disruption of one or more of these neurons, for example by expression of a toxic gene, may result in C. elegans variants which can be useful in mating screens .
  • the egg laying assay can also be performed using transgenic C. elegans which exhibit altered egg laying behaviour as a result of the expression of a toxic gene in a specific tissue or cell type.
  • Suitable transgenic C. elegans can be constructed according standard techniques known in the art using one of the toxic genes listed above under the control of an appropriate tissue- or cell type-specific promoter. Promoters which may be useful for this purpose include the lin-31, egl-17, unc-17 and unc-53 promoters .
  • the defecation assays can also be performed using transgenic C. elegans which exhibit altered defecation behaviour as a result of the expression of a toxic gene in a specific tissue or cell type.
  • Suitable transgenic C. elegans can be constructed according standard techniques known in the art using one of the toxic genes listed above under the control of an appropriate tissue- or cell type-specific promoter. Promoters which may be useful for this purpose include the unc-43 and unc-25 promoters.
  • Table 8 Enhancers of the C. elegans pharynx pumping rate isolated from the pharmacopoeia.
  • Table 9 Inhibitors of the C. elegans pharynx pumping rate isolated from the pharmacopoeia.
  • Table 10 partial list of hits obtained when screening 800 compounds from a pharmacopoeia library using a movement assay with C. elegans .
  • the hit compounds were scored as causing a detectable change in the movement behaviour of C. elegans .
  • Table 11 Enhancers of C. elegans pharynx pumping found from screening the Tocris compound library (Bristol, UK) using a pharynx pumping assay.

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