Note: Descriptions are shown in the official language in which they were submitted.
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Compound screening method
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. Specifically, 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 lmm long in three
days or so. As it is fully transparent at all stages
in 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 types 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.
These characteristics of C. elegans make it an
extremely useful tool in the drug discovery process.
In particular, 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
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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
Biology, Chapter 8, volume 84, Caenorhabditis elegans:
Modern Biological analysis of an Organism Ed. Epstein
and Shakes, Academic Press, 1995 and J. Ahringer in
Curr. Op. in Gen. and Dev. 7, 1997, 410-415.
-Rand and Johnson in particular describe compound
screening assays in which varying concentrations of
the compound to be tested are added to nutrient agar
or broth which is subsequently seeded with bacteria
and then inoculated with worms. Any phenotypic
changes in the worm as a result of exposure to the
compound are then observed.
Although the nematode, and in particular
C. elegans, is proving a powerful and efficient tool
in the identification or discovery of
pharmacologically active molecules, the presently
known techniques for compound screening do not readily
lend themselves to high throughput screening. This is
largely because the known assay techniques rely on
visual inspection of worms exposed to the compound
under test in order to determine whether the compound
has-ari effect on the phenotype of the worms.
Consequently, even if an assay were to be performed in
the multi-well assay format necessary for high
throughput screening it would be necessary to score
each individual well by eye in order to determine the
outcome of the assay.
There is thus a need for reliable and
reproducible screening methods using live C. 212gans
which do not require scoring by visual inspection and
are therefore more suitable for use in automated high
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throughput screening. The availability of such
screening methods would dramatically increase the
usefulness of C, elegans as a screening tool, enabling
researchers to exploit the enormous potential of C.
elegans as a whole animal system for drug discovery
and development.
Accordingly, in a first aspect the invention
provides a method of identifying chemical substances
which have potential pharmacological activity using
nema~ode worms, which method comprises the.steps of:
(a) dispensing substantially equal numbers of
nematode worms into each of the wells of a multi-well
assay plate;
(b) contacting the nematode worms with a sample
of a chemical substance;
(c) detecting a signal indicating phenotypic,
physiological, behavioural or biochemical changes in
the nematode worms using non-visual detection means.
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.
However, depending on the type of activity to be
detected, 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.
Typically, compound screening assays involve running a
plurality of assay mixtures in parallel with different
concentrations of the chemical substance under test.
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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.
In a second aspect the invention provides,a
method of determining the mode of action of a chemical
substance using nematode worms, which method comprises
the -steps of
(a) dispensing substantially equal numbers of a
panel of different mutant nematode worms into each of
the wells of a multi-well assay plate;
(b) contacting the nematode worms with the
chemical substance; and
(c) detecting a signal indicating phenotypic,
physiological, behavioural or biochemical changes in
the nematode worms using non-visual detection means.
In this method, 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.
In a third aspect 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
(a) subjecting a population of nematode worms to
random mutagenesis;
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(b) dispensing one mutager.ized Fl nematode worm
into each of the wells of a multi-well assay plate;
(c) allowing the F1 nematode worms to generate
F2 offspring;
(d) contacting the nematode worms with the
compound; and
(e) detecting a signal indicating phenotypic,
physiological, behavioural or biochemical changes in
the nematode worms using non-visual detection means.
This method of the invention is, in effect, a
classic a genetic suppressor screen performed in a
multi-well format. In 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. Therefore, to
identify a suppressor mutant one effectively looks for
mutants which exhibit no or minor changes in
phenotypic, physiological, biochemical or behavioural
characteristics in part (e) following exposure to the
compound.
There are many advantages to be gained from
performing genetic suppressor screens in a multi-well
format, as described by the inventors. In particular,
less compound is required to perform an assay in a
multi-well plate, as compared to a standard agar plate
assay. Furthermore, as the assay in multi-well plates
is performed in liquid, compounds to be tested are
taken up more efficiently by the nematodes than in a
standard plate assay and also compounds tend to
precipitate less in liquid than on agar plates, due to
the lower concentration.
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In a fourth aspect 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:
(a) dispensing substantially equal numbers of
nematode worms into each of the wells of a multi-well
assay plate;
(b) contacting the nematode worms with the first
compound;
(c)contacting the nematode worms with a further
chemical substance; and
(d) detecting a signal indicating phenotypic,
physiological, behavioural or biochemical changes in
the nematode worms using non-visual detection means.
This method may be used to screen for antagonists
of a given compound. This principle is illustrated in
the accompanying Example 8.
The methods of the invention are all performed in
a multi-well plate format and are therefore
particularly suitable for use in mid-to-high
throughput screening. In a preferred embodiment, the
multi-well plates have 96 wells, but the invention is
also applicable to multi-well plates with another
number of wells, which include but is not restricted
to plates with 6, 12, 24, 384, 864 or 1536 wells. The
terms "multi-well plate" and "microtiter plate" are
used interchangeably throught.
As with all the screening methods described
herein the above-described methods are preferably
performed using nematode worms from the genus
Caenorhabditis, most preferably C. elegans or C.
briggsae. Although C. elegans and C. briggsae are
preferred, it will be appreciated that the screening
methods described herein could be carried out with
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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 lmm 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. .
All of the methods of the invention require the
detection of a signal which indicates phenotypic,
physiological, behavioural or biochemical changes
occurring in the nematode worms in the presence of the
compound under test. It is an essential feature of
the methods of this invention that this signal (also
referred to as the read-out) is detected using a non-
visual detection means. As used herein the term "non-
visual detection means" refers to any means of
detecting a signal which does not require visual
inspection by the human eye.
The use of a 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 signal generated as a result of phenotypic,
physiological, behavioural or biochemical changes in
the nematode worms can be of various types including,
for example, a fluorescent, luminescent or
colorimetric signal generated in the nematode worms
themselves or a change in optical density in a whole
suspension of worms.
In one embodiment of the methods a signal is
generated by a marker molecule which is added to the
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worms following contact with the chemical substance
under test. The marker molecule is taken up by the
worms and the activity of the chemical substance on
the nematode worms can then be monitored either
directly or indirectly by detecting signal resulting
from a change in the properties of the marker molecule
as a result of phenotypic, physiological, behavioural
or biochemical changes in the worms.
There are various ways in which the worms can
take- up the marker molecule. For example,.worms may
take up the marker as a result of the action of a
chemical substance under test. Another possibility is
that 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.
Alternatively, the marker molecule can be a
genetically encoded marker which is expressed in cells
of the nematode worms themselves. Routine methods for
the construction of transgenic C. elegans are well
known in the art and with the use of appropriate
promoter sequences transgenic C. elegans can be
constructed which express a genetically encoded marker
molecule in all cells, in a particular tissue or in
one-or more specified cell types. Suitable
genetically encoded marker molecules include
autonomous fluorescent proteins (AFPs) such as green
fluorescent protein (GFP)and blue fluorescent protein
(BFP), aequorin, alkaline phosphatase, luciferase, ~i-
glucuronidase, ~i-lactamase, ~3-galactosidase,
acetohydroxyacid synthase, chloramphenicol
acetyltransferase, horseradish peroxidase, nopaline
synthase or octapine synthase.
The marker molecule can also be added to the
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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. Examples of such precursor marker
molecules include calcein-AM , fluorescein diacetate
(FDA) and BCECF-AM which are cleaved by esterases,
alkaline phosphatase substrates such as fluorescein
diphosphate and AMPPD, aminopeptidase substrates such
as CMB-leu, and glucuronidase substrates such as X-
gluc.
In order to assist in the measurement of a signal
generated using a marker molecule, fluorescence
quenchers or luminescence quenchers may be used. For
example, a quencher could be added to the medium in
order to quench any background fluorescence in the
medium, this may make it easier to visualise a
fluorescence signal from the gut of the nematodes.
Suitable non-visual detection means include
muiti~aell plate readers, also '.known as microtiter
plate readers or elisa plate readers. The use of
microtiter plate readers facilitates high throughput
screening to select for active chemical substances
with potential pharmacological activity. Suitable
mufti-well plate readers are commonly used in the art
and are available commercially. Such plate readers
can be used with a wide range of detection methods
including fluorescence detection, luminescent
detection, colorimetric detection, spectrophotometric
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detection, immunochemical detection, radiation
detection and optical density detection.
The advantage of mufti-well plate readers is that
they can be used to make quantitative measurements of
the signal generated as a result of the activity of
the chemical substances on the nematode worms. The
ability to make quantitative measurements means that
it is possible to construct quantitative dose response
curves of the activity of a chemical compound on the
nematodes. Using these dose response curves one can
determine the IC50 and ED50 of compounds in nematodes
such as C. elegans, and hence determine optimal
concentrations. Furthermore, the dose response curves
enable the determination of any toxic effects of the
compound and may also give an indication of possible
secondary targets and side-effects of the compound.
Non-visual detection systems other than multi-
well plate readers can also be used in the methods of
the invention. An example of such a detection system
is based on 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. For screening assays
to be performed with a small number of nematodes or
for assays that give a faint signal, or for assays for
which the presence of food can be a disadvantage in
the measurement of the signal, a FANS is a preferred
detection instrument. However, the use of a FANS is
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not limited to these experimental conditions, FArdS
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. In short, worms are
contacted with the chemical substances with or without
the addition of a marker molecule. After the
appropriate time, the multi-well plates are submitted
to t~Fie 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.
In order to generate quantitative results using
the methods of the invention it may be important to
ensure that substantially equal numbers of individual
nematodes are added to each of the wells. The precise
number of 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.
rious 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
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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. 0.250 low
melting point agarose), glycerol etc.
As an alternative to the above-described approach
an equal distribution of worms over the wells of the
mufti-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. In addition, 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, Ll, 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 mufti-well
plate. The mufti-well screens described by the
inventors are performed in liquid medium. However, as
the natural environment of nematodes such as C.
elegans is 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. to
produce a viscosity greater than that of normal liquid
medium for nematode culture, such as M9). Use of a
viscous liquid medium retains the advantages of liquid
culture, i.e. ease of handling nematodes in liquid,
whilst maintaining the health of the worms. Preferred
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types of polymer are low melting point agarose,
carboxymethyl cellulose and polyethylene glycol
(especially PEG8000). The optimum amount of polymer
to be added may be determined by routine experiment
and may vary depending on the nature of the read-out
of the assay. For example, in the 'movement assay'
described below addition of 0.3o medium viscosity
carboxymethyl cellulose has been determined to be
optimal. The inventors have used several viscosity
variants of carboxy methyl cellulose cellulose to
determine the optimal conditions to perform the
screens described herein. In one experiment, three
variants of carboxy methyl cellulose, namely low,
medium and high viscosity carboxymethyl cellulose
provided by Sigma (St. Louis, M0, USA) were tested at
a concentration of 0.3% (see Figure 11). It was
observed that at this concentration the medium and the
high viscosity carboxymethyl cellulose showed the best
results in the screens. For practical reasons, it is
preferred to use medium viscosity carboxymethyl
cellulose. A concentration of about 0.30
carboxymethyl cellulose is suitable for the majority
of screens. The addition of a water polymer to
increase the viscosity of the assay medium 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 multiwell
(microtiter) plate.
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
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concentration sufficient to prevent the nematodes from
sticking to the wells of the microtiter plate.
Due to the nature of the plastic material used
for the construction of microtiter plates the surfaces
of such plates are generally hydrophobic. Hence,
because the outer surface of nematodes such as C.
elegans is also hydrophobic the nematodes have a
preference to stick to the walls of microtiter plates,
significantly hampering the performance of assays
carried out in microtiter plates. This problem might
be avoided with the use of different types of
microtiter plates but to date there are no microtiter
plates available on the market which sufficiently
reduce this problem. The present inventors have now
found that the problem of nematodes sticking to the
walls of microtiter plates can be overcome by the
addition of a suitable concentration of a water
soluble polymer to the assay medium.
Preferred types of water soluble polymer are
polyethylene glycol (PEG), particularly PEG8000,
polyvinyl alcohol (PVA) and polyvinyl pyrrolidone
(PVP), with PEG8000 being the most preferred. For a
given type of polymer in a given type of screening
assay the optimal concentration of polymer to be added
to the medium can readily be determined by routine
experiment. For PEG8000, a concentration. of 0.1~
gives good results in most types of screens. Polymer
concentrations in the range 0.01% to loo may also be
suitable, depending on the type of assay.
Addition of a polymer to the assay medium results
in a particular improvement for assays performed in
mufti-well plates having more than 96 wells by
preventing the worms from sticking to the walls of the
wells. Moreover, the presence of the polymer in the
medium generally facilitates manipulations of
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nematodes in liquid culture, such as pipetting and
operation of automated dispensing systems such as the
FANS device, the Qfit2 device from Genetix (Dorset,
UK) and other automated systems used to fill
microtiter plates.
The addition of 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.
All of the screening methods described herein can
be performed using various kinds of C elegans,
including wild-type worms, selected mutants,
transgenic worms and humanized worms. The transgenic
strains can be strains expressing a transgene in the
whole organism, or in a part of the organism, in a
single tissue, in a sub-set of cell types, in a single
cell type or even in one cell of the organism. The
mutant worms may carry a mutation in a single gene or
in two or more different genes. Humanized worms are
particularly useful for the identification of
compounds with potential therapeutic activity in the
human pharmaceutical field as they can be used to
perform screens which are specifically directed at
human target proteins but which have all the
advantages of the nematode biology and ease of
manipulation.
Standard methods for culturing nematodes are
described in Methods in Cell biology Vol. 48, 1995,
ed. by Epstein and Shakes, Academic press. Standard
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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
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 C. elegans 1998, Nature 391, 860-811.
A population of worms can be subjected to random
mutagenesis by using EMS, TMP-UV or radiation (Methods
in Cell Biology, Vol 48, ibid). Several selection
rounds of PCR could then be performed to select a
mutant worm with a deletion in a desired gene. In
addition, a range of specific C. elegans mutants are
available from the C. elegans mutant collection at the
C. elegans Genetic Center, University of Minnesota, St
Paul, Minnesota.
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. For example, the worm may be exposed
to a chemical substance/compound listed in a
pharmacopoeia with known pharmacological activity.
Alternatively, 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
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proteins are not excluded.
In one embodiment the methods of the invention
are performed using transgenic C. elegans expressing a
transgene which comprises a 'toxic gene'. In this
context 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
(hun~ingtin), 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. Using appropriate tissue-
specific or cell type-specific promoters 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.
Many suitable tissue-, cell type- or developmentally-
specific promoters are known for use in C. elegans.
All of the screening methods described herein can
also be performed using synchronized worm cultures.
Synchronized worms are worms that are in the same
growth stage. The various growth stages of nematode
worms such as C. e~egans are eggs, the L1 stage, L2
stage, L3 stage, L4 stage and adult stage.
Furthermore, in a preferred embodiment of the
invention, 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
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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). To generate C. elegans dauers,
several techniques have been described (Elegans II,
ibid). Mainly, a temperature sensitive daf-c mutant
of C. elegans is used to generate dauers, although
other possibilities exist such as daft-is mutants
which produce 100% dauers at 25°C.
In a particular embodiment of the methods of the
invention, 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. These
methods may be hereinafter be collectively referred to
as 'pharynx pumping assays'.
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 cf taking up
liquid and subsequently spitting it out is called
pharyngeal pumping or pharynx pumping.
Because the process of pharynx pumping involves
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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.
In particular, 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. As esterases are present in the
gut of nematodes such as C. elegans, the pharynx
pumping rate can be measured indirectly by measuring
calcein fluorescence.
In the examples given herein, 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:
- Esterase substrates. Calcein-AM, FDA, BCECF-AM
-Alkaline phosphatase substrates: Fluorescein di
phosphate FDP)
-Endoprotease; Aminopeptidase substrates: CMB-leu
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With a luminescent read out:
-Alkaline phosphatase substrates: AMPPD
With a colour read out:
-Glucuronidase substrates: X-gluc
This list is not exclusive, marker molecule
precursors can also be found or developed which are
cleavable by other enzymes present in the C. elegans
gut, such as DNAses, ATPases, lipases, amylases, etc.
Once such a marker precursor enters the gut, it is
cleaved to release the detectable marker which can
then be monitored. Thus, it is possible to measure the
rate of pharynx pumping indirectly by measuring the
accumulation of a detectable marker molecule in the
gut.
The pH of the C. elegans gut is low, therefore
molecules that becomes fluorescent at a low pH are
useful tool to assess the rate of pharynx pumping.
LysoSensor green from Molecular probes exhibits such
properties and has been successfully used to assess
pharynx pumping. The fluorescence observed in the gut
is similar, but less bright, to the fluorescence
obtained with calcein-AM. However, 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 i~erfere with the read-out as it is dependent
only on a pH change and is not enzyme related.
LysoSensor marker molecules or probes are weak
bases that are selectively concentrated in acidic
organelles as a result of protonation. This
protonation also relieves the fluorescence quenching
of the dye by its weak-base side chain. Thus, the
LysoSensor dyes become more fluorescent in acidic
environments . The blue-fluorescent LysoSensor Blue
and green-fluorescent LysoSensor Green probes are
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available with optimal pH sensitivity in either the
acidic or neutral range (pKa ~5.2 or ~7.5). With their
low pKa values, LysoSensor Blue DND-167 and
LysoSensor Green DND are almost nonfluorescent except
when inside acidic compartments.
The pharynx pumping assays can also be performed
using mutant C. elegans strains which have a
constitutively pumping pharynx or by using transgenic
strains which also exhibit this phenotype. By using a
wild-type strain or the constitutive pharynx pumping
strain, it is possible to identify chemical substances
that enhance, inhibit or modulate pumping rate,
respectively.
As the pharynx of the nematode C. elegans is a
muscle and the pumping rate is mainly governed by some
selected neurons, measuring changes in the pharynx
pumping rate is a good tool to study neurotransmitter
signals and the stimulation of muscles. As the rate
of pharynx pumping can be measured quantitatively and
a method has been developed to screen for chemical
substances which influence this pumping rate, the
present invention is a method to screen and isolate
chemical substances with potential pharmacological
activity.
Chemical substances that influence the pharynx
pumping rate will most probably be substances that
have an activity on general muscle biology, and/or cn
neurotransmitter pathways. Examples of proteins which
can be the target of these chemical substances are
neurotransmitter receptors such as muscarinic
receptors, glutamate receptors, hormone receptors and
5-HT receptors, cannabinoid receptors, adrenergic
receptors, dopaminer'~ic receptors, opioid receptors,
GABA receptors, adenosine receptors, VIP receptors and
nicotinic receptors, proteins involved in
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neurotransmitter synthesis, neurotransmitter release
pathway proteins, G-protein coupled receptor proteins.
Furthermore proteins for G-protein coupled second
messenger pathways such as adenylate cyclase, protein
kinase A, cAMP responsive element binding proteins,
IP3, diacylglycerol, protein kinase C phospholipase A-
D, phosphodiesterases, and proteins encoding for
functions in gap junctions, proteins involved in
oxidative phosphorylation in mitochondria and proteins
involved in other energy-related pathways,. 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,
-molecules that enhance, inhibit or modulate the
synthesis of a neurotransmitter,
- molecules that have a function in the depletion of
the transmitter,
- molecules that prevent or stimulate the release of
the transmitter from the synaptic vesicles in the
synaptic cleft,
- molecules that function as a receptor inhibitor or
stimulator,
- molecules that mimic the transmitter molecules that
function as conduction inhibitors or activators,
-molecules that function as an activator or inhibitor
of the conduction blockade,
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-molecules that prevent or stimulate the re-uptake of
transmitter after firing of the neuron,
-molecules that function as a false transmitter (+/-),
-molecules that prevent or stimulate receptor
clustering,
-molecules that act in novel pathways.
Thus, 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.
Furthermore, 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.
-V~ith the pharynx pumping assay there is the
potential to screen for 10 to 15 modes of action and
for 2 to 6 neurotransmitter pathways and ion channels.
As both activation as inhibition can be observed, this
screening method will make it possible to screen for
to 180 targets in a single screen.
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
35 chemical substance, or to select for chemical
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substances which act on a specific target
In order to perform a screen to identify the
mode of action of a compound substantially equal
numbers of a panel of different defined mutant,
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. For each of
the mutant, transgenic or humanized strains 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.
The examples given herein list several 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 encoding for
functions in gap junctions, ion channel proteins and
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ion pump proteins. A non-exhaustive list of well
known mutants which are suitable for use in this
aspect of the invention is provided in the examples
given herein.
Using such mutant, 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,
anxiolytics, 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.
Furthermore, 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. Using appropriate transgenic,
mutant or modified strains, it is possible to screen
for chemicals which act in specific targets and thus
identify also insecticides and nematocides.
These 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.
In a particularly important embodiment of the
invention, the pharynx pumping screen may used to
screen for compounds having potential insecticidal
activity. The inventors hays observed that exposure
of C. elegans to compounds having pesticidal activity,
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such as herbicides, insecticides, nematocides or
fungicides, has an effect on the pharynx pumping rate.
This is illustrated in the accompanying Figures 18 to
21 which show the effects of known insecticides on the
pharynx pumping rate of C. elegans, as measured using
the pharynx assay methodology. Hence, the pharynx
pumping screen can readily be adapted to screen for
compounds having pesticidal activity.
In another embodiment of the invention 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 mufti-well plates
thousands of worms can be screened at once.
First, a random pool of mutant worms is
generated. Several techniques such as EMS mutagenesis,
TMP-UV mutagenesis and radiation mutagenesis have been
described to generate mutant worms (Methods in Cell
biology, Vol. 48, ibid). One mutagenized F1 nematode
is then dispensed into each of the wells of the multi-
well plate and the F2 generation are allowed to
produce offspring in the wells. A sample of a
compound that has a known activity on the nematode
worms is then added to the F2 worms. Changes in the
pharynx pumping rate are then monitored as described
above, for example using a marker molecule or a marker
molecule precursor.
Mutant worms are scored in which the effect of
the compound on the pharynx pumping rate is
suppressed, enhanced or modulated. These mutant worms
will have mutations in one or more genes that are
affected by the compound. The mutated gene or genes
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can then be isolated using standard genetic and
molecular biology techniques. These genes, and their
corresponding proteins, are considered to be important
genes and proteins of the affected pathway, and hence
are putative new targets for the further development
of screens in the drug discovery process. As with all
the methods described herein, this method is
preferably performed using microscopic nematodes,
particularly worms of the genus Caenorhabditis and
most preferably C. elegans.
In still another embodiment of the invention the
pharynx pumping assay methodology can also be used to
screen for chemical substances that are enhancers,
suppressors or modulators of a selected chemical
compound having a defined effect on nematode worms.
In this assay worms are placed in multi-well
plates with a compound that has known effect on the
pharynx pumping rate of nematode worms. A second
chemical substance is then added to each of the wells
and chemical substances which enhance, reduce or
modulate the effect of the selected compound are
identified by detecting changes in the pharynx pumping
rate of the nematode worms using the methods described
above. This method is useful to screen for chemical
substances that are active in a selected biochemical
pathway. The chemical substances thus isolated can be
putative therapeutics, or can be considered as hits
for further drug development.
In a still further embodiment of the pharynx
pumping assay, 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
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the cell.
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.
In a still further embodiment of the pharynx
pumping assay 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. In addition, 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.
The examples given below list known C. elegans
mutants that exhibit an altered pumping rate as a
result of an altered pharyngeal morphology. In
addition, it is possible to generate C. el2gans worms
which exhibit a defect in one or more of the cell
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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. In this context 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. For example, 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
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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. Furthermore, by altering the nature of the
toxic gene expressed in cells of the
pharynx/pharyngeal neurons chemical substances will be
isolated that are useful in the development of
treatments for Parkinson's disease, Alzheimer's
disease, Lewy body disease, Best macular dystrophy,
age-related macular dystrophy and polyglutamine-
induced diseases such as Huntington's disease,
Kennedy's disease and ataxia. 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.
Tfie performance of the pharynx pumping assays
described herein may be improved by adding a water
soluble polymer to the assay medium in order to
increase the viscosity of the assay medium. Preferred
types of polymer are carboxymethyl cellulose,
polyethylene glycol (especially PEG8000) and low
melting point agarose. For any given type o. polymer
and type of pharynx pumping assay, the optimum
concentration of polymer added to the medium may be
determined by routine experiment. As illustrated in
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the accompanying Figures 14 and 15, addition of a
polymer to increase the viscosity of the assay medium
results in increased pharynx pumping in C, elegans.
It is thought that the use of a more viscous medium
may mimic the more solid conditions in which C.
elegans naturally lives.
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 mufti-well plate. Preferred types of polymer
are polyethylene glycol, particularly PEG8000, PVA and
PVP, with PEG8000 being most preferred. For any given
type of polymer and type of pharynx pumping assay, 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. During the setting up of the assay 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. One may conclude that addition
of PEG8000 to the medium, results in more pumping and
less variation in the pharynx pumping assay (see
Figure 10).
In a further embodiment of the invention the step
of detecting a signal indicating a phenotypic,
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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.
In this particular embodiment of the invention,
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. For example, changes in intracellular
levels of Ca2+ 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. Thus, when
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
mufti-well plate reader. Other genetically encoded
sensor molecules could be used whose fluorescent or
luminescent properties are altered in the presence of
secondary messengers such as, for example, cAMP,
diacyl~glycerol or inositol triphosphates (IP3).
Preferably this aspect of the invention is
carried out using 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
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cells of the pharynx, the vulva muscles, the body wall
muscles and neurons. As in previous examples, the
transgenic worm can be of wild-type genetic
background, a mutant transgenic or a humanized strain.
As 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.
In a still further embodiment of the invention,
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.
The movement of nematode worms is mainly the
result of the action of the body wall muscles and is
regulated by neuronal activity. Accordingly, screens
based on detection of altered movement behaviour can
be developed to identify chemical substances which may
have an effect on muscle and/or neuronal activity.
Advanced multi-well plate readers are able to
detect sub-regions of the wells of multi-well plates.
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By using these plate readers it is possible to take
measurements in selected areas of the surface of the
wells of the mufti-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) or optical
density can be observed in the wells containing
nematodes that move normally as compared to wells
containing nematodes that are defective fo.r movement.
For the wells containing the nematodes that move
normally, 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.
In an adaptation of the movement assay,
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
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.
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
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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
(summarised in Table 10) 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.
The inventors have observed that this problem may
be overcome with the addition of a polymer to the
liquid medium in order to increase the viscosity of
the medium. The increased viscosity allows for more
resistance to the moving worm and hence better
suspension in the medium. The inventors have tested
low, medium and high viscosity variants of
carboxymethyl cellulose (from Sigma, St. Louis, rIO,
USA and described hereinbefore) to determine optimal
conditions for the movement assay. A concentration of
0.3% medium viscosity carboxymethyl cellulose was
determined to be optimal. This effect is not,
however, limited to carboxymethyl cellulose and
similar improvement in the movement assay can be
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achieved using other types of water soluble polymer,
for example low melting point agarose, PEG etc. The
precise 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. For
any liven type of polymer and type of assay, the
concentration of polymer required for optimum
performance of the assay can readily be determined by
routine experiment.
The effect of viscosity in the movement assay has
been determined for various C. elegans mutants in a
comparative study, the results of which are
illustrated in Figures 12 and 13. In this study C.
elegans Unc and Ace mutants having movement defects
were compared with each other and with wild-type C.
elegans N2, in M9 medium and media with varying
viscosity. The results of this experiment illustrate
that medium to high concentrations of carboxymethyl
cellulose improve the movement assay.
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.
Preferred types of polymer are polyethylene glycol,
particularly PEG8000, PVA and PVP, with PEG8000 being
most preferred. For any given type of polymer and
type of movement assay, the optimum concentration o.
polymer added to the medium may be determined by
routine experiment. For PEG8000, a concentration of
O.lo is particularly preferred.
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As with the other screening methods described
herein the movement assay methods are preferably
carried out using microscopic nematode worms,
particularly those of the genus Caenorhabditis and
most preferably C. elegans. 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
doub3e mutant, is particularly suitable fo.r 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.
In a still further embodiment of the invention 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 such as C.
elegans is very complex, involving at least following
steps: recognition, backing, tail curling, vulva
locat-ion and copulation. To perform this behaviour,
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
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meeting abstract 15 1, 199 1). Due to the complex
nature of the mating behaviour, several conditions and
mutants have been described to enhance mating
behaviour in C. elegans. One of these is the use of
hermaphrodites with a decreased movement, such as the
unc-52 (e444) mutant which shows paralysed behaviour
at adulthood.
Because mating involves the activity of both
muscles and neurons, screens based on detecting
chart-ges in the mating behaviour of nematode worms(the
mating assay) can be used to identify chemical
substances which may modulate muscle and/or neuronal
activity. The mating assay can be used to isolate
chemical substances that modulate mating, or to
isolate chemical substances that modulate the activity
of a compound that effects mating behaviour, or to
isolate genes and pathways that are active in the
mating behaviour, or to isolate genes and pathways
that modulate the activity of a compound that affects
mating behaviour. In other words, the mating assay
can be used for all the same purposes as the pharynx
pumping assays and movement assays described above.
C. elegans are not able to perform mating in
liquid media. The high-throughput screens based on
mating behaviour are therefore performed in semi-
liquic4<conditions. A low-melting agarose solution of
approximately 0.5% is suitable for this purpose. This
semi-liquid medium gives sufficient support for the
nematodes to move toward each other and to perform
mating. Addition of other polymers to obtain suitably
viscous medium may also be used in the mating assay.
Mating performance is measured by measuring the
number of eggs or offspring produced from a mating
experiment. In a particular embodiment of the
invention specific strains are used which are not able
to generate offspring by self-fertilization. Such so-
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called hermaphrodite 'non-selfers' cannot generate
offspring, but hermaphrodites that have mated will
generate offspring. The offspring can be measured
directly by the previous described movement test, or a
marker dye can be added to the medium such as calcein-
AM so that the previously described pharynx pumping
screen can be performed. Alternatively, specific
antibodies and fluorescent antibodies can be used to
detect the offspring. Any specific antibody that only
recognizes eggs, or L1 or L2 or L3 or L4 stage worms,
will only recognize offspring, by way of example an
antibody that recognizes an antigen on the surface of
C. elegans L1 larvae has been described by Hemmer et
al., (1991) J Cell Biol, 115(5): 1237-47. Finally,
the number of eggs or offspring in each well can be
counted directly using a FANS device.
In another embodiment of the invention 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. The offspring generated from mating of
these transgenic worms will also express the marker
molecule and hence can be easily measured using a
mufti-well plate reader or a FANS device. In the case
that-the male worms are the transgenic worms
expressing the marker then 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. In the case that the hermaphrodite
worm is the transgenic strain expressing the marker
molecule the hermaphrodite strain is preferably also a
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'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
phar=ynx pumping assays. By using 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 hey-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-T1 promoter which directs gene expression
in 60 cells of the spermatheca.
In a still further embodiment of the invention 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
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behaviour of the nematode worms.
The vulva of hermaphrodite C. elegans nematodes
contains at least 24 cells and several neurons, of
which the HSN neurons are considered to be the most
important in egg-laying. Furthermore at least 8
uterine muscles have been described. Several mutants
have been described in gonad development, egg-laying,
vulva development and function ( The nematode
Caenorhabditis elegans, ibid.; C. elegans II, ibid.),
accordingly, high-throughput screening.assays can
be developed which use a read-out based on detection
of changes in the egg laying behaviour of nematodes
such as C. elegans. Again, assays based on detection
of egg laying can be used for the same purposes as the
pharynx pumping and movement assays described herein.
In these assays 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. In certain embodiments, 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. In further,embodiments, the methods may
compri-se detecting the numbers of eggs produced with
the use of specific dyes which recognize the eggshell.
In one specific embodiment 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. During the hatching process the enzyme
chitinase is released into the medium. The enzyme
recognizes the substrate
4-methylumbelliferyl =-D-N,N,N,-triacetylchitotrioside
(or 4-Methylumbelliferyl
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~i-p-N, N'-diacetylchitobioside) which is a fluorescent
precursor molecule (provided by Sigma, St. Louis, MO).
Hydrolysis of the synthetic substrate
4-methylumbelliferyl triacetylchitotrioside is
followed by measuring the fluorescence of the
liberated 4-methylumbelliferone in a microtiter plate
reader. Other substrates which comprise a dye, which
may be a luminescent, fluorescent or coloured dye,
linked to a chitin moiety may be used in such screen.
An example of this is Resorufin
N-acetyl-Z-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 80u1 of M9
medium. Add the compound to be tested at appropriate
concentrations in l0ul. Add the chitinase substrate
at an appropriate concentration in 10u1. 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.
In a still further embodiment of the invention the
step ~f 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 caorms.
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
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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. Since
defecation requires the activity of both muscles and
neurons high-throughput screening assays can be
developed which use a read-out based on detection of
changes in the defecation behaviour of nematodes such
as C: elegans. Again, assays based on detection of
defecation can be used for the same purposes as the
pharynx pumping and movement assays described herein.
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. 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; Refiner et al., Genetics
141: 961-976, 1995). However, 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. As
defecation in C. elegans requires the activity of
muscles and neurons, compounds which alter the rate of
defecation may potentially have CNS-related
pharmacological activity.
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 ~.T211s of the multi-well
plate the worms will take up the compound which is
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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.
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.
The terbium present in the pre-loaded bacteria
added to the wells will result in a low level of
background luminescence. 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.
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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 nematode 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).
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. When adapted to a short incubation time,
the method may allow monitoring of defects in the
defecation process.
The invention will be further understood with
reference to the following experimental examples
together with the accompanying Figures in which:-
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
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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 0.5uM
calcein-AM. They were handled with or without the
addition of 0.1% PEG.
Figure 11 illustrates the results of experiment to
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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
variQ-us 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. Fig 18-Picrotoxin, Fig 19-
Rotenone, Fig 20-Dieldrin, Fig 21-Ivermectin. 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
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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.
Preferentially, synchronized worms are used. The
production of large amounts of synchronized worms has
been described in (Methods in cell biology, Vol. 48,
ibid). After the worms have grown to the preferred
stage, they are washed in M9 buffer prior to further
use,_and re-suspended in an assay buffer (40mM NaCl,
6mM Kcl, 1mM CaClz, 1mM MgCl2). (10 X M9 buffer: 30g
KHZPO4, 60 g Na2HP04, 50 g NaCl, 10 ml MgS04 1M, made
up to 1 litre with Hz0). 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. In a preferred
set-up of this experiment 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
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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 /.cM up to 100/.cM.
The concentration of the DMSO should not be too high
and preferentially should not exceed lo, more
preferentially the concentration of the DMSO should
not exceed 0.5% and even more preferentially, the
concentration of the DMSO is lower than 0.30.
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Example 2
Conditions for a pharynx pumping assay.
Depending on the specific assay which it is desired
to perform, different 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.
To select for chemical substances that enhance the
pumping of the C. elegans pharynx 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. As wild-type worms show a
reduce-~ pumping rate in this assay, enhancement of the
pumping rate can easily be scored.
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.
2legans gut to release calcein, which is a
fluorescent molecule. The pumping rats of the pharynx
will determine how much medium will enter the gut of
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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. Moreover, using a multi-well plate reader,
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.
To perform the pharynx pumping screen with
calcein-AM, a concentration of between 1 and 100,uM
calcein-AM is added into the medium. Preferably 5 to
lO,uM calcein-AM is used. Fluorescence is measured
using a multi-well plate reader (Victor2, Wallac Oy,
Finland) with following settings: Ex/Em = 485/530.
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~uitable for other precursors. The precursor can
be cleaved by esterases, but could also be a substrate
for other enzymes in the nematode gut. Furthermore,
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:
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With a fluorescent read out:
-Esterases substrates: Calcein-AM, FDA, BCECF-AM
-Alkaline phosphatase substrates: Fluorescein
diphospate (FDP)
-Endoproteases; Aminopeptidase substrates: CMB-leu
With a luminescent read out:
-alkaline phosphatase substrates: AMPPD
With a colour read out.
-Glucuronidase substrates: X-gluc
Other target enzymes present in the gut for which
substrates can be found or developed are DNAses,
ATPases, lipases and amylases. An overview of various
marker molecules, mainly fluorescent can be found in
"Handbook of fluorescent probes and research
chemicals, molecular probes, ed. by R. P. Haughland"
Example 3 Testing the pharynx pumping assay with
compounds from the pharmacopoeia
160 well known drugs selected from the
pharmacopoeia were used in a screen to test the
performance of the pharynx pumping method. The drugs
tested belong to a variety of categories, which
included analgesics, antidiabetics, antiarrythmics,
calcium channel blockers, diuretics, cholinesterase
inhibitors, proton pump inhibitors and
antidepressants.
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
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pumping phenotype, and a mutant C. elegans strain with
a constitutively pumping pharynx was used to detect
inhibitors of pumping.
In a first assay, 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.
In a variation of this protocol, 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.
Although a broad range of chemicals have been
selected from the pharmacopoeia with a variety of
actions, most if not all of the compounds that had an
activity on the pharynx pumping rate belong to the
family of CNS drugs, calcium channel inhibitors and
muscle relaxants, indicating that the C. elegans
pharynx assay is a good model system to screen for
compounds that have activity in the above described
areas.
The variation of the protocol resulted in the
detection of some new compounds, next to the compounds
that have previously been detected; these include the
chemicals metrifonate, physostigmine, atropine,
L-Hyoscyamine, diphenylhydantoin and ZAPA. All these
compounds are known as CNS drugs or are used to treat
Alzheimer's disease or are used as antipsychotic,
antidepressant or antiepileptic drugs.
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Example 4
Selecting for the mode of action of a compound and
selecting compounds which act on specific tar a
Amongst 14 types of pharyngeal neurons, at least
the neurons, 11, 12, 13, M3, MC, NSM, M1, 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. The main neurotransmitters
involved in neuronal signal transduction in the
nematode C. 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).
From the drugs selected in the basic pharynx
screen (Example 3~) it is clear that 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.
To overview the present CNS related drugs, it is
best to classify them according to their biochemical
function in the neurotransmitter pathway cascade. In
brief, CNS related drugs can at least have influence
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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
(_/+) .
Next to these features that are all related to
the neurotransmitter pathways, a lot of CNS related
drugs can be found in the classes of chloride channel
blockers, sodium/calcium channel blockers, calcium
blockers, and other ion channel biockers.
To screen for CNS related drugs, several "in
vitro" screening assays have been developed in the
prior art. These screening methods, designated as "in
vitro binding assays" or "cloned transporter assay
systems" are well known to persons skilled in the art.
For these assays, cell membranes harboring a specific
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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. Depending on the type of
receptor that is present in the membrane,
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.
When the receptor ligand (being the neurotransmitter
in most cases) is radioactive labelled, it is possible
to measure the binding rate of the ligand to the
receptor. 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. Several
variations of this methodology have been developed,
some of which are able to isolate compounds that
inhibit re-uptake of the ligand such as serotonin,
norepinephrine and dopamine (Koppel et al., Chem.
Biol. 1995, Jul 2:7 483-7; Beique et al., Eur. J.
Pharmacol. 1998, May 15 349:1 129-32)
Other systems that have been developed for the
screening of CNS related drugs involve isolated
tissues or organs from mammals. Furthermore systems
have been described to isolate CNS related drugs, with
living animals such as mice.
Although 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. L~Ioreover for every
potential target in the neurotransmitter pathway
cascade, an "in vitro binding assay" needs to be
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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.).
Furthermore it is possible to determine the mode of
action of newly isolated chemical substances and
screen selectively in a certain pathway for chemical
substances with potential pharmacological activity.
A collection of 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. Examples of
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
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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.
These 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.
In addition to these mutants, 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). Once again, both 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 car. be
exp-re~s~ed 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.
To screen for and to isolate chemical substances
that are active in the area of CNS related drugs, 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,
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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.
Furthermore 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. Preferably 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.
An extensive list of mutants which may be
suitable for use in pharynx pumping assays can be
found in C. elegans II, CSHL press and in neurobiology
of the C. elegans genome, C. I. Bargmann, Science
282:2028-2033. A complete list of G-proteins can be
found in " the complete family of G-protein genes of
Caenorhabditis elegans, Jansen G. et al., the Worm
Breeders Gazette, Vol. 15 (5), Feb. 1999.
Some examples of 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. 2legans wild-
type or C. elegans mutant strains resulting in
transgenic and humanized worms respectively:
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Table 1
Neurotransmitters/pathwayC. elegans mutants
Acetylcholine eat-18, eat-2, chat-1, unc-17
Acetylcholine esterasesace-i, ace-2, ace-3
Nicotinic acetylcholineunc-29,unc-38, lev-l,deg-3, acr-2
receptors
Dopamine cat-2,cat-4,bas-l,cat-l,cat-3,cat-5
Serotonin bas-l,mod-5,goa-1
Glutamate avr-l5,eat-4,glr-1
GABA unc-47,unc-25,unc-46,unc-49,exp-1
Na+/K+ATPases subunits eat-6
Calcium channels eat-l2,unc-2,unc-36,unc-13
Others eat-5,unc-7,unc-l8,rab-3,snt-l,ric-
4,snb-l,unc-64,unc-50,unc-74
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. Alternatively, 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.
Example 5
Dosaae response
To determine the sensitivity of the pharynx
pumping assay, dilution series were made for some
chemical substances. These include the chemicals
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clomipramine, tamoxifen, BP554, pimazide and
thapsigargin. A concentration range was made from
less than 1 ,uM up to 100 ~cM, and the pumping assay was
repeated as described in previous examples.
From these results distinct dose-response curves could
be drafted.
This experiment shows clearly that the pharynx
pumping assay is quantitative and can be used to
determine the IC50 and ED50 of chemical substances.
Furthermore from this experiment the toxic effect
of the chemical substance can be detected. The dosage
response curve of the enhancer clomipramine shows
clearly the toxic effect of the solvent DMSO at higher
concentrations (Figure 5).
Finally it is possible to detect the effect of a
chemical substance on secondary targets or detect side
effects of a chemical substance at various
concentrations. This can be seen in the dosage
response curve of thapsigargin, known to be an
inhibitor of SERCA, which results in a decrease of the
pumping rate of the C. elegans pharynx (Figure 6).
Nevertheless, at low concentration an enhancement of
the pumping can be observed. This is the first
observation of this feature of thapsigargin. Although
further research is necessary to explain this
behaviour, which could be caused by a still unknown
secondary target of thapsigargin or another side
effect, this experiment shows clearly the sensitivity
of the pharynx assay, and the use of the pharynx assay
to edit dosage response curves.
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Example 6
Detecting the activity of chemical substances with
genetic techniaues.
Other techniques exist to measure the pumping rate
of the C. elegans pharynx. As the pharynx is muscle,
the contractility of the pharynx is dependent on the
internal calcium levels, These calcium levels can be
measured using specific calcium-sensitive reporter
genes.
~ t has been reported by Kerr et al. (West coast
Worm meeting abstract 77, 1998) that increased
electrical activity can he detected indirectly by
measuring the calcium levels in the C. elegans
pharynx. The calcium sensitive reporter proteins
described therein are Aequorin and GFP-calmodulin
(Miyawaki et al., Nature 388:882-887).
In this study 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 enhances
of pumping such as serotonin influence the observed
fluorescence of the GFP-calmodulin in a predicted way.
Analogous transgenic worms expressing GFP-
calmodulin can be used to screen for chemical
substances that influence the pumping rate of the
nemat-ade pharynx using the pharynx pumping assay
methodology. Analogous to the pumping assay described
for calcein-AM in the previous examples, the
transgenic worms are placed in multi-well plates and
chemical substances are added. The fluorescence of the
GFP-calmodulin is then measured rather than calcein
fluorescence using the same multi-well plate reader
instrument.
With the use of appropriate promoter sequences,
expression of aequorin or GFP-calmodulin can be
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engineered in other muscle tissues, or even in neurons
in order to monitor the calcium levels in these cells.
Such transgenics can be used in a screen as described
above.
Example 7
Genetic enhancer and suppressor screens,
Genes and hence biochemical pathways can be found
that enhance, suppress or modulate the activity of a
gives compound. When applying a compound to the
nematode C. elegans, phenotypic changes can be
observed, however, the target of the compound or its
mode of action can be known or be unknown. The
screening method described below can be used to
identify genes which suppress or enhance the activity
of a compound which has a defined effect on the
phenotype of C. elegans.
The compound 2,5-diphenyloxazole is an inhibitor
of the pumping rate of the pharynx both in wild-type
worms and in constitutive pumping worms. It is used
herein as an example of a compound which has a defined
effect on C. elegans.
C. elegans worms are subjected to random
mutagenized using standard techniques such as EMS,
TMP-UV or radiation (Methods in Cell Biology, Vol.
48)-. -'tee F1 generation of these mutagenized worms are
placed worm by worm in the wells of mufti-well plates
and the worms allowed to grow and generate offspring.
When the offspring have reached the desired growth
stage, 2,5-diphenyloxazole and calcein-AM is added.
The plates were further incubated for approximately
one hour and fluorescence of the generated calcein was
measured using a mufti-well plate reader. Wells that
had a higher fluorescence read-out were scored. The
worms in these wells were used for further analysis,
z
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as they harbor a mutation in a gene or a pathway that
suppresses the activity of 2,5-diphenyloxazole.
An analogous screen was performed with the
compound doxepin, which is an enhancer of pharynx
pumping. Mutants were scored that show a reduced
pumping phenotype in the presence of the compound
doxepin.
Example 8
Scre~ninq for antagonists of a compound (thapsiaarain)
The compound thapsigargin is known to inhibit the
activity of the sarco/endoplasmic reticulum calcium
ATPase (SERCA). The SERCA protein pumps calcium into
the sarco/endoplasmic reticulum and provides the cell
with an internal storage of calcium. The internal
storage of calcium is important for muscle activity.
In C. elegans, inhibiting SERCA activity by applying
thapsigargin to the worm results in a decrease in the
pharynx pumping rate. Another feature observed by the
action of thapsigargin on the nematode worm C. elegans
is decreased movement, which is a result of the
inhibition of SERCA activity of the body wall muscles.
A pharynx pumping screen has been developed to
screen for chemical substances that suppress the
activity of thapsigargin on SERCA. C. elegans
nematodes, both wild-type nematodes and nematodes with
a constitutive pumping pharynx are placed in the wells
of multi-well plates as previous described.
Thapsigargin is added to the worms at an inhibitory
concentration and calcein-AI~I is added at a
concentration of 5-10 ~M as previous described.
Finally the chemical substa~ces to be selected are
added. Control wells are also set up containing
thapsigargin alone with no second chemical substance.
Analogous to the pharynx pumping screen,
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fluorescence is measured using a multi-well plate
reader. Wells harboring a chemical substance where the
measured fluorescence is higher than in the control
wells containing no chemical substance are scored.
These wells harbor a chemical substance that is an
antagonist of the thapsigargin activity, as the
inhibitory activity of tbapsigargin is suppressed.
Chemical substances thus identified may inhibit
directly the activity of thapsigargin, or stimulate
the activity of SERCA, or have an enhancer"activity on
the SERCA pathway, and hence on the calcium biology of
the organism.
Chemical substances selected in this screen 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 organism. Examples of disease
areas for which these therapeutics are useful are
cardiac hypertrophy, cardiac failure, arterial
hypertension, Type H diabetes and Brody disease.
In the example given above, thapsigargin is used
as an example of a compound having a defined
phenotypic effect on C. elegans and any compound that
has an inhibitory activity on the pharynx pumping rate
can be used in an analogous screen.
-Sr~eens to select for chemical substances that
have an antagonist activity on compounds are known to
enhance the pumping rate of the pharynx can also be
performed. In such an experiment, a chemical substance
is scored if it reduces the pumping rate of the
pharynx in the presence of the compound known to be an
enhancer of pharynx pumping.
Analogous experiments can be done with compound
inhibiting other calcium pumps and even other ion
pumps.
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Example 9
Screening for chemical substances in transaenic,
mutant and humanized animals (SERCA-PLB)
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.
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.
There are several SERCA genes and isoforms which
are associated with different types of diseases;
SERCA2 and PLB are associated with cardio-vascular
dis~a~s, SERCA1 and sarcolipin are associated with
skeletal-muscle diseases, and three SERCA genes have
been associated with non-insulin-dependent diabetes
mellitus.
In order to perform screens to identify chemical
substances which modulate the activity of SERCA
pathways SERCA genes and PLB have been expressed in C.
elegans. The expression of these genes can be
regulated under the control of several specific
promoters with the following activities:
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a) The C. elegans myo-2 promoter which promotes
expression in the pharynx
b) The C. elegans SERCA promoter which promotes
expression in the C. elegans muscles,
including the pharynx, the vulva muscles and
the body wall muscles.
The following transgenics were constructed:
--a) pig and/or human SERCA under the,SERCA
and/or myo-2 promoter.
b) 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).
c) pig and/or human PLB under the SERCA and/or
the myo-2 promoter.
d) 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).
e) pig and/or human PLB-GFP fusion under the
SERCA and/or the myo-2 promoter.
f) pig and/or human PLB-GFP fusion under the
SERCA and/or the myo-2 promoter in a C.
w w elegans mutated for the C. elegans SERCA
(Knock-outs and selected mutants).
g) pig and/or human SERCA under the SERCA
promoter and pig and/or human PLB under the
myo-2 promoter.
h) pig and/or human SERCA under the SERCA
promoter and pig and/or human PLB under the
myo-2 promoter in a C. elegans mutated for
the C. elegans, SERCA (Knock-out and
selected mutants).
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i) pig and/or human SERCA under the SERCA
promoter and pig and/or human PLB-GFP under
the myo-2 promoter.
j) pig and/or human SERCA under the SERCA
promoter and pig and/or human PLB-GFP under
the myo-2 promoter in a C. elegans mutated
for the C. elegans SERCA (Knock-out and
selected mutants).
dome of these constructed 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. As described in the
previous examples, the transgenic and mutant animals
were placed in the wells of mufti-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 mufti-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-~eutic use, or as hits for the further
development of therapeutics.
A analogous experiment can be performed with the
SERCA1 gene and its regulator Sarcolipin (SLN), to
detect chemical substances that alter their activity
and/or regulation.
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Example 10
Screenina for chemical substances in transaenic and/or
mutant animals (neurodeaeneration)
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 I1, I2, I3, M3, MC,
NSM, M1, 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 (the pharyngeal muscles, the pharyngeal
epithelial cells, the pharyngeal glands, and the
pharyngeal neurons) 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.
Expression of toxic genes in one of the cells involved
in pharynx anatomy and pharyngeal function will result
in degeneration, dysfunction or abnormal development
of-the respectively cells. As a result the pumping
rate of the pharynx will be altered, most probably the
pumping rate will be decreased.
Examples of toxic genes that could be used to for
this purpose are listed above. Transgenic C. elegans
can be constructed which express these genes in a
tissue specific way. For example, the myo-2 promoter
will induce expression in the pharynx muscles, the
unc-129 promoter will induce expression in the
neuronal cells. For every cell type or tissue, a cell
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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.
Mutants and transgenics that have an altered
pharynx anatomy or pharynx pumping can then be used in
a pharynx pumping screen to select for chemical
substances that restore or rescue the genetic or
morphological defect. If the mutant or transgenic
animal has a decreased pumping rate the screen will
preferentially identify chemical substances that
enhance the pumping rate. If the mutant or transgenic
shows an increased pumping rate, the screen will
preferentially identify chemical substances that
reduce the pumping rate of the pharynx
Table 2: examples of mutants which may be used in
pharynx
Gene allele Pharyngeal phenotype Other phenotype
dig-1 n1321 Twisted
eat-6 ad467 relaxation defective ATPase
eat-13 ad522 relaxation defective Slow growing
goa-1 sy192 increased pumping hyperactive
mig-4- rh51 Twisted
mlc-2 pumping defects Larval lethal
pha-2 ad427 misshapen pharynx Larval lethal
pha-3 ad607 misshapen pharynx Slow growing
phm-2 ad538 relaxation. de~ective
cha-1 p1152 slow pumping Unc
clk-1 e-2519 slow pumping Slow
eat-1 ad427 irregular pu-ping Long and thi.~,
eat-2 ad451 slow pumping hypers. to
cholin.agonist
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Gene allele Pharyngeal phenotypeOther phenotype
eat-3 Very Slow pumping Misformed
eat-4 pumping defects.
eat-5 unsynchronized pumping
eat-7 sleeping
eat-8 brief pumping
eat-9 irregular pumping slightly starved
eat-14 relaxation defects motion defects
eat-i8 slow pumping starved
eat-x pumping defect
osm slow pumping chemotaxis
defects
snt-1 pumping defects Unc
unc-11 slow pumping Kinker
unc-13 irregular pumping Paralysed
unc-17 slow irregular pumpingSmall
unc-26 slow pumping little movement
unc-31 constitutive pumpingSlow
unc-36 irregular pumping Paralysed
unc-57 slow pumping Small
unc-58 sticky pumping Shaker
2 0 unc-90 sticky pumping Short
unc - sticky pumpi.~.g poor growth
105 _~
sma-1 pharynx de~e~~s
sma-2 reduced pumping
2 5 sma-3 pharynx defec~s
sma-4 pharynx de=2~~s
exo-2 sa26/+ fas~ shal?~w p~~mpingjerky, egl,
constipated
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Example 12:
Specific example of the assay with dauers, neuro-
deaeneration and the use of the daf-7 promoter
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
kill=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.
In an experiment analogous to example 11, the ASI
neuron has been killed, disrupted or altered in its
properties. More specifically toxic genes have been
expressed in this neuron by inducing their expression
under the control of the daf-7 promoter. Disrupting
the ASI neuron in such a way results in the formation
of dauers.
Such strains were used in screens as previously
described. In a first example the resulting dauers
were used in a pharynx pumping assay. Dauer worms do
not have or have only a reduced pharynx pumping.
Chemical substances were identified that cause the
worms to bypass the dauer phenotype and hence restore
the pharynx pumping. As before, the rate of pharynx
pumping was measured using calcein-AM.
In a second example the dauers were submitted to
the movement assay. As dauer worms do not move, and
hence precipitate in the wells, they can be used in
the movement assay to identify chemical substances
that cause the worms to bypass the dauer phenotype
and hence alter the movement of the worms. The
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movement behaviour of the worms was detected using
autofluorescence over the centre of the wells.
Example 13:
Specific example of the assay, with dauers.
Daf-2 is is a nematode mutant, which grows
normally at 15°C but generated 100 o 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.
To perform such an assay synchronized L1 Daf-2 is
worms are distributed over the wells of microtiter
plates. Synchronized eggs could also have been used.
The worms were supplied with food and grown further
at 25°C, resulting in dauer formation. After
approximately 4 days the chemical under test and
calcein-AM is added and fluorescence is measured at
selected time intervals, varying form 1 hour to 4
days, keeping the temperature at 25°C. Chemical
compounds were scored that caused the worms to bypass
the dauer phenotype. Due to the presence of the food
substrate, it may be difficult to detect fluorescence
using a mufti-well plate reader. The FANS device may
alternatively be used to measure fluorescence in this
ins_tan.ce.
An analogous experiment can be performed in which
the chemical under test is added to the wells,
approximately together with the L1 worms.
In an other variant of this experiment, large
quantities of Daf-2 is dauers were cultivated. The
dauers were then dispensed over the wells of multi-
well plates and chemical substances were added. The
mufti-well plates were placed in a mufti-well plate
reader set up to perform a movement assay (i.e. to
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measure autofluorescence). Autofluorescence
measurements were recorded at several time intervals
varying from 1 hour to 4 days, keeping the wells at
25°C.
Example 14
Screening for chemical substances and compound
antagonists with the movement assay.
=The nematode mutant (ace-l; ace-2) does not show
any movement and has a spasm-like phenotype. The worm
does not show any sinusoidal shape, but is straight
shaped. This is because the mutant is mutated in the
acetylcholine esterases,.resulting in high
concentrations of acetylcholine in the synapses.
Neostigmine, a well known acetylcholine esterase
inhibitor, was added to wild-type worms distributed
over the wells of a multi-well plate and submitted to
the movement assay after approximately 2 hours. As
Figure 8, panel 1 shows clearly worms exposed to
neostigmine showed a clear decrease in movement.
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. As receptor antagonist,
hexamethonium will also result in a decrease of
movement, as it prevents proper signalling. In last
panel of Figure 8, it is clearly shown that
hexamethonium represses the movement of wild-type
worms, but significant less than neostigmine (1000
represents normal movement of wild-type worms).
In an other experiment, wild-type worms were
contacted with inhibitory concentrations of
neostigmine to prevent movement. After a small
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incubation period, various concentrations of
hexamethonium were added and the wells were.
submitted to the movement assay (measurement of
autofluorescence). As Figure 8 shows, increasing
concentrations of hexamethonium resulted in more
movement as predicted (hexamethonium is an
antagonist), but the upper limit seems to be
determined by the inhibitory activity of
hexa-methonium. At very high concentrations of
hexamethonium (although lower than the concentrations
shown in last panel) a toxic effect is observed,
resulting in a decrease in movement. This toxic
effect is probably due to the presence of high
concentrations of both neostigmine and hexamethonium.
An analogous experiment was performed with the
ace- lace-2 double mutant. In this experiment,
increasing concentrations of hexamethonium were added
to the wells in the absence of neostigmine. The
results of both experiments were comparable.
This experiment shows clearly the applicability of
the movement assay to select for chemical substances
and antagonists of selected compounds.
Example 15
Example of a mating assay using hermaphrodite non-
selfers.
High throughput analysis of the nematode mating
behaviour could be performed by counting the
offspring of the mating experiment. First, equal
amounts of male worms were distributed over the wells
of multi-well plates. Hermaphrodites were then added
over the wells in such a way that the every well
contains an equal amount of hermaphrodites. The
ratio between males and hermaphrodites can be varied
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from experiment to experiment.
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.
Example 16
Example of a mating assay hermaphrodite non-selfers
expressing GFP
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
expre~.-sing other makers such as luminescent markers
can be used in an analogous experiment.
Example 17
Example of a mating assay males expressing GFP
In another variant of the mating assay the
hermaphrodites were chosen. in following combinations:
a) The hermaphrodites were wild-type
hermaphrodites, or hermaphrodites showing a
reduced movement phenotype
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b) The male nematodes were wild-type, transgenic,
mutant or humanized nematodes, expressing GFP.
In this experiment, 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.
Example 18
Male-specific neurons.
The following 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.
Table 3
Neuron Structure Class Role
CAn ventral cord motor ?
CPn ventral cord motor turning
CEMn head sensory ?
DXn motor ?
DVE inter ?sperm activation
or
transfer
DVF inter ?sperm activation
or
transfer
EFn turning
HOA hook sensc~y vulva location
HOB hook sensory vulva location
PCA p.c.s. sensory vulva location
PCB p.c.s. sensory vulva location
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Neuron Structure Class Role
PCC p.c.s. sensory vulva location
PGA p.a.g. inter ?
PGA p.a.g. inter ?
PW p.a.g. inter ?
S PVY p.a.g. inter backing
R1A ray sensory dorsal response?
R1B ray sensory dorsal response?
R2A ray sensory ventral response?
R2B ray sensory ventral response?
1 ~ R3A ray sensory ?
R3B ray sensory ?
R4A ray sensory ventral response?
R4B ray sensory ventral response?
R5A ray sensory dorsal response?
turning?
1 5 RSB ray sensory dorsal response?
R6A ray sensory ?
R6B ray sensory ?
R7A ray sensory dorsal response?
R7B ray sensory dorsal response?
turning?
R8A ray sensory ventral response?
turning?
R8B ray sensory vent=al response?
turn'_ng?
R9A ray sensory turning?
R9B ray sensory turning?
SPC spicule motorJp=opriospicule insertion
SPD spicule sensoy spicule insertion
SPV spicule sensory inh~its ejaculation
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Example 19
Further mutant and transcrenic C. e1e ac~ns.
The following Table 4 lists C. elegans mutants
which show abnormalities in male mating behaviour
which may be used in the mating assays:
Table 4
Gene Defect
(Mutant)
cat=~1,cat-2,cat-4,cod-5 Turning
che-2,che-3,che-4,cod-10 Response to contact
cod-1,cod-2,cod-4,cod-6, cod-7, cod-8Spicule insertion
cod-12,cod-13, Vulva location
cod-14,
cod-15
ram-1,ram-2,ram-3,ram-4, ram-5 Ray morphology
The following Table 5 lists mutant C.elegans which
may be used in the egg laying assays:
Table 5
Gene (Mutant) Defect
2 0 egl-1, egl-43 HSN function migration and
differentiation
egl-l, sem-1, sem-4 vulva muscle development
egl-15, egl-17 sex myoblast migration
egl-10, egl-30 synaptic transmission
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
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purpose include the lin-31, egl-17, unc-17 and unc-53
promoters.
The following Table 6 lists mutant C. elegans
which may be used in the defecation assays:
Table 6
Gene (Mutant) Defect
aex-1; aex-2, aex-3, aex-4;aBoc and expulsion
aex ~, aex-6
unc-25; unc-47; exp-1; exp-2constipated (expulsion)
pho-1 to pho-7, egl-8 aBoc specific
dec-1, dec-2, dec-4, dec-7,defecation cycle
dec-11, dec-12
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.
-Tl~e following Table 7 lists mutant C. elegans
which may be used in the movement assays:
Table 7
Gene (Mutant) Defect
unc-17 ace~ylc?oline receptor; coiler
ace-1; ace-2 ace~yic'.~.oline esterase; loopy
head
moveme~~
unc-25; unc-47 GABA; shrinker
unc-15; unc-54 paramyosin, myosin; paralysed
unc-36 Ca channel; paralysed
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Table 8: Enhancers of the C. elegans pharynx pumping
rate isolated from the pharmacopoeia.
Compound Mode of Action Disease area Positives
name after
1 hour
incubation
Clomipramine ser uptake inhibitorAntidepressant
Amitriptylineser uptake inhibitorAntidepressant
Desipramine ser uptake inhibitorAntidepressant
Fluvoxamine ser uptake inhibitorAntidepressant
Nortriptylineser uptake inhibitorAntidepressant
Imipramine ser uptake inhibitorAntidepressant
Fluoxetine ser uptake inhibitorAntidepressant+
Doxepin unknown Antidepressant,+
Antipruritic
Nordoxepin unknown Antidepressant,+
Antipruritic
Mianserin 5HT antagonist +
Norclomipramineser uptake inhibitorAntidepressant
Cyproheptadineser receptor Antihistaminic;
antagonist antipruritic;
appetite
stim.
Cyclobenzaprinet Psychomotor
depressant;
muscle
relaxant
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Table 9: Inhibitors of the C. elegans pharynx pumping
rate isolated from the pharmacopoeia.
Compound nameMode of Action Disease area Positives
after 1
hour
incubation
Pimozide D2 antagonist Antipsychotic
Haloperidol D2 antagonist Alzheimer,
- Antipsychotic
Trazadone serotonin uptake Alzheimer,
blocker Antidepressant
metabolite D2 antagonistAntipsychotic
5HT1-agonist
BP554 5HT1-agonist
Ivermectin chloride channel Antihelmintic
blocker
Levamisole Antihelmintic
Metrifonate cholinesterase Antihelmintic,+
inhibitor Alzheimer
Physostigminecholinesterase Alzheimer +
inhibitor
Tamoxifen chloride channel Antihistamine
blocker
Flunarizine Na/Ca channel blockerAntipsychotic
Thapsigargin "calcium channel
blocker"
alpha NETA choline acetyl
transferase inhibitor
Atropine cholinergic antagonist t
L-Hyoscyaminecholinergic antagonistActive form +
of
atropine
Diphenylhydantoin Anticonvulsant,+
antiepileptic
2 0 ZAPA GABA-antagonist +
2,5-
diphenyloxazole
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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.
Harmane HCI 9 B4 18431 555256085,208 1773,442-
15 1
TPMPA 8 H6 21792 1207771,94823 385,7584HIT
8 .
Prazosin 4 E9 94689 11360312,6242 362,8578HIT
8
Vigabatrin 6 H7 20828 6141068,7655 196,1402HIT
(2S,3R)-Chloropheg 6 H8 21514 4873671,03039 155,6601HIT
MSOPPE 6 E7 23760 4461478,44576 142,4947HIT
(n)-Acetylcarnitine2 C10 23457 4415677,44537 141,0319HIT
DPPE 5 G11 20365 4362967,23686 139,3487HIT
Indole-2-carboxylic2 A4 19362 4340363,92537 138,6268HIT
acid
N-desisopropylpropanolol6 A7 23737 4310278,36982 137,6654HIT
YS-035 3 E3 21415 4245770,70353 135,6054HIT
L-AP5 1 G2 21446 4239370,80588 135,4009HIT
2,4-Dihydroxyphenylacetyl-2 G4 18057 4220359,61679 134,7941HIT
2 L-asparagine
0
L-AP3 ! 2 G2 22073 4185872,87597 133,6922HIT
D-AP5 1 F2 19717 4169765,09743 133,178 HIT
O-Phospho-L-serine 1 A3 20292 4108666,99584 131,2265HIT
Clofibric acid 6 D9 22933 4091675,71534 130,6835HIT
cis-Azetidine-2,4- 7 D8 19503 4056864,39089 129,572 HIT
dicarboxylic acid
L-AP4 1 C2 19824 3952365,4507 126,2343HIT
Spaglumic acid 3 E6 20295 3841767,00575 122,7018HIT
Arecaidine but-2-ynyl2 F5 20002 3767566,03838 120,3319HIT
ester
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Cycloleucine 2 E4 1923436560 63,50276116,7707HIT
S(-)-Atenolol 3 G6 1817636336 60,00968116,0552HIT
Propanolol glycol 6 D7 1713434849 56,56943111,3058HIT
GF 109203X 5 E11 1568534448 51,78542110,025 HIT
Ketoconazole 8 G11 1580333531 52,17501107,0962HIT
DL-2-Aminosuberic 1 H2 1714133518 56,59254107,0547HIT
acid
HU 210 7 B9 1548133514 51,1119 107,0419HIT
GR 46611 7 F8 1581533199 52,21463.106,0358HIT
7-(Dimethylcarbamoyloxy)-6 C2 1427832900 47,14009105,0808HIT
6-phenylpyrrolo
GBLD 345 6 G3 1262032858 41,66605104,9467HIT
L-701,324 7 E6 1464732476 48,35837103,7266HIT
tADA 7 E8 1609632342 53,14238103,2986HIT
RS 17053 8 B10 1443932050 47,67164102,366 HIT
N-Benzylnaltrindole6 G6 1493231760 49,29933101,4397HIT
Table 11: Enhancers of C. elegans pharynx pumping
found from screening the Tocris compound library
(Bristol, UK) using a pharynx pumping assay.
Name Known pharmacological activity
Clomipramine serotonin uptake inhibitor
6-Nitroquipazine serotonin uptake inhibitor
2 5 Fluvoxamine Serotonin uptake inhibitor
Methiothepin 5HT1,2 antagonist
5-Nonyloxytryptamine oxalate5HT1 B agonist
N-Desmethylclozapine 5HT2C antagonist
3-Methoxycarbonylamino-b-carbolinebenzodiazepine receptor
inhibitor
7-(Dimethylcarbamoyloxy)-6- benzodiazepine receptor
phenylpyrrolo inhibitor
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Nimodipine Ca channel blocker
CP 55,940 cannabinoid agonist
WIN 55,212-2 cannabinoid agonist
WIN 64338 cannabinoid agonist
HU 210 cannabinoid agonist
Bromocriptine D2 agonist
1-(2-Benzo[b]thienyl)-N- dopamine uptake inhibitor
butyleyclohexanamine
1-[1-(2- dopamine uptake inhibitor
Benzo[b]thienyl)cyclohexyl]pyrrolidine
2-amino-4-methylpyridine iNOS inhibitor
17-ODYA leukotryiene B4 hydrolase
inhibitor
Etazolate PDE4 inhibitor
cis-(n)-N-methyl-N-[2-(3,4- sigma receptor ligand
dichlorophenyl)-
N-exo-Bicyclo[2,2,1]hept-2-yl-N'-(2-sigma receptor ligand
iodophenyl)- (haloperidol
sensitive)
L-732,138 substance P receptor antagonist
Cyclosporin A calcineurin phosphatase
activity
inhibitor
Dioctanoylglycol diacyl glycerol kinase
inhibitor
LY 225910 CCKB receptor antagonist
a-NETA- choline acetyl transferase
inhibitor
4-Naphthalimidobutyric acid aldose reductase inhibitor
Ergotamine antimigraine oxytocic
