Note: Descriptions are shown in the official language in which they were submitted.
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NEW USE OF THE JAK-STAT SYSTEM
BACKGROUND OF THE INVENTION
Cellular stress is believed to be an important part of a pathological cellular
response. Such
cellular stress can be inflicted by several different factors/situations such
as oxygen depletion,
metabolic dysfunction or chemical compounds. A cellular stress causes the cell
to alter its
cellular function by an interference with signalling molecules. A prolonged
cellular stress can
cause disease manifestations in e.g. cardiovascular and metabolic disorders.
One of the key signaling pathways in many cells consists of the JAK-STAT
pathway. Janus
Kinases (JAKs) are intra cellular tyrosine kinases and Signal Transducers and
Activators of
Transcription (STATs) are transcription factors activated by tyrosine
phosphorylation and
again different molecular forms. The JAK-STAT pathway is preferentially
activated by the,
so-called, cytokine system. Cytokines include e.g. growth hormone (GH),
prolactin (Prl),
leptin, erytropoetin (Epo), colony stimulation factors, different interleukins
and interferons.
All of these activate the JAK-STAT pathway through specific membrane bound
receptors and
it its relevant to note that different forms of JAK molecules exist such as
JAKI, JAK2, JAK3
and TYKI. Also different STAT molecules have been isolated, e.g. STATI, STAT2,
STAT3,
STAT4, STATS, STATSa, STATSb and STAT6. Individual JAK-STAT combinations can
be
used in a specific signal transduction chain (for reviews, see references 1
and ?). The JAK-
STAT pathway also includes other signaling molecules e.g. MAP kinases and IRS.
According
to this invention the JAK-STAT pathway is defined as cellular effects induced
by JAK.
An important aspect of cytokine signaling by receptors that use the JAK-STAT
pathway is the
mode of shutting the receptor signal off. Recently a new class of molecules,
Suppressors of
Cytokine Signaling (SOCS), has been described. SOCS proteins are induced as a
consequence
of a JAK-STAT activation and function to turn individual cytokine receptors
off. As with
other molecules in the JAK-STAT pathway several different SOCS have been
described e.g.
SOCSI, SOCS2, SOCS3 and CIS (references 3 and 4).
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THE PRESENT INVENTION
As one outcome of our long investigations of cytokine signalling, we have
surprisingly, be-
come aware of that the JAK-STAT pathway can be modulated by a number of
different
chemical compounds. This fact became evident after a detailed investigation of
shut off
mechanisms of GH signals. By said research work we found that the JAK-STAT-
SOCS path-
way is extremely sensitive to chemical compounds that cause a cellular stress.
A probable rea-
son that this pathway displays such a sensitivity resides in the fact that the
shut off mechanism
i.e. the SOCS function is executed by a very rapid turnover of the SOCS
proteins.
The present invention relates to an assay system, based on the JAK-STAT-SOCS
pathway,
which can be used to monitor compounds that cause a cellular stress. Cellular
stress can be
monitored in several other ways e.g. by measuring heat shock response proteins
or apoptosis.
However, the analysis of the JAK-STAT-pathway in relation to actions of
chemical com-
pounds is advantageous as the JAK-STAT-pathway is an important intra cellular
pathway and
pharmacological compounds can influence the JAK-STAT pathway without causing
overt
heat shock responses and apoptosis.
The technology described in the present invention can be used to screen
chemical, biological,
and physical entities, and particularity established drugs or potential drug
candidates, for their
effects on the JAK-STAT-SOCS pathway. If a particular compound scores positive
in this as-
say it may cause cellular stress, which might lead to side effects if intact
organisms are treated
with said compound.
In the prior art the existence and biological importance of the JAK-STAT-SOCS
pathway is
well documented. We have, however, not been able to find any documentation in
the prior art
showing that this pathway can be used as a read out for side effects of
biologically active enti-
ties such as drugs. It is becoming increasingly important, at an early stage,
to identify whether
biologically active entities or other entities give side effects or not. It
might be any compound,
biological or chemical or physical agent to which a living organism can be
exposed. Biologi-
cally active entities are defined as any molecule that have or in the future
may have a thera-
peutic, nutritional or environmental utility or on the contrary be detrimental
to the organisms
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health. Said entities include drugs used in the clinic, candidate drug
compounds with a poten-
tial clinical use, natural or synthetic nutritional or environmental chemicals
that causes bio-
logical effects, radiation and similar means that might influence a living
organism.
As exemplified below an analysis of the JAK-STAT-SOCS pathway can be used to
reveal
potential side effects of test compounds. The bearing principle is to first
activate a JAK-
STAT-SOCS pathway in a cell and then add a test compound. As stated above,
many factors
can activate the JAK-STAT pathway in cultured cell and the actual nature of
these, as well as
doses required, are known to any person skilled in the art. A non-limiting
list of factors that
activate the JAK-STAT pathway include growth hormone, prolactin, erytropoetin,
leptin,
erythropoetin, a variety of interleukins, colony stimulation factors and
interferons. A person
skilled in the art also knows that there are several different methods
available to detect com-
ponents of the JAK-STAT pathway, these include protein analysis of activated,
i.e. phospho-
rylated, JAKs or STATs as well as the measurement of levels of SOCSs with
antibody tech-
niques or related protein-binding techniques. Alternative assays are gel shift
analysis or simi-
lar DNA binding techniques that can be used to detect STAT DNA binding and
different types
of hybridisation techniques that can be used to analyse SOCS mRNA levels. In
addition ge-
netic tagging techniques where components in the JAK-STAT-SOCS pathway are
modified
with a tag to simplify detection in the cell are applicable. The present
invention can thus be
carried out using a variety of methods. The terms JAK-STAT pathway or JAK-STAT
system
is herein defined as any cellular event that depends on the activation of JAK.
There are also
other cellular parameters than JAKs or STATs that can be used to measure a
cytokine signal
such as activation of MAP kinases and IRS. The principle of the invention
relies in the use of
isolated cells in which the JAK-STAT pathway is activated. In case a cell is
exposed to a test
compound the interference of such a test compound with the JAK-STAT pathway
will be de-
tected by a temporal analysis of JAK phosphorylation or by STAT-5 DNA binding
(or alter-
native methods) or by a temporal analysis of SOCS protein/mRNA levels. If a
compound in-
teracts in the JAK-STAT pathway it will score positive if the time for JAK-
STAT activation
is prolonged or if levels of SOCS are reduced.
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The term "temporal analysis" means a comparison of the duration of the JAK-
STAT signal at
the activation of the JAK-STAT pathway in the presence of a biologically
active entity with
the duration of a JAK-STAT signal caused by a cytokine in the absence of a
biologically ac-
tive entity. A significant change, normally a prolongation of the duration of
the JAK-STAT
pathway is assayed for. The use of the different end point read outs, here
exemplified as
STAT activation and SOCS levels can be converted into high through-put assays
by a person
skilled in the art. In practice, cells can be grown, preferably in a 96 well
format, then exposed
to a cytokine such as growth hormone and either the STAT activation or levels
of SOCS
measured. Initial experiments are needed to establish the time kinetics for
cytokine activation
of the JAK-STAT pathway with regard to the particular cytokine used and the
particular cell
line used. It is to be expected that the activation is transient which means
that one time point
where the JAK-STAT pathway is maximally active can be identified and another,
later, time
point, where the signal has been significantly reduced. If GH is used as the
stimulatory cyto-
kine in liver cells the activation of the JAK-STAT pathway is maximal within a
time period of
one hour (1h) and the signal is significantly reduced after four hours (4h).
This information
may vary between different cells and cytokines. After the initial parameters
have been estab-
lished, test compounds can be added in conjunction with the activation of the
JAK-STAT
pathway. A suitable time period to analyse components of the JAK-STAT pathway
in the GH
system can be 4h. At this time period one can assay for a prolongation of the
JAK-STAT
pathway consisting of e.g. maintained STAT DNA binding and/or JAK
phosphorylation
and/or levels of SOCS mRNA or SOCS protein.
The use of different cell types i.e. derived from different cell linages,
makes it possible to per-
form a risk assessment if a test compound has a potential to cause side
effects through the
JAK-STAT system. With different cell linages is meant that cells can be of
different embryo-
logical origin such as from endoderm, ektoderm, mesenchyme and so forth.
Primary cell cul-
tures as well as established cell lines can be used. The main features will be
to use cells com-
monly used in the laboratory and a list of such cells can be obtained from the
ATCC (Ameri-
can Tissue Culture Collection). In certain cases it might be useful to analyse
cells, e.g. fibro-
blasts or tumor cells, from individual patients to characterise the response
to chemical com-
pounds. In the examples below, the model has been JAK2 activation of STAT-5 in
liver cells;
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a prolongation of STAT-5 DNA binding has been the read out for test compounds
or a reduc-
tion of SOCS 2, SOCS 3 and CIS. The time kinetics as well as the model used in
the examples
are only meant as illustrations of the invention and are not intended to limit
the use of this in-
vention. Variations in experimental designs such as the used cell, the time
kinetics applicable
and/or the component of the JAK/STAT/SOCS pathway used as an end point
measurement,
fall within the scope of the present invention as long as the screening system
is cell based, a
cell in which the JAK-STAT-SOCS system is activated used and a temporal
analysis of the
effects of the test compounds on this pathway conducted.
All of the features disclosed in the claims are herewith included by
reference.
Figure 1 relates to the transient nature of STATS activation by GH measured
using Gel mo-
bility shift assay.
BRL4 cells were grown for 8 hours under serum free conditions. Recombinant hGH
(SOnM)
was then added, the cells were harvested at the indicated points of time and
nuclear extracts
were prepared. STATS and STATI binding activity were determined using
electrophoresis
mobility shift assay with a 32P-labeled SPIGLE1 probe. Relative DNA binding
levels were
measured by autoradiography. This experiment shows that GH activates STATS DNA
binding
in a transient fashion. After GH stimulation a STAT DNA binding activity is
rapidly increased
followed by a rapid decrease to background level after 2 hours.
Table 1. Effect of various drugs on GH induced STAT-5 activation
Cell treatment STATS DNA bindnin _activity
30 minutes 4 hours
No treatment - -
GH + -
GH + D609 + +
GH + H7 + +
GH + BAPTA-AM + +
GH + Verapamile + +
GH + A23187 + +
GH + Actinomycin D + +
GH + Calphostin C - -
GH + Bvsindolmaleimide - -
I
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6
Cells were grown to 90% confluence, starved for 8h and incubated for 30 min
with different
drugs: D609 (50 mg/ml) (B) H7 (200uM), BAPTA-AM (25nM), Verapamile (100 uM),
A23187 (5nM), Actinomycin D(lug/ml) Calphostin C (1mM) and Bisindolylmaleimide
I
(2mM). Recombinant hGH (50 nM) was then added, the cells where harvested at
the indicated
times (30 min or 4h) and nuclear extracts were prepared. STATS binding
activity determined
using electrophoresis mobility shift assay with 32P-labeled SPIGLE 1 probe.
Activated STATS
was visualized and quantitated by autoradiography. The table summarized the
results obtained
with different drugs. Significant detection of STATS DNA binding is denoted as
+. None of
the drugs caused STAT activation when added alone. When added in combination
with GH
the drugs didn't have major effects on the binding activity when measured
after 15 min but
some of said drugs could prolong the GH-induced STATS activation at the 4h
period.
Table 2. Effect of various drugs on GH-induced activation of SOCS
Cell treatment SOCS
Anal
sis
SOCS mRNA SOCS rotein
No treatment - ND
GH + ND
GH + D609 reduced reduced
GH + H7 reduced ND
GH + BAPTA-AM increased reduced
Cells were grown to 90% confluence, starved for 8h and incubated for 30 min
with different
drugs as described in Table 1. Recombinant hGH (50 nM) was then added and
cells were har-
vested at different times and used to prepare total RNA. SOCS-2, SOCS-3 and
CIS mRNA
was measured using solution hybridization assay in a time course study. SOCS
proteins were
analysed using Western blots. The table summarized the results obtained with
different drugs.
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EXAMPLES
1. An assay to monitor drug induced effects on STAT activation
BRL-4 cells (a rat liver cell) that respond to GH were used (5). GH was added
to these cells at
a concentration of 100 mg/ml. As shown in Fig 1 this cause a transient
activation of STAT-5
measured in a gel shift analysis (6); a maximum STAT-5 binding was observed
after 30 min-
utes and after 4h the response was lost. A large variety of chemical compounds
were tested for
their ability to prolong STAT-5 activation and a surprising finding was that
so may com-
pounds could prolong a STAT-5 activation in the presence of GH. In the absence
of GH these
compounds did not exert any effect. As listed in Table 1 such compounds
included CHX,
D609, Bapta and H7. These compounds have very different mechanisms of action
and such
actions include inhibition of protein translation, tyrosine phosphorylation,
calcium and lipases.
The finding that all of these prolong STAT-5 activation indicates a relation
between the action
of the compounds as well as a prolongation of STATS and cellular stress. By
the use of this
cellular system a compound can be categorized for its ability to prolong STATS
or not. Such
information is important in the drug development process. One embodiment of
the present
invention is a cell based drug screen by which a drug might be categorized for
a possible in-
teraction with the JAK-STAT-SOCS pathway. A cytokine receptor would here be
activated in
a cell by one cytokine or a combination of different cytokines. Such cvtokines
could consist of
proteins from varying sources and of varying purity, protein variants of
cytokines or possibly
of analogues of cytokines. The drug of interest is added in conjunction with
(before, together
or after) the cytokine stimulation. Subsequently the effect of said drug could
be detected by
analysing the STAT proteins in a period of time. Interference in the pathway
will result in a
prolongation of a cytokine response on STAT DNA binding activity or a
prolongation of the
presence of STAT tyrosine phosphorylation. As is well known to a person
skilled in the art,
such assays can be conducted in an automated or semi-automated fashion
suitable for screen-
mg purposes.
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2. An assay to monitor drug induced effects on SOCS expression
The effect of different drugs to cause a prolongation of STAT-5 activation in
example I is
most easily explained by the possibility that SOCS proteins are reduced by
drugs, a reduced
level of SOCS would subsequently lead to a diminished "cytokines receptor shut
off'. This
concept was tested by analysing different SOCS mRNAs using the technique of
RNase pro-
tection/solution hybridisation (7,8). As shown in Table 2 several different
drugs could reduce
SOCS mRNA. In one case (BAPTA) the effect was, however, the opposite, namely
an in-
crease of SOCS mRNA. For this reason the effect of BAPTA on protein synthesis
was meas-
ured and was found to be reduced. Consequently, all treatments described in
table 2 will ulti-
mately result in a reduction of SOCS proteins. According to one embodiment of
the present
invention the SOCS protein levels is subsequently measured. This can be
achieved with dif
ferent types of e.g. protein binding assays. In a screening assay, test
compounds will score
positive if they cause a reduction of SOCS proteins including SOCS1, SOCS2,
SOCS3 and
CIS.
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9
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2.Heim MH. The Jak-STAT pathway: cytokine signalling from the receptor to the
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Recept Signal Transduct Res 1999 Jan-Ju1;19(1-4):75-120
3. Stan R, Willson T A, Viney E M, Murray L J L, Rayner J R, Jenkins B J,
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