Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02222409 1997-11-16
W 096/38586 PCT~EP96/02291
Method for the detectlon of compounds that modulate the effects of the
obese protein
The invention relates to a novel method and more partieularly to a method for
the detection of eompounds that mimie, potentiate or inhibit the physiologieal effeets
5 of the ob-protein
The ob-protein (or leptin) is a seereted hormone that aets as signal from
adipose tissue to other organs to regulate weight and energy balanee (Zhang et. al.,
Nature, 1994, 372, 425). Additional roles for the ob-protein in hematopoietie and
reproduetive funetion have been suggested (Cioffi et. al. Nature Medicine, 1996, 2(5),
10 585). Protein moleeules that eontain a eore eomposed of four o~-heliees forming a
bundle of up-up-down-down topology eomprise of a family of eytokines and growth
factors. Proteins of this family cause homo- and hetero-oligermerisation of membrane
receptors known to activate kinase cascades resulting in gene transcription. Receptors
of the family whieh are aetivated by oligermt-ric~tion fall into two broad elasses; those
15 sueh as epidermal growth faetor, whieh possess integral tyrosine kinase activity in their
intracellular ~ m~inc (A. Ullrich & J. Schleccing~r, Cell, 1990, 61, 203-212), and
those such as IL4 and erythropoietin, which lack this activity and m~ te their
response by way of an associated protein tyrosine kinase (J.N. Ihle et al., TIBS, 1994,
19, 222-227). Both receptor subtypes are activated by eytokines, but the 4-helix20 bundle proteins aetivate only the non-integral tyrosine kinase subtype. The non-
integral protein tyrosine kinase reeeptors generally aet through a pathway involving
Janus kinase (JAK) and their assoeiated signal transdueers and aetivators of
transeription (STAT) proteins. On aetivation STAT proteins bind to DNA response
elements thereby eontrolling gene transcription. Oligonucleotide sequences
25 cnmpricing DNA regulatory elements of the general sequence TT(N)nAA have beenidentified (Seidel et al., Proc. Nat. Acad. Sci. USA., 1995, 92, 3041) as STAT
response elem~nt~ These elements bind STAT proteins in response to sign~ling
molecules such as eytokines.
In eopending United Kingdom patent applieation number 9509164.1 we have
30 deseribed our diseovery that the ob-protein is eharaeterised by a four helix bundle
tertiary strueture. We now believe that the ob-protein interaets with a membranebound reeeptor that aetivates a JAK-STAT kinase easeade and henee forms the basis
for an assay system for the deteetion of eompounds that mimie, potentiate or inhibit
the physiologieal effeets of the ob-protein. Sueh an assay has utility in seleeting
35 eompounds for the treatment of weight, energy balance, hematopoietic, fertility and
other disorders modulated by the "ob-protein". The assay is especially useful for
selecting compounds for the treatment of those disorders related to obesity, anorexia,
cachexia and diabetes.
Accordingly, the invention provides a method for the detection of a compound
40 that mimies, potenti~tes or inhibits the physiological effect of the ob-protein, which
method comprises:
(a) for a compound which mimics the physiological effect of the ob-protein, assessing
the effect of the compound upon an ob-protein activated signal transducer and
CA 02222409 1997~ 16
W 096/38586 PCT~P96/02291
activator of transcription (STAT) DNA response element coupled to a reporter gene;
or
(b) for a compound which pott-nti~tes or inhibits the physiologicalleffect of the ob-
protein, ~3~se~ing the effect of the con~,ulld upon the response provided by ob
5 protein upon an ob-protein activated STAT DNA response element coupled to a
reporter gene;
the response element and the l~p~lL~I being expressed in an ob-protein responsive cell
line. ,,
Suitably, the response element is coupled to a ~lullloL~I gene, preferably a
minim~l ~lumoL~l.
A suitable response element is a nucleotide of formula TT(N)n AA, where N is
any nucleotide and n is 4, 5 or 6.
A favoured response el~m~nr is selectively activated by the intracellular eventsme~ ted the by the ob-protein interacting with its receptor. Such selective response
el--ment~ can be determined by ~nilling the relative activation of a range of response
element-reporter gene constructs when transfected into an ob-responsive cell line by
the ob-protein versus other cytokines.
A favoured response elt-ment is a nucleotide of formula TT(N)n AA, where N
is any nucleotide and n is 5.
A further suitable response element is TTCCCGGAA.
A further suitable response element is that region of the promoter of a gene
regulated by the ob-protein that is required for STAT interactions. This gene will
depend on the particular therapeutic use of the compounds to be selected by the assay.
A suitable reporter gene is firefly luciferase or chloramphenicol
acetyltransferase enzyme.
A suitable promoter is a minim~l promoter such as the herpes simplex virus
thymidine kinase or SV40 promoter.
An example of an "ob-responsive" cell line is a liver or liver hepatoma derived
cell line. Liver or liver hepatoma cell lines are available from either the American Type
Culture Collection (ATCC) or the Eurpean Collection of Animal Cell Cultures
(ECACC). An example of one such cell line is Hep G2 (hep~tocçll~ r carcinoma,
human) which is available from the ATCC (EIB-8065). The Hep G2 cell line is
disclosed and claimed by US patent 4393133.
A further example of an "ob-responsive" cell line is the rat-l fibroblast (Kroder
et. al., Exp. Clin. Endocrinol. Diabetes, 1996, 104 (suppl 2), 66). A ratl fibroblast
cell line is available from the ATCC (CRL-2210).
Other responsive cell lines can be identified using a displacement binding assay.
Although binding may not be to a functional long form of the receptor, which is the
form that transmits a signal to the cytoplasm. Td.-ntific~tion of a functional long form
of the receptor may be by PCR or Northern blot analysis (eg. Human ob-receptor:
Tartaglia et al., Cell, 1995, 83, 1263). Ultimately responsive cells are detected by
monitoring cellular events in the presence of varying concentrations of leptin.
CA 02222409 l997-ll-l6
W 096/38S86 PCT~EP96/02291
Potential methods for identifying c~nfli~l~te cell lines or monitoring these cellular
events include the following:-
1. Microphysiometer: This method detects small changes in pH res-lltin~ from
biochemical changes in the cell. Ob-protein responsive cells upon s*m~ tion may
S undergo biochemical changes that cause a small change in the extracellular
~rlific~tion rate which can be detected by a silicon microphysiometer. The
microphysiometer biosensor methodology has been reviewed by McConnell, Science,
=i 1992, 257, 1906.
2. Electrophoretic mobility shift assay (EMSA): Nuclear extracts from cells
10 after tre~tmçnt with ob-protein are mixed with radiolabeled oligonucleotides
cnnt~ining a promiscuous or specific STAT response element DNA sequence.
Extracts from cells that respond to the ob-protein may cause a gel shift of the
oligonucleotide for the STAT response element.
References: Book "Recombinant DNA", 2nd Edition, Watson et al., 1992, Page 158;
Lamb et al., Blood, 1994, 83, 2063;
3. Measurement of protein phosphorylation assay: The coupling of receptor
activation to the final response through tyrosine phosphorylation of intr~ee~ r
proteins may be assayed by the use of antibodies recognising phosphorylated tyrosines.
More specifically since the leptin receptor may srim~ te tyrosine phosphorylation of
20 the JAK/STAT pathway this method provides a method of detecting leptin response
cell lines. Specific JAK/ STAT antibodies may be used alongside antibodies for
tyrosine phosphorylation to detect leptin activation in a leptin responsive cell line.
Inhibition as well as stimlll~tion of protein phosphorylation may occur. In particular,
inhibition by the ob-protein of insulin stim~ tell phosphorylation of the insulin
25 receptor and insulin receptor substrate- 1 has been shown in rat-l fibroblasts
ov~lc~ ssing insulin receptors (Kroder et. al 1996, Exp. Clin. Endocrinol. Diabetes,
104, suppl 2, p66)
4. Displ~em~nt binding: After incubation of cell lines with radiolabelled
leptin, for example [125Il-leptin, the non-specific binding versus specific binding of
30 leptin can studied by the addition of unlabelled leptin. A high specific to non-specific
ratio binding suggests that the cell line may contain the leptin receptor.
5. Detection of the protein for a functional form, preferably a functional long
forrn, of the ob-receptor by use of selective antibodies.
6. Detection of mRNA for a functional form, preferably a functional long
35 form, of the ob-receptor by Northern, RT-PCR or "slot blot" analysis.
Cell lines known to be involved in controlling aspects of the particular diseasestate for which compounds are being sought are preferred.
Cells lines derived from liver, brain, or pancreatic tissue and fibroblasts are
particularly useful for "ob-responsive" cells for the assaying of compounds directed at
40 obesity and diabetes. Certain areas of the brain are the focus of weight controlling and
energy baIance regulating effects of the ob-protein. The liver controls many metabolic
processes that modulate lipid and glucose levels. Cells derived from particular regions
of these organs cont~ining the applu~liate endogenous JAKs, STAT proteins and
I
CA 02222409 1997-11-16
W 096/38586 PCTAEP96/02291
other intracellular proteins which are required for m~ ing the effects of the leptin
are ~lere~led.
The response element, the reporter, and preferably the promoter, are suitably
incorporated into a vector capable of transfecting the ob-responsive cell line.
Suitable vectors are commercially available vectors, such as pGL2-basic
luciferase vector (Promega). I
A suitable configuration of the vector is the STAT DNA response element
upstream of a promoter and a reporter gene. A more suitable configuration of thevector is the STAT DNA response ele.mt-nt in multiple tandem repeats (2-10) upstream
of a thymidine kinase promoter and a luciferase reporter gene
Vectors are constructed containing a reporter gene for exam; ple firefly
luciferase or chloramphenicol acetylLIdn~.r~;ldse enzyme linked to a;minim~l promoter
for example the herpes simplex virus thymidine kinase or SV4Q promoter. The DNA
fragments for the STAT response element are inserted into the vector using
apl-lv~liate restriction enzyme sites upstream of the minim~l promoter.
The response element, the reporter and the promoter, as required, are
incorporated into the vector using conventional expression techniques, for example the
DNA fragments for the response element may be inserted into the vector using
a~lv~liate restriction enzyme sites upstream of the minim~l promoter.
STAT response elem~nt-luciferase enzyme reporter systems can be constructed
as described by Lamb et al., Blood, 1994, 8, 2063 and Seidel et al., Proc. Nat. Acad.
Sci. usa., 199S, 92, 3041.
Ob-responsive cells are transfected with the STAT response element-minim~l
promoter-luciferase reporter constructs using standard methodology for example the
calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456). To
correct for differences in transfection efficiency, the cells can be co-transfected with a
reference pl~cmi~l expressing ,B-galactosidase activity. After a perlod of transfection
(12-24 hours) the cells are treated with varying concentrations of compound and then
harvested and lysed. The lysates are assayed for luciferase, and if a~ liate ~-
galactosidase, activity. Potentiation or antagonist activity can be assayed by pre- or
co-addition of an a~pl~ iate concentration of ob-protein to the compound under
evaluation and measuring the potentiation or reduction in luciferase response relative
to that of ob-protein alone. Standard methods exist for assaying lu, ciferase enzyme
activity for example Ow et al., Science, 1986, 234, 856 and de Wçt et al., Mol. Cell
Biol., 1987, 7, 725. as well as several comrnercial kits.
Stable cell lines can be generated by transfecting an "ob-responsive" cell line
with the reporter construct and a selectable marker. Selectable markers are routinely
used to generate stable cell lines as described in Recombinant DNA, 2nd edition, J.D.
Watson et. al., 1992, page 216. These stably transfected cell lines can be used to
generate high throughput assays for compounds that mimic, potentiate or block the
physiological effects of the ob-protein.
The invention also extends to a compound that mimics, potentiates or inhibits
the physiological effect of the ob-protein, when i(lenhfie.l by the method disclosed
herein.
CA 02222409 1997-11-16
W 096/38586 PCT~EP96/02291
The invention also extends to a kit of parts adapted for use in the method
disclosed herein.
When used herein 'a compound which mimics the physiological effects of the
o~protein' refers to a compound which is capable of acting in the absence of the ob-
S protein to either stimnl~te the ob-protein receptor to provide substantially the same
physiological effect as the ob protein or to activate a response down stream of this
receptor (post-receptor).
When used herein 'a compound that potentiates the physiological effect of the
o~protein' refers to a compound which enhances the potency and/or maximal
10 physiological effect of the ob-protein.
When used herein 'a compound that inhibits the physiological effect of the ob-
protein' refers to a compound which reduces or subst~nti~lly blocks the physiological
effect of the ob protein.
The following example illustrates the invention
~v
~: ;
CA 02222409 1997-11-16
W 096/38586 PCT~P96/02291
F,Y~mrle
General Procedure:
Ob-responsive cells are transfected with a reporter plasmid cont~ining a STAT
response element, in multiple tandem copies upstream of a minim~l~ promoter for
example herpes simplex thymidine kinase and a luciferase gene reporter constructusing
standard methodology for example the calcium phosphate method (Graham and Van t
Der Eb, Virology, 1973, 52, 456). To correct for differences in transfection efficiency,
10 the cells can be co-transfected with a reference pl~mi~l expressing ~-galactosidase
activity. After a period of transfection (12-24 hours) the cells are treated with varying
concentrations of compound and then harvested and lysed. The Iys tes are assayed for
luciferase, and if appropriate ~3-galactosidase, activity. Antagonist ctivity can be
assayed by pre- or co-addition of an a~lu~liate concentration of o~-protein to the
15 compound under evaluation and measuring the reduction in lucifer$e response relative
to that of ob-protein alone. Standard methods exist for assaying luc,iferase enzyme
activity for example Ow et al., Science, 1986, 234, 856 and de Wet et al., 1987, 7,
725. as well as several commercial kits.
Example
A liver hepatoma derived cell line is transfected with a reporter plasmid, pGL2-basic
luciferase vector (promega) cont:~ining an insert of an oligonucleotide corresponding
to a four fold tandem repeat of the STAT response element, TTCCCGGAA, upstream
of the minim~l promoter for herpes simplex thymidine kinase (-35 to +10) using
standard methodologyfor example the calcium phosphate method (Graham and Van
Der Eb, Virology, 1973, 52, 456). To correct for differences in transfection efficiency,
the cells can be co-transfected with a reference pl~mi~l expressing ~-galactosidase
activity. After a period of transfection (12-24 hours) the cells are treated with varying
concentrations of compound and then harvested and lysed. The Iysates are assayed for
luciferase, and if a~ liate ,13-galactosidase, activity. Antagonist activity can be
assayed by pre- or co-addition of an ap~ liate concentration of ob-protein to the
cc,.ll~oulld under evaluation and measuring the reduction in luciferase response relative
to that of ob-protein alone. Standard methods exist for assaying luciferase enzyme
activity for example Ow et al., Science, 1986, 234, 856 and de Wet et al., Mol. Cell
Biol., 1987, 7, 725. as well as several commercial kits.
