U.S. patent application number 10/436137 was filed with the patent office on 2003-10-30 for screening method for srebp pathway-specific inhibitors.
This patent application is currently assigned to Chugai Seiyaku Kabushiki Kaisha. Invention is credited to Hamakubo, Takao, Kawabe, Yoshiki, Kodama, Tatsuhiko, Ta-Yuan, Chang.
Application Number | 20030203392 10/436137 |
Document ID | / |
Family ID | 22495407 |
Filed Date | 2003-10-30 |
United States Patent
Application |
20030203392 |
Kind Code |
A1 |
Ta-Yuan, Chang ; et
al. |
October 30, 2003 |
Screening method for SREBP pathway-specific inhibitors
Abstract
This invention provides a screening method for sterol regulatory
element binding protein (SREBP) pathway-specific inhibitors using a
mutant cultured cell, as well as therapeutic agents for
hyperlipemia, arterial sclerosis, obesity or cancer containing an
SREBP pathway-specific inhibitor selected by said screening
method.
Inventors: |
Ta-Yuan, Chang; (Etna,
NH) ; Kodama, Tatsuhiko; (Tokyo, JP) ;
Hamakubo, Takao; (Tokyo, JP) ; Kawabe, Yoshiki;
(Shizuoka-ken, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
Chugai Seiyaku Kabushiki
Kaisha
Trustees of Dartmouth College
|
Family ID: |
22495407 |
Appl. No.: |
10/436137 |
Filed: |
May 13, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10436137 |
May 13, 2003 |
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09141371 |
Aug 27, 1998 |
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6602710 |
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Current U.S.
Class: |
435/6.13 ;
435/320.1; 435/7.2; 514/1 |
Current CPC
Class: |
G01N 33/5008 20130101;
G01N 2800/044 20130101; C12Q 1/6897 20130101; G01N 33/5041
20130101; G01N 2500/00 20130101; G01N 33/502 20130101; G01N 33/92
20130101 |
Class at
Publication: |
435/6 ; 435/7.2;
435/320.1; 514/1 |
International
Class: |
A61K 031/00; C12Q
001/68; G01N 033/53; G01N 033/567 |
Claims
What is claimed is:
1. The screening method for sterol regulatory element binding
protein (SREBP) pathway specific inhibitors, which comprises using
a cell in which a chimeric gene of a reporter gene and the gene
encoding the C-terminus of SREBP has been expressed.
2. The screening method of claim 1, which comprises the steps of:
(a) introducing a chimeric gene of a reporter gene and the gene
encoding the C-terminus of SREBP into a cell in which SCAP has not
lost response to sterols, (b) culturing said cell in the presence
of a test drug for SREBP pathway inhibitor to allow the cell to
express said chimeric gene, and (c) measuring any signal generated
by said reporter gene.
3. The screening method of claim 1, which comprises screening for
SREBP pathway-specific inhibitors using a cell in which a chimeric
gene of a reporter gene and the gene encoding the C-terminus of
SREBP has been expressed, and then screening for sterol-like SREBP
pathway-specific inhibitors using a cell in which SCAP has lost
response to sterols and the same chimeric gene cell has been
expressed.
4. The screening method of claim 3, which comprises the steps of:
(a) introducing a chimeric gene of a reporter gene and the gene
encoding the C-terminus of SREBP into a cell in which SCAP has not
lost response to sterols, (b) culturing the cell of step (a) in the
presence of a test drug for sterol-like SREBP pathway inhibitor to
allow the cell to express said chimeric gene, (c) measuring any
signal generated by said reporter gene, (d) introducing the same
chimeric gene as used in said step (a) into a cell in which SCAP
has lost response to sterols, (e) culturing the cell of step (d) in
the presence of a test drug for sterol-like SREBP pathway inhibitor
to allow the cell to express said chimeric gene, (f) measuring any
signal generated by said reporter gene, and (g) comparing the
signal measured in step (c) and the signal measured in step
(f).
5. The screening method of claim 1, which comprises screening for
S2P-specific inhibitors using a cell in which a chimeric gene of a
reporter gene and a gene encoding the stretch from the first
transmembrane domain of SREBP to the cleavage site with SREBP Site
1 protease (S1P) has been expressed.
6. The screening method of claim 5, which comprises the steps of:
(a) introducing a chimeric gene of a reporter gene and a gene
encoding the stretch from the first transmembrane domain of SREBP
to the cleavage site with SREBP Site 1 protease (S1P) into a cell
which does not lack S2P, (b) culturing said cell in the presence of
a test drug for SREBP pathway inhibitor to express said chimeric
gene, and (c) measuring any signal generated by said reporter
gene.
7. The screening method of claim 1, wherein the reporter gene is
green fluorescence protein gene carrying a nucleus localization
signal (NLS) sequence.
8. The screening method of claim 1, wherein the reporter gene is
GAL4/VP16 fusion gene.
9. The screening method of claim 1, wherein the mutant cultured
cell is a mutant cultured cell derived from CHO cells.
10. A vector obtained by inserting a chimeric gene of a reporter
gene and the gene encoding the C-terminus of SREBP into a reporter
gene expression vector.
11. A vector obtained by inserting a chimeric gene of a reporter
gene and a gene encoding the stretch from the first transmembrane
domain of SREBP to the cleavage site with SREBP Site 1 protease
(S1P) into a reporter gene expression vector.
12. A SREBP pathway-specific inhibitor obtained by the screening
method of claim 8.
13. A therapeutic composition comprising the inhibitor of claim 12.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to a screening method for
SREBP (sterol regulatory element binding protein) pathway-specific
inhibitors. The present invention also relates to therapeutic
agents for hyperlipemia, arterial sclerosis, obesity or cancer
containing an SREBP pathway-specific inhibitor selected by said
screening method.
[0002] Cholesterol is an essential substance for living bodies as a
component of cell membranes or a precursor for syntheses of steroid
hormones or bile acids. However, excessive accumulation thereof is
fatal for cells, and therefore, homeostatic maintenance of cellular
cholesterol level is a very important physiological mechanism. At
the level of living bodies, excessive accumulation of cholesterol
is also known to cause various diseases such as hyperlipemia or
atherosclerosis. The results of a recent large-scale clinical test
on cholesterol-lowering drugs showed that the mortality of patients
of cardiac diseases is improved by lowering serum cholesterol
level, revealing the importance of the maintenance of a proper
cholesterol level in living bodies (Scandinavian Simvastatin
Survival Study Group, Lancet (1994) 344:1383-1389; Gotto, A. M.,
Am. J. Med. (1998) 104:6S-8S).
[0003] Homeostatic maintenance of cellular cholesterol level is
known to occur at various stages such as the level of
transcription, translation, enzyme, or the like. Recent discovery
of factors involved in transcriptional control of a group of
cholesterol synthases or low-density lipoprotein (LDL) receptors
dramatically accelerated an understanding of the mechanism of
homeostatic regulation of cholesterol at transcriptional level.
[0004] LDL receptors or cholesterol synthases such as HMG-CoA
reductase are directly responsible for the maintenance of
cholesterol level as a gate for cholesterol uptake into cells or a
source of newly synthesized cholesterol. It is well known that
expression of these factors is feedback-regulated depending on the
cholesterol level. Recently, sterol regulatory element binding
proteins (SREBPs) that are transcriptional activation factors
binding to a sterol regulatory element (SRE) in the promoter domain
of these genes was shown to be involved in said regulation (Brown,
M. S. and Goldstein, J. L. Cell (1997) 89:331-340).
[0005] SREBP has two isoforms of closely related structures
expressed from different genes, SREBP-1 and SREBP-2. Both are
expressed as membrane proteins spanning the membrane twice with
both ends oriented to the cytosol in the endoplasmic reticulum
membrane or nuclear membrane. Upon decrease of cellular cholesterol
level, the envelope protein SREBP is thought to be cleaved with
proteases in two steps to release the activated N-terminal
DNA-binding domain from the membrane, which migrate into the
nucleus to activate transcription of target genes (FIG. 1). The
protease-catalyzed two-step cleavage mechanism has been mostly
unknown. It has been shown that the first step of cleavage is
sensitive to cholesterol while the second step (site 2) of cleavage
automatically occurs as a result of the first step (site 1) of
cleavage.
[0006] In M19, which is a mutant cell line of Chinese hamster ovary
(CHO)-K1 cells (ATCC: CRL-9618) with lowered cholesterol synthesis
and LDL receptor activities (Hasan, M. T. et al., Somat. Cell Mol.
Genet. (1994) 20:183-194), a study has shown that no cleavage
occurs at site 2 of SREBP (Sakai, J. et al. Cell (1996)
85:1037-1046), and a gene for a protease responsible for cleavage
at site 2, S2P has been cloned by complementation using said cell
line (Rawson et al., Mol. Cell (1997) 1:47-57). The SREBP sequence
cleaved with S2P has not been determined but it is known to lie
near the N-terminal first membrane-spanning domain.
[0007] On the other hand, the cleavage at site 1 is known to occur
between Leu and Ser, but the enzyme per se has not been known.
Another mutant cell line of CHO-K1 cells, 25RA (Chang, T. Y. and
Limanek, J. S., J. B. C. (1980) 255:7787-7795) is resistant to
25-hydroxycholesterol and does not lead to cell death by
cholesterol overload, differing from CHO-K1 cells. On the basis of
dominant sterol resistancy of 25RA cells, an SREBP
cleavage-activating protein (SCAP) was cloned by using an
expression library prepared with 25RA cells. An analysis of the
SCAP gene of 25RA cells and CHO-K1 cells revealed that one of two
point mutations on the SCAP gene of 25RA cells involved a change
from aspartic acid to asparagine at position 443 to enhance SREBP
cleavage activity at site 1 without being regulated by sterols. The
other mutation was a silent mutation. In other words, cholesterol
synthesis or LDL receptor activity is not lowered by sterols in
25RA cells carrying mutations on SCAP because SREBP is cleaved at
site 1 even in the presence of excessive sterols, revealing that
SCAP has an important function in the SREBP cleavage regulation by
cholesterol.
[0008] In this way, establishment of mutant cells derived from
CHO-K1 promoted an understanding of the mechanism of
transcriptional control of cholesterol metabolism. This mechanism
is thought to be common to mammalian cells such as human or murine
cells.
[0009] Target genes for SREBPs as reported include enzymes for the
cholesterol synthesis system such as HMG-CoA synthase, HMG-CoA
reductase, farnesyl diphosphate synthase, squalene synthase, as
well as enzymes for the fatty acid synthesis system such as acetyl
CoA carboxylase or fatty acid synthase, enzymes for the
triglyceride synthesis system such as glycerol-3-phosphate
acyltransferase, and the SREBP-2 gene itself (FIG. 1). This
suggests that inhibition of the SREBP pathway may result in
inhibition of synthesis of lipids such as cholesterol. This
possibility was experimentally verified using sterols such as
25-hydroxycholesterol in cultured cells. However, direct
demonstration on individual level has not been made. Moreover,
recent reports have shown diversity of target genes for SREBPs,
suggesting that the SREBP pathway may be involved in various
physiological phenomena, as will be described later.
[0010] Despite attempts to develop various cholesterol synthesis
inhibitors, many of them have not been successful because of
toxicity or other reasons. HMG-CoA reductase inhibitors are among
rare cases of success, but their effect to lower serum cholesterol
was not evaluated as sufficient (Illingworth, D. R., Am. J.
Cardiol. (1993) 72:54D-58D). At present, HMG-CoA reductase
inhibitors and bile acid reabsorption inhibitors are mainly used
for therapy of hyperlipemia. Both drugs decrease serum cholesterol
by lowering cholesterol in the liver and activating low-density
lipoprotein (LDL) receptors. However, the effect is attenuated by
cholesterol uptake into the liver following activation of LDL
receptors and no potent cholesterol-lowering effect can be obtained
in any case, because these drugs rely on an indirect mechanism of
lowering cholesterol in the liver to activate LDL receptors. On the
other hand, the results of a large-scale clinic test on serum
cholesterol-lowering drugs based on said inhibitors indicated
benefits of lowering serum cholesterol in patients with cardiac
diseases and raised expectations for more potent
cholesterol-lowering drugs.
[0011] From this background, we assumed that lipid-lowering agents
inhibiting lipid synthesis such as cholesterol synthesis by
inhibiting the SREBP pathway may be very useful therapeutic agents
for hyperlipemia and also useful for therapy of arterial sclerosis.
In view of the fact that intermediate products or metabolic
products of the cholesterol synthesis system expressed at a level
controlled by SREBPs have been reported to be ligands for PPARs
(peroxisome proliferator activated receptors), orphan receptors
(Forman, B. M. et al., Cell (1995) 81:687-693; Janowski, B. A. et
al., Nature (1996) 383:728-731; Lala, D. S. et al., Proc. Natl.
Acad. Sci. U.S.A. (1997) 94:4895-4900), it is useful to use said
mechanism for therapy of diseases caused by a decrease in these
molecules. Inhibition of the SREBP pathway also seems to be useful
for therapy of obesity, since PPAR.sub.65 was shown to be an
important determinant for differentiation to adipocytes and
activation of PPAR (Peroxisome Proliferator Activated Receptor)
potentially induced by activation of SREBP was also reported (Kim,
J. B. et al., Proc. Natl. Acad. Sci. U.S.A. (1998) 95:4333-4337).
Inhibition of the SREBP pathway also seem to be applicable as
anticancer agents since some intermediate products of the
cholesterol synthesis system are used to modify Ras or other genes
playing an important role in cell growth. Thus, inhibition of the
SREBP pathway may be promising for therapy of various diseases.
[0012] However, any convenient screening method for inhibitors of
this pathway has not been known up to the present. Noting the
above-mentioned 25RA and M19 cells as well as DM7 cells carrying
mutations of said two cell lines (Hasan, M. T. et al., Somat. Cell
Mol. Genet. (1994) 20:481-491), we examined the potential of a
screening system for SREBP pathway-specific cholesterol synthesis
inhibitors using these cells having mutations in the SREBP pathway
and designed a convenient screening system.
SUMMARY OF THE INVENTION
[0013] The present invention provides a screening method for SREBP
pathway-specific inhibitors using a mutant cultured cell.
[0014] A preferred first embodiment of the screening method of the
present invention is a screening method for SREBP pathway-specific
inhibitors, comprising assaying both cholesterol synthesis and LDL
receptor activities using a cell in which SREBP cleavage activating
protein (SCAP) has lost response to sterols.
[0015] A preferred second embodiment of the screening method of the
present invention is a screening method for sterol-like SREBP
pathway-specific inhibitors using a combination of a cell in which
SCAP has lost response to sterols and a cell in which SCAP has not
lost response to sterols.
[0016] A preferred third embodiment of the screening method of the
present invention is a screening method for SREBP pathway-specific
inhibitors using a combination of a cell lacking SREBP Site 2
protease (S2P) and a S2P-carrying cell.
[0017] A preferred fourth embodiment of the screening method of the
present invention is a screening method for SREBP pathway-specific
inhibitors using a cell in which a chimeric gene of a reporter gene
and the gene encoding the C-terminus of SREBP has been
expressed.
[0018] A preferred fifth embodiment of the screening method of the
present invention is a screening method for sterol-like SREBP
pathway-specific inhibitors, comprising screening for SREBP
pathway-specific inhibitors using a cell in which a chimeric gene
of a reporter gene and the gene encoding the C-terminus of SREBP
has been expressed, and then screening for sterol-like SREBP
pathway-specific inhibitors using a cell in which SCAP has lost
response to sterols and the same chimeric gene has been
expressed.
[0019] A preferred sixth embodiment of the screening method of the
present invention is a screening method for S2P-specific inhibitors
using a cell in which a chimeric gene of a reporter gene and the
gene encoding the stretch from the first transmembrane domain of
SREBP to the cleavage site with SREBP Site 1 protease (S1P) has
been expressed.
[0020] The present invention also provides a vector obtained by
inserting a chimeric gene of a reporter gene and the gene encoding
the C-terminus of SREBP into a reporter gene expression vector.
[0021] The present invention also provides a vector obtained by
inserting a chimeric gene of a reporter gene and the gene encoding
the stretch from the first transmembrane domain of SREBP to the
cleavage site with SREBP Site 1 protease (S1P) into a reporter gene
expression vector.
[0022] The present invention also provides SREBP pathway-specific
inhibitors obtained by the screening method of the present
invention.
[0023] The present invention also provides therapeutic agents for
hyperlipemia containing thus obtained SREBP pathway-specific
inhibitors.
[0024] The present invention also provides therapeutic agents for
arterial sclerosis containing thus obtained SREBP pathway-specific
inhibitors.
[0025] The present invention also provides therapeutic agents for
obesity containing thus obtained SREBP pathway-specific
inhibitors.
[0026] The present invention also provides therapeutic agents for
cancer containing thus obtained SREBP pathway-specific
inhibitors.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1. Schematic representation of the sterol regulatory
element binding protein (SREBP) pathway.
[0028] FIG. 2. A sequential cleavage scheme of SREBPs with S1P and
S2P as well as a sequential cleavage scheme of SREBPs with a fusion
protein produced by introducing and expressing a chimeric gene
according to an embodiment of the present invention.
[0029] FIG. 3. A graph showing the relation between the
concentration of amphotericin B and survival of 25RA cells in the
presence or absence of simvastatin and DM7 cells in the absence of
simvastatin.
[0030] FIG. 4. A graph showing the effect of 25-hydroxycholesterol
on cholesterol synthesis activity in CHO-K1 cells and 25RA cells.
Cholesterol synthesis activity was measured by the amphotericin B
cytotoxicity assay.
[0031] FIG. 5. A graph showing cholesterol synthesis activity in
CHO-K1 cells and M19 cells in the presence or absence of
simvastatin. Cholesterol synthesis activity was measured by the
.sup.14C-acetate incorporation assay.
DETAILED DESCRIPTION OF THE INVENTION
[0032] Suitable cells for the screening method of the present
invention are first described. Cells used in the screening method
of the present invention are, for example, the above-mentioned
Chinese hamster ovary (CHO)-K1 cells (ATCC: CRL-9618) and mutant
cultured cells thereof. However, the SREBP pathway is considered as
a common mechanism to mammals as described above, and therefore,
the invention is not limited to these cells derived from
CHO-K1.
[0033] Suitable cells in which SCAP has lost response to sterols
include but are not limited to, for example, the above-mentioned
25RA that is a mutant cell line of CHO-K1 cells. 25RA cells
carrying mutated SCAP always keep SREBP in activated state so that
they are not regulated in response to sterols. That is, SREBP is
not inactivated in such cells even when cholesterol is
excessive.
[0034] Suitable cells lacking SREBP Site 2 protease (S2P) include
but are not limited to, for example, the above-mentioned M19 that
is a mutant cell line of CHO-K1 cells. In M19 cells lacking S2P,
SREBP is not activated and no response to sterols occurs. That is,
SREBP is not activated even when cholesterol is lacked. Suitable
cells having similar properties include SRD6 (Evans, M. J. and
Metherall, J. E., Mol. Cell Biol. (1993) 13:5175-5185).
[0035] Suitable cells having both of these mutations include but
are not limited to, for example, the above-mentioned DM7 derived
from CHO-K1 cells. That is, DM7 is a cell line in which SCAP has
lost response to sterols and S2P is lacked.
[0036] Properties of typical cultured cells used in the present
invention are collated in the following Table 1, wherein +
represents the presence of mutation and - represents the absence of
mutation.
1 TABLE 1 Enhanced SCAP Cell line Lack of S2P (non-responsive to
sterols) CHO-K1 - - M19 + - 25RA - + DM7 + +
[0037] In the screening method of the present invention, these
mutant cell lines and CHO-K1 cells with no mutation (ATCC:
CRL-9618) can be appropriately used alone or in combination. For
example, the above-mentioned second embodiment of screening can be
accomplished by a combination of 25RA cells and CHO-K1 cells, and
the third embodiment can be accomplished by a combination of M19
cells and CHO-K1 cells or a combination of DM7 cells and 25RA
cells.
[0038] 25RA cells, M19 cells and DM7 cells have been deposited with
the National Institute of Bioscience and Human-Technology of the
Agency of Industrial Science and Technology residing at 1-3,
Higashi 1-Chome, Tsukuba-city, Ibaraki-prefecture, 305-8566, Japan)
on May 19, 1998 under accession numbers FERM BP-6361, FERM BP-6362
and FERM BP-6363, respectively.
[0039] (1) Screening According to the First Embodiment
[0040] The first embodiment of the present invention comprises
screening for SREBP pathway inhibitors by assaying both cholesterol
synthesis and LDL receptor activities using a cell in which SCAP
has lost response to sterols.
[0041] The screening method for SREBP pathway-specific inhibitors
according to this embodiment comprises the steps of:
[0042] (a) culturing a cell in which SCAP has lost response to
sterols in the presence and absence of a test drug for SREBP
pathway inhibitor, and
[0043] (b) measuring both cholesterol synthesis activity and LDL
receptor activity of said cell.
[0044] Any compound that inhibits both cholesterol synthesis
activity and LDL receptor activity in cells in which SCAP has lost
response to sterols is highly likely to be an SREBP pathway
inhibitor.
[0045] Suitable cells in which SCAP has lost response to sterols
may include 25RA cells, for example. In SP2-deficient M19 cells in
which both cholesterol synthesis activity and LDL receptor activity
are extremely lowered, both activities also seem to be lowered by
SREBP pathway inhibitors. Although both activities can also be
assayed in ordinary cultured cells, it is difficult to remove
false-positive samples that indirectly inhibit the SREBP pathway
because the activity of the SREBP pathway varies with intracellular
cholesterol level in these cells. For example, acyl-CoA:
cholesterol acyl transferase (ACAT) inhibitors lower both
activities by increasing cellular free cholesterol. However, such
false positive samples are less likely to occur in cells in which
SCAP has lost response to sterols, such as 25RA cells, which are
therefore suitable for screening according to this embodiment.
[0046] (2) Screening According to the Second Embodiment
[0047] The second embodiment of the present invention comprises
screening for sterol-like SREBP pathway inhibitors using a
combination of a cell in which SCAP has lost response to sterols
and a cell in which SCAP has not lost response to sterols.
[0048] The screening method for SREBP pathway-specific inhibitors
according to this embodiment comprises the steps of:
[0049] (a) culturing a cell in which SCAP has lost response to
sterols and a cell in which SCAP has not lost response to sterols
cell in the presence of a test drug for sterol-like SREBP pathway
inhibitor, and
[0050] (b) measuring the cholesterol synthesis activity or LDL
receptor activity of said both cells.
[0051] For example, suitable cells in which SCAP has lost response
to sterols include 25RA cells and suitable cells in which SCAP has
not lost response to sterols include CHO-K1 cells.
[0052] In ordinary cells that are responsive to sterols,
cholesterol synthesis or LDL receptor activity is lowered by
sterol-like SCAP inactivation effect. In cells that have lost
response to sterols, however, cholesterol synthesis or LDL receptor
activities is not lowered even under sterol-like SCAP inactivation
effect because SCAP is not inactivated. Properties of such cells
can be used to screen for test drugs inhibiting the SREBP pathway
like sterols by measuring the cholesterol synthesis activity or LDL
receptor activity of a cell in which SCAP has lost response to
sterols and a cell in which SCAP has not lost response to sterols.
According to this method, cholesterol synthase inhibitors that are
positive in ordinary cells also inhibit cholesterol synthesis in
cells that are not responsive to sterols so that they can be
readily discriminated from each other.
[0053] (3) Screening According to the Third Embodiment
[0054] The third embodiment of the present invention comprises
screening for SREBP pathway inhibitors using a combination of an
S2P-deficient cell and a S2P-carrying cell.
[0055] The screening method for SREBP pathway-specific inhibitors
according to this embodiment comprises the steps of:
[0056] (a) culturing an S2P-deficient cell and a S2P-carrying cell
in the presence of a test drug for SREBP pathway inhibitor, and
[0057] (b) measuring the cholesterol synthesis activity or LDL
receptor activity of said both cells.
[0058] Suitable combinations of an S2P-deficient cell and a
S2P-carrying cell include, for example, a combination of M19 cells
and CHO-K1 cells or a combination of DM7 cells and 25RA cells.
[0059] In ordinary cells carrying S2P, cholesterol synthesis or LDL
receptor activity is lowered by S2P inhibitors. In S2P-deficient
cells, however, cholesterol synthesis or LDL receptor activity is
no more lowered even by inhibition of the SREBP pathway because S2P
has already been inactivated. Properties of such cells can be used
to screen for test drugs inhibiting S2P by measuring the
cholesterol synthesis activity or LDL receptor activity of ordinary
cells and S2P-deficient cells. According to this method,
cholesterol synthase inhibitors that are positive in ordinary cells
also inhibit cholesterol synthesis in S2P-deficient cells so that
they can be readily discriminated from each other.
[0060] (4) Screening According to the Fourth Embodiment
[0061] Although screening methods according to the first to third
embodiments as described above can be used to conveniently screen
for SREBP pathway inhibitors, the SREBP pathway has been explained
only recently and still unknown in many aspects. Therefore, it is
more useful to use SREBP itself for screening for inhibitors
specific to the SREBP activation mechanism. In this respect, the
reporter gene assay using SRE is effective, but SREBP alone weakly
binds to SRE and should act synergistically with co-activators such
as transcriptional activator SP1 (specific protein 1) or
transcriptional activator NF-Y (nuclear factor Y) for stronger
binding, which causes false positive reactions.
[0062] If the N-terminal DNA-binding domain (DBD) that is
unnecessary for cleavage of SREBP with S1P and S2P is replaced by
another reporter to detect release of SREBP from the membrane,
SREBP activation can be detected more specifically and more
sensitively.
[0063] Accordingly, the fourth embodiment of the present invention
comprises screening for SREBP pathway-specific inhibitors using a
cell in which a chimeric gene of a reporter gene and the gene
encoding the C-terminus of SREBP has been expressed.
[0064] The screening method for SREBP pathway-specific inhibitors
according to this embodiment comprises the steps of:
[0065] (a) introducing a chimeric gene of a reporter gene and the
gene encoding the C-terminus of SREBP into a cell in which SCAP has
not lost response to sterols,
[0066] (b) culturing said cell in the presence of a test drug for
SREBP pathway inhibitor to allow the cell to express said chimeric
gene, and
[0067] (c) measuring any signal generated by said reporter
gene.
[0068] Suitable reporter genes include, for example, green
fluorescence protein gene carrying a nucleus localization signal
(NLS) sequence or GAL4/VP16 fusion gene. Suitable cells into which
the chimeric gene of a reporter gene and the gene encoding the
C-terminus of SREBP is expressed include, for example, cells in
which SCAP has not lost response to sterols, such as CHO-K1 cells
or M19 cells.
[0069] Human SREBP-2 has been shown to undergo sterol-regulated
cleavage even if the amino acids up to position 472 are replaced by
Ras (Sakai et al., Cell (1996) 85 1037-1046). It has also been
shown that 8 amino acids in the region 455-483 and the region
402-477 in SREBP-1a and SREBP-2, respectively, are unnecessary for
sterol-regulated cleavage (Hua et al., J. Biol. Chem. (1996) 271
10379-10384). This indicates that any protein fused to the
C-terminal region from position 484 in human SREBP-1 or position
478 in human SREBP-2 may be cleaved with S1P and S2P. One of such
reporters is a protein fused to a fluorescence protein, green
fluorescence protein (GFP) (Hamakubo et al., Atherosclerosis (1997)
134:350). However, this fusion protein is not suitable for
quantitative assay of cleavage activity, because it is released
from the endoplasmic reticulum membrane and diffused into the
cytoplasm after cleavage. Thus, we designed a more sensitive fusion
protein by introducing a nucleus localization signal (NLS) sequence
into GFP so that released GFP migrates to the nucleus, which in
turn generates fluorescence.
[0070] Namely, a gene in which DBD of SREBP is replaced by
NLS-containing GFP is inserted into an appropriate expression
vector and the vector is transferred into a cell by a known method
to allow it to express said gene. When SREBP is cleaved with S2P,
fluorescence migrates to the nucleus. Thus, screening can be
accomplished by measuring change of fluorescence amount in the
nucleus of a cell treated with a test drug.
[0071] If DBD of SREBP is replaced by a factor having a stronger
transcriptional activity alone, a reporter gene corresponding to
this transcription factor can be introduced to measure the activity
of the SREBP pathway as reporter activity. For example, a fusion
protein of DBD of GAL4 and the activation domain (AD) of VP16 is
known to strongly activate genes downstream of a yeast-specific
GAL4-specific sequence (UAS.sub.G) (Sadowski et al., Gene (1992)
118:137-141), and suitable for this purpose. That is, a gene in
which DBD of SREBP is replaced by said protein is introduced with a
reporter gene such as UAS.sub.G-luciferase and expressed in cells,
so that luciferase activity increases when SREBP is cleaved with
S2P. Thus, screening can be accomplished by measuring change of
luciferase activity after treatment with a test drug.
[0072] FIG. 2 shows a sequential cleavage scheme of SREBPs by S1P
and S2P as well as a sequential cleavage scheme of SREBPs by a
fusion protein produced by introducing and expressing a chimeric
gene according to the present embodiment.
[0073] (5) Screening According to the Fifth Embodiment
[0074] Although the SREBP chimeric gene used in the above fourth
embodiment may be usefully introduced and expressed in ordinary
cells in terms of response to sterols, i.e. a cell in which SCAP
has not lost response to sterols, it is more preferable to express
said chimeric gene into a cell in which SCAP has lost response to
sterols, such as 25PA cells, because the SREBP activation mechanism
of ordinary cells is influenced by cholesterol as described
above.
[0075] Accordingly, the fifth embodiment of the present invention
allows more reliable screening for sterol-like SREBP
pathway-specific inhibitors, by performing screening according to
the fourth embodiment, then expressing the same chimeric gene in a
cell in which SCAP has lost response to sterols to perform
screening according to the fourth embodiment, and comparing the two
screenings. That is, any test drug that was positive in ordinary
cells but negative in cells non-responsive to sterols seems to have
a sterol-like SREBP pathway-specific inhibitory effect.
[0076] The screening method for SREBP pathway-specific inhibitors
according to this embodiment comprises the steps of:
[0077] (a) introducing a chimeric gene of a reporter gene and the
gene encoding the C-terminus of SREBP into a cell in which SCAP has
not lost response to sterols,
[0078] (b) culturing the cell of step (a) in the presence of a test
drug for sterol-like SREBP pathway inhibitor to allow the cell to
express said chimeric gene,
[0079] (c) measuring any signal generated by said reporter
gene,
[0080] (d) introducing the same chimeric gene as used in step (a)
into a cell in which SCAP has lost response to sterols,
[0081] (e) culturing the cell of step (d) in the presence of a test
drug for sterol-like SREBP pathway inhibitor to allow the cell to
express said chimeric gene,
[0082] (f) measuring any signal generated by said reporter gene,
and
[0083] (g) comparing the signal measured in step (c) and the signal
measured in step (f).
[0084] (6) Screening According to the Sixth Embodiment
[0085] The sixth embodiment of the present invention comprises
screening for S2P-specific inhibitors using a cell in which a
chimeric gene of a reporter gene and the gene encoding the stretch
from the first transmembrane domain of SREBP to the cleavage site
with SREBP Site 1 protease (S1P) has been expressed.
[0086] As already described, cleavage of SREBP occurs in two steps
and the second step of cleavage with S2P is said to essentially
involve cleavage with S1P and automatically occur after cleavage
with S1P. Thus, screening for S2P-specific inhibitors can be
accomplished by expressing a chimeric gene of a reporter gene (such
as GFP-NLS, GAL4-VP16) fused to a gene encoding the stretch from
the first transmembrane domain of SREBP to the cleavage site with
SREBP Site 1 protease (S1P) as described above in a cell to use it
in the same manner as described for screening according to the
fourth or fifth embodiment.
[0087] The screening method for SREBP pathway-specific inhibitors
according to this embodiment comprises the steps of:
[0088] (a) introducing a chimeric gene of a reporter gene and a
gene encoding the stretch from the first transmembrane domain of
SREBP to the cleavage site with SREBP Site 1 protease (S1P) into a
cell which does not lack S2P,
[0089] (b) culturing said cell in the presence of a test drug for
SREBP pathway inhibitor to allow the cell to express said chimeric
gene, and
[0090] (c) measuring any signal generated by said reporter
gene.
[0091] Means for Measuring Cholesterol Synthesis Activity
[0092] In order to measure cholesterol synthesis activity, the
examples of the present invention use:
[0093] (1) amophotericin B cytotoxicity assay, or
[0094] (2) .sup.14C-acetate incorporation assay.
[0095] The amphotericin B cytotoxicity assay involves measuring
cholesterol synthesis activity, based on the property of
amphotericin B to bind to cholesterol in cell membranes and
cytotoxically act in proportion to cholesterol level in cell
membranes whereby cells can survive at low cholesterol synthesis
activity but die at high synthesis activity. That is, the lower the
cell survival after treatment with amphotericin B, the higher the
cholesterol synthesis activity.
[0096] On the other hand, the .sup.14C-acetate incorporation assay
involves measuring .sup.14C-cholesterol level increasing as cells
exploit .sup.14C-acetate added to the culture medium for
cholesterol synthesis.
[0097] Alternatively, cholesterol content in cells may be directly
assayed, and the present invention is not limited to the two assays
used herein.
[0098] Means for Measuring LDL Receptor Activity
[0099] In order to measure LDL receptor activity, the examples of
the present invention use the .sup.125I-LDL binding assay. However,
other suitable means may include incorporation of LDL labeled with
a fluorescent lipid DiI into cells or ELISA-mediated measurement of
the amount of LDL receptors expressed on the surface of cell
membranes, and the screening system of the present invention is not
specifically limited to those shown herein.
[0100] SREBP pathway-specific inhibitors obtained by the screening
method of the present invention are useful as therapeutic agents
for hyperlipemia, arterial sclerosis, obesity, cancer, etc. The
administration route is suitably oral for therapy of hyperlipemia,
arterial sclerosis and obesity, or oral or intravenous as
anti-cancer agents. The daily dosage may vary from 0.1 mg to 500
mg, preferably 1 mg to 100 mg per adult. However, the dosage and
administration route are not specifically limited to those
indicated herein, but should be appropriately chosen according to
various factors such as the disease to be treated, medical
condition of the patient, physical properties of the resulting
SREBP pathway-specific inhibitor or the strength of inhibitory
action.
[0101] The following examples further explain the present invention
in detail, but are not construed as limiting the scope thereof.
Various modifications or changes are obvious for those skilled in
the art on the basis of the disclosure herein and information
available in the field of art, and those are also encompassed in
the scope of the present invention.
EXAMPLES
Example 1
[0102] Screening Method According to the First Embodiment
[0103] In order to assess the availability of this method, an
inhibitor of a rate-limiting enzyme for cholesterol synthesis
system HMG-CoA reductase, simvastatin, was examined as a test drug.
In addition to 25RA cells, DM7 cells were also examined as a cell
model treated with an SREBP pathway inhibitor.
[0104] Influences on cholesterol synthesis activity of 25RA cells
in the presence or absence of simvastatin and DM7 cells in the
absence of simvastatin were first determined by the amphotericin B
cytotoxicity assay.
[0105] On day 0, 25RA and DM7 cells were seeded in 96-well plates
at a density of 5.times.10.sup.3 cells/well, and incubated
overnight in F12 containing 10% LPDS (L-F12) for 25RA or Ham's F12
medium containing 10% FBS (F-F12) for DM7. On day 1, the media were
replaced by L-F12 containing simvastatin at various concentrations
(0-0.3 .mu.M) and incubation was continued for 24 hours. On day 2,
cells were washed with PBS, and then incubated in PBS containing
amphotericin B at various concentrations. After incubation for 4
hours, the cells were washed with PBS and protein content in each
well was measured by the Lowry method to assay amphotericin B
sensitivity. Amphotericin B sensitivity was expressed as a value
(%) when the remaining protein content in each well after the same
treatment in the absence of amphotericin B was counted as 100%.
[0106] Then, influences on LDL receptor activity of 25RA cells in
the presence or absence of simvastatin and DM7 cells in the absence
of simvastatin were determined by the .sup.125I-LDL binding assay.
.sup.125I-LDL was prepared as described by Kawabe et al. (Kawabe,
Y. et al., Arch. Biochem. Biophys. (1994) 310:489-496). On day 0,
cells were seeded on 6-well plates at a density of 3.times.10.sup.4
cells/well, and incubated in F-F12. On day 3, the medium was
replaced by L-F12 (containing 20 .mu.M oleic acid) each containing
(a) 1 .mu.g/ml 25-hydroxycholesterol and 10 .mu.g/ml cholesterol,
(b) 0.1% EtOH or (c) 1 .mu.M simvastatin, and incubation was
continued for 24 hours. On day 4, the cells were incubated at
4.degree. C. for 2 hours, and then washed twice with PBS. Then,
incubation was continued at 4.degree. C. in L-F12 (containing 10 mM
Hepes-NaOH, pH 7.4) containing 10 .mu.g/ml .sup.125I-LDL or 10
.mu.g/ml .sup.125I-LDL and 400 .mu.g/ml LDL. After 5 hours, the
cells were washed with buffer B (150 mM NaCl, 50 mM Tris-HCl, 2
mg/ml BSA, pH 7.4), then buffer C (150 mM NaCl, 50 mM Tris-HCl, pH
7.4) each three times, and incubated with 1 ml of buffer D (50 mM
NaCl, 10 mM Hepes-NaOH, 4 mg/ml dextran sulfate, pH 7.4) at
4.degree. C. After one hour, buffer D was recovered and assayed for
its radioactivity. The protein level in cells remaining in wells
was measured by the BCA method to calculate .sup.125I-LDL binding
per mg of cells.
[0107] As the results in FIG. 3, survival of 25RA cells decreased
depending on the concentration of amphotericin B. However, DM7
showed remarkable resistance to amphotericin B as compared with
25RA. Simvastatin inhibited amphotericin B-induced decrease of
survival of 25RA cells concentration-dependently.
[0108] As to LDL receptor activity, 25RA showed higher
.sup.125I-LDL binding than CHO-K1 in the presence of sterols, as
shown in Table 2. .sup.125I-LDL binding of CHO-K1 was increased by
simvastatin treatment, but .sup.125I-LDL binding of 25RA was
unchanged.
2TABLE 2 Cell surface binding of [.sup.125I]LDL Sterols + - -
Simvastain -- -- + CHO-KI 12.5 18.1 30.1 25PA 19.0 17.9 20.9 ng/mg
protein
[0109] As a result, a HMG-CoA reductase inhibitor, simvastatin, had
an inhibitory effect against cholesterol synthesis activity but no
effect to decrease LDL receptor activity in 25RA. On the other
hand, a cell model treated with an SREBP pathway inhibitor, DM7,
showed a lower cholesterol synthesis activity than 25RA. As has
been already reported, DM7 shows a remarkably low .sup.125I-LDL
binding such as 1/5.5 of 25RA (Hasan, M. T. et al., Somat. Cell
Mol. Genet. (1994) 20 481-491). Thus, an experimental system
assaying cholesterol synthesis and LDL receptor activities using
25RA cells proved to be a useful system that can specifically
detect SREBP pathway inhibitors.
Example 2
[0110] Screening Method According to the Second Embodiment
[0111] The effects of 25-hydroxycholesterol on cholesterol
synthesis activity in CHO-K1 and 25RA were examined. Cells were
treated with 0.1 .mu.g/ml of 25-hydroxycholesterol for 24 hours
before amphotericin B was added. Cholesterol synthesis activity was
measured by the amphotericin B cytotoxicity assay in the same
manner as the preceding example.
[0112] As shown in FIG. 4, 25-hydroxycholesterol inhibited
cholesterol synthesis activity in CHO-K1 but not in 25RA. This
demonstrated that a cholesterol synthesis activity assay using
these two cell lines is a useful system that can specifically
detect sterol-like SREBP pathway inhibitors.
Example 3
[0113] Screening Method According to the Third Embodiment
[0114] Cholesterol synthesis activity in CHO-K1 cells and M19 cells
was examined using simvastatin as a test drug. The cholesterol
synthesis activity was measured by the .sup.14C-acetate
incorporation assay.
[0115] On day 0, 5.times.10.sup.5 cells were seeded in 25 cm.sup.2
flasks and incubated in F-F12 containing 10% FBS. On day 2, the
medium was replaced by L-F12 containing 1 .mu.M simvastatin (final
concentration: 0.2% DMSO) or L-F12 free from simvastatin, and K1
was incubatted for 24 hours while M19 was incubated for 12 hours.
On day 3, 50 .mu.l of 1 mM [2-.sup.14C] sodium acetate (20
.mu.Ci/flask) was added and after incubation for 6 hours, cells
were washed with phosphate buffered saline (PBS) and solubilized in
2 ml of 0.1N NaOH. 50 .mu.l of the cell solution was used for
protein assay and the remainder was used for .sup.14C-cholesterol
assay. For .sup.14C-cholesterol assay, the cell solution was first
combined with 2 ml of ethanol solution containing 0.36 N NaOH and
heat-treated at 80.degree. C. for one hour. After cooling, the
mixed solution was extracted with petroleum ether and concentrated
under N.sub.2 stream, thereafter cholesterol was separated by thin
layer chromatography to measure the radioactivity of
.sup.14C-cholesterol on the plate as photostimulated luminescence
(PSL) using BAS2000 (Fuji Film). Cellular protein level was
measured by the BCA method and the cholesterol synthesis activity
was expressed as the synthesized cholesterol amount in PSL per mg
of the protein per 6 hours.
[0116] As the results in FIG. 5, simvastatin inhibited cholesterol
synthesis activity in both cell lines. Since S2P inhibitors are
ineffective in S2P-deficient cells, a cholesterol synthesis
activity assay using the two types of cell lines proved to be a
useful system that can specifically detect S2P inhibitors,
Example 4
[0117] Screening Method According to the Fourth Embodiment
[0118] PCR-amplified human SREBP-2 fragment (475-1141) having an
XhoI site at its both ends was inserted into the XhoI site of a
green fluorescence protein expression vector, pEGFP-C1 (Clontech).
A sequence having a modified BspEI site at each end of T-large
antigen-derived NLS (5'-cctaagaagaagaggaaggtt-3') was inserted into
the BspEI site of the resulting pEGFP-SREBP2 (475-1141) to prepare
pGNR. This was transferred into CHO-K1 or M19 by the FuGENE6 method
(Boeringer Mannheim). 9 hours after transfection, the medium was
replaced by L-F12 (in the presence or absence of 1 .mu.g/ml
25-hydroxycholesterol and 10 .mu.g/ml cholesterol), and after
incubation overnight, fluorescence intensity of the nucleus was
observed by a confocal laser scanning microscope.
[0119] As the results in Table 3, fluorescence was detected in the
absence of sterol in the nucleus derived from CHO-K1 cells but not
in M19. This demonstrated that SREBP pathway inhibitors could be
specifically detected by treating cells carrying pGNR with a test
drug and then observing fluorescence intensity of the nucleus.
3TABLE 3 Comparison of fluorescence intensities of the nuclei of
cells carrying pGNR K1 M19 In the presence of sterol - - In the
absence of sterol + -
[0120] In the same manner as in Example 4, pGNR was transferred
into CHO-K1 and 25RA, grown and observed.
[0121] As the results in Table 4, attenuated fluorescence was
detected in the nucleus of CHO-K1 in the presence of sterol, but
not in 25RA. This demonstrated that sterol-like SREBP pathway
inhibitors can be specifically detected by observing 25RA and
CHO-K1-derived cells in which PGNR has been introduced.
4TABLE 4 Comparison of fluorescence intensities in the nuclei of
cells carrying pGNR K1 25RA In the presence of sterol - + In the
absence of sterol + ++
Example 6
[0122] Screening Method According to the Sixth Embodiment
[0123] Into PGNR digested with Bgl2 and Hind3 was inserted human
SREBP-2 having a Bgl2 site and a Hind3 site with a stop codon at
the PCR-amplified 5' end and 3' end (475-522), respectively, to
prepare pGNT. This was transferred into 25RA and DM7 and incubated
overnight in F-F12, and then fluorescence intensity of the nucleus
was observed by a confocal laser scanning microscope in the manner
as described in Example 4.
[0124] As the results in Table 5, intense fluorescence was detected
in the nucleus of 25RA while the fluorescence in the nucleus of DM7
was weak. This demonstrated that S2P inhibitors can be specifically
detected by observing fluorescence of the nucleus carrying PGNT and
treated with a test drug.
5TABLE 5 Comparison of fluorescence intensities of the nuclei of
cells carrying pGNT 25PA DM7 +++ +/-
* * * * *