JPH1156397A - Screening method for hyperlipidemia drug - Google Patents

Abstract

(57) [Summary] [Object] To provide a method for screening a safe therapeutic agent for hyperlipidemia not accompanied by fatty liver. Kind Code: A1 A method for screening an agent that selectively increases the action of SREBP (sterol responsive element binding protein) -2, and a therapeutic agent for hyperlipidemia containing an agent that selectively increases the action of SREBP-2 .

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for screening a therapeutic drug for hyperlipidemia. More specifically, the present invention relates to a method of screening for an agent that selectively increases the action of SREBP (sterol responsive element binding protein) -2. Such a drug is promising as a therapeutic agent for hyperlipidemia.

[0002]

2. Description of the Related Art Currently, HMG-CoA reductase (hydroxymethylglutaryl-CoA reductase: hydroxymethylglutaryl C) is mainly used for the treatment of hypercholesterolemia.
An enzyme that catalyzes the reaction of reducing oA in the presence of NADPH to produce mevalonic acid) and bile acid reabsorption inhibitors have been used. Both drugs lower blood cholesterol by lowering liver cholesterol and activating low density lipoprotein (LDL) receptors. However, in all cases, the LDL receptor is activated by an indirect mechanism of hepatic cholesterol lowering, so the effect of the cholesterol uptake by the liver due to LDL receptor activation is attenuated and it is not possible to exert a strong cholesterol lowering effect .

[0003] LDL responding to this decrease in liver cholesterol
As a receptor activation mechanism, a sterol-responsive element (SRE: s
sterol-responsive element binding protein (SRE), which is a transcription activator that binds to terolreguratory element
BP) has been shown to be involved. When the amount of cholesterol in the cell decreases, SREBP bound to the endoplasmic reticulum becomes activated SREBP-2 released from the membrane by a specific protease, translocates into the nucleus, and binds to SRE, whereby the LDL receptor gene It is thought to activate transcription (Wang e
tal., Cell 77: 53-62, 1994). Therefore, it is thought that by activating the release of SREBP from the membrane, etc., the LDL receptor can be activated irrespective of intracellular cholesterol, and a drug with such a mechanism was searched. It has not been put to practical use.

[0004] SREBP has two types of isoforms, SREBP-1 and SREBP-2, and SREBP-1 has 1a, 1a by alternative splicing at its 5 'or 3'.
b, 1c (Yokoyama et al.,
Cell 75 (1): 187-98, 1993). FIG. 1 shows the structure of the SREBP isoform and a schematic diagram of each nuclear translocation type, and the translocation of SREBP to the nucleus by excision from the endoplasmic reticulum membrane.

[0005] Although SREBP-1 was found in a human cDNA library, ADD1 which is considered to be a homologue thereof from a rat adipocyte cDNA library was first found as an important transcription factor for adipocyte differentiation. (Tontonoz et al., Mol. Cell. Biol. 13: 4753-9, 199
3).

As described above, SREBP has been discovered as a transcriptional activator of the human LDL receptor gene. However, subsequent studies have shown that cholesterol synthase HMG-Co
A synthase (Yokoyama et al., Cell 75 (1): 187-98, 1
993), HMG-CoA reductase (Vallettet al., J. Biol.
Chem. 271 (21): 12247-53, 1996), farnesyl diphosphate synthase (Jackson et al., J. Biol. Chem. 37 (8):
1712-21, 1996), squalene synthase (Guan et al.,
J. Biol. Chem. 270 (37): 21958-65, 1995),
Acetyl-CoA carboxylase, a fatty acid synthesis enzyme
(Lopez et al., Proc. Natl. Acad. Sci. USA 93 (3): 104
9-53, 1996), fatty acid synthase (Bennett et al., J. B.
iol. Chem. 270 (43): 25578-83, 1995), glycerol-3-phosphate acetyltransferase which is a triglyceride synthesis enzyme (Ericsson et al. SREBP-2, J. Biol. Ch.
em. 272 (11): 7298, 1997). S
ato et al., J. Biol. Chem. 271 (43): 26461-4, 1996).

[0007] More recently, SREBP-1a or 1c
Transgenic mice have been developed that overexpress the nuclear translocation region in the liver, and these mice exhibited fatty liver symptoms.Thus, activation of SREBP-1 is a possible mechanism of LDL receptor activation. Was considered inappropriate (Shimano, H. et al.,
J. Clin. Invest. 98: 1575-1584, 1996; Shimano, H.et
al., J. Clin. Invest. 99: 846-854, 1997).

[0008]

DISCLOSURE OF THE INVENTION The present invention focuses on the possibility of SREBP as an activation mechanism of LDL receptor, focusing on the possibility that SREBP has a different action depending on the isoform, regardless of intracellular cholesterol, Search for an SREBP isoform capable of activating transcription of the LDL receptor gene,
An object of the present invention is to provide a method of screening for an agent that selectively increases a REBP isoform. The present invention further aims at treating hyperlipidemia with such agents.

[0009]

Means for Solving the Problems In order to achieve the above object, the present inventors have conducted intensive studies and have found that the selective action of SREBP-2, an SREBP isoform, can be enhanced without increasing fatty acid synthesis. The present inventors have discovered that LDL receptor activity can be increased, and completed the present invention.

[0010] That is, the present invention provides a method of screening for an agent that selectively increases the action of SREBP (sterol responsive element binding protein) -2. In a preferred embodiment of the present invention, a test agent is allowed to act on cells expressing two of SREBP-1 and SREBP-2, and the effect of SREBP-2 is greater than that of SREBP-1. This is done by identifying the drug.

[0011] The present invention further provides a therapeutic agent for hyperlipidemia containing an agent that selectively increases the action of SREBP-2. Such therapeutics can be identified by the screening methods described above.

[0012] Human SREBP-2 consists of 1141 amino acids and is 47% more than SREBP-1a (1147 amino acids).
Homologous. SREBP-1a and SREBP-2 are both acidic N
H ₂ terminal, highly conserved bHLH-Zip (basic helix - loop - helix - leucine zipper)
It has a motif (71% homology), as well as a very long sequence of 740 amino acids at the COOH terminus of the bHLH-Zip region. SREBP-2 differs from SREBP-1a by having a glutamine-rich region (27% glutamine of 121 residues) (US Pat. No. 5,527,690).

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In developing the screening method of the present invention, first, SREBP isoform SRE
We searched for a target gene for BP-2 and constructed a cell line that could examine changes in lipid metabolism of cells resulting from transcriptional activation by SREBP-2.

This cell line is a human-derived cell, HeLa S
3 cells, LacI expression vector p3'SS and activated human
Two types of plasmids (pOPI3BP2) expressing SREBP-2 (containing 1-481 amino acids) under the control of a lac operator were introduced (a schematic diagram is shown in FIG. 2). In this cell, the SREBP-2 (1-481)
Expression is suppressed by LacI expression. However, the addition of isopropylthiogalactopyranoside (IPTG) induces SREBP-2 (1-481) expression from this vector. Therefore, activated SREBP-2 increases LDL receptor activity without increasing fatty acid synthesis activity by measuring changes in the expression level of various target genes or lipid metabolic activity in this cell after IPTG addition. It can be clarified whether or not to do so.

Using the above cells, the effects of SREBP on the regulation of expression were examined for their effects on the transcriptional regulation and metabolic activity of various genes as described above. As a result, as shown in Table 1, LDL receptor mRN was added by adding IPTG.
A and LDL receptor activities increased about 2.5 and 3.5 fold, but fatty acid synthase mRNA did not change and fatty acid synthase activity did not increase.

From these results, SREB induces fatty liver.
Unlike P-1, enhanced action of SREBP-2 has been shown to increase LDL receptor activity without increasing fatty acid synthesis. Therefore, it was revealed that SREBP-2 selective enhancement of action is useful as a safe mechanism of action that does not accompany fatty liver caused by activation of SREBP action. In addition, SREBP-1 and SRE
FIG. 3 shows the mechanism of regulation of LDL receptor expression by BP-2.

In the present invention, “selectively increasing the effect of SREBP-2” means that the effect of SREBP-2 is greater than that of SREBP-1. S
The increase in the action of SREBP-2 is preferably 1.5 times or more, more preferably 2 times or more, as compared with the increase in the action of REBP-1.

Therefore, compared to the increase in the action of SREBP-1,
If a drug that greatly increases the effect of BP-2 can be screened, this drug can be used to lower blood cholesterol and be used for the treatment of hyperlipidemia.

In the screening method of the present invention, SREBP-
Cells expressing both 1 and SREBP-2 are used. SREBP-
Cells expressing two of 1 and SREBP-2 are SREBP-1 and SREBP-2.
The two REBP-2s may be expressed in the same cell, or may be expressed in separate cells.

[0020] Enhancement of SREBP-2 action refers to nuclear localization type SREBP-2.
To enhance the nuclear activation of LDL receptors and enhance the transcriptional activation of LDL receptors. Enhancement of SREBP-2 action means increasing the amount of SREBP-2 mRNA and expression of SREBP-2 protein and increasing the translocation into the nucleus by cleaving the SREBP-2 precursor expressed in the endoplasmic reticulum or nuclear membrane. Further, it becomes possible by enhancing the binding between SREBP-2 and a transcriptional activator on the LDL receptor gene promoter. Therefore, SREBP-
The action point of the drug that enhances the action of 2, for example, SRE
Increased transcription efficiency of BP-2 gene, increased stability of SREBP-2 mRNA, increased translation efficiency of SREBP-2 mRNA, increased stability of SREBP-2 protein, increased cleavage of SREBP-2 protein into nuclear translocation, increased SREBP- (2) Increase in nuclear translocation of protein, increase in transcriptional activity of SREBP-2, enhancement of transcriptional activator binding to SREBP-2 in the nucleus, etc. Therefore, when screening for a drug that selectively enhances SREBP-2, one of these action points is considered.
More than one can be used as an index. In particular, SREBP-2
It is preferable to use an increase in gene transcription efficiency and / or an increase in SREBP-2 transcription activity as an index. By comparing the effect of the drug to be screened on SREBP-1 and SREBP-2, a drug having an increased effect of SREBP-2 greater than that of SREBP-1 is identified.

As an example of the screening method of the present invention, for example, after a test agent is added to a cell culture medium and incubated, the cells are recovered and mRNAs of SREBP-1a, -1b, -1c and SREBP-2 are recovered. Amount or SREBP-1a, -1b, -1c and SREBP
-2 It can be performed by measuring the amount of protein. SR
A test drug in which increase in SREBP-2 mRNA or SREBP-2 protein is larger than increase in EBP-1 mRNA or SREBP-1 protein is defined as positive.

In measuring the action of SREBP-1 or SREBP-2, not only the gene encoding SREBP-1 or SREBP-2 but also the fusion protein prepared by replacing the SRE binding region with another protein is encoded. The SREBP-1 fusion protein gene or SREBP-2 fusion protein gene may be used. This is due to the enhanced release of nuclear-relocated SREBP by SREBP-2
In the case of a mechanism that enhances the effect, it is thought that it is not involved in nuclear translocation-type release (Sakai, J. et al., Cell 85: 1037-1046,
1996) Even if the SRE binding region is replaced with another protein, cleavage release from the membrane binding region occurs in the same manner as in the wild type. However, such a gene is required to include in the fusion protein a sequence encoding the amino acid sequence of a specific cleavage site required for releasing nuclear localization type SREBP by SREBP. For example, in SREBP-1a,
A fusion protein having a sequence of 455-1147 amino acids and of SREBP-2 has a sequence of 463-1141 amino acids. All of these proteins are considered to be cleaved by caspase-3 at the 460th (SREBP-1a) or 480th (SREBP-2) A
sp. (Wang, X. et al., J. Biol. Chem. 27
0: 18044-18050, 1995), which also contains sequences necessary for a sterol-responsive two-step cleavage reaction occurring at the carboxyl terminus from this cleavage position (Sakai, J. et al., Cell 8).
5: 1037-1046, 1996). The biggest feature of such a method using a fusion protein gene is that SREBP-1 and SREBP-2
It is possible to distinguish between SREBP-1 and SREBP-2, which cannot be achieved by a method for directly or indirectly detecting a reaction expressed through binding to SRE. Furthermore, by fusing a protein that is easier to detect, a highly sensitive and simple screening system can be constructed.

For example, a cell is prepared in which a plasmid having a binding sequence for a transcription factor such as GAL4 (UAS _{G in} the case of GAL4) is introduced upstream of a reporter gene such as luciferase. In addition, 1) a transcriptional activator that binds to an upstream sequence introduced into cells such as GAL4, or an artificial transcriptional activator in the form of a fusion, and a specific transcriptional activator required to release nuclear localization SREBP. A gene that expresses a protein fused to a membrane-binding region containing the amino acid sequence of the cleavage site and its carboxyl-terminal, for example, a fusion protein having a sequence of 463-1141 amino acids in SREBP-2, or 2) human
Prepare two cells, each of which has been introduced into the previously prepared cells, a gene expressing a protein fused with a transcription activator as shown in 1) on the N-terminal side of SREBP-1a (455-1147 amino acids). . After adding the drug to the culture of each cell and incubating, the cells are collected and the reporter activity of each is measured.

Specific examples include the systems shown in the following examples. However, the screening method of the present invention is not limited thereto, and various changes and modifications can be made by those skilled in the art.

The agent identified by the screening method of the present invention, which increases only the action of SREBP-2 without increasing the action of SREBP-1, promotes the expression of LDL receptor. Increased LDL receptor on the cell surface is due to L in plasma
This would reduce DL levels, and thus such agents are promising as therapeutic agents for hyperlipidemia.

[0026]

【Example】

Example 1: Construction of a cell line capable of activating only SREBP-2 After cutting the expression vector pOPI3CAT containing the Lac operator sequence in the RSV promoter with NotI, blunt-ending with Klenow fragment, and further removing phosphorus Oxidize.
The SREBP-2 (1-481) expression vector pSREBP-2 (1-481) was cut with XhoI and NotI and the 1.5 kb fragment was blunt-ended with Klenow fragment, and the previously obtained pOPI3CAT-derived DNA insert. Plasmid constructed in this way
It was confirmed by decoding the base sequence that pOPI3BP2 had SREBP-2 (1-481) inserted in a direction in which expression could be induced by IIPTG.

HeLa S3 cells were transfected with p3'SS, a Lac repressor (LacI) expression vector, by electroporation.
Surviving cell lines were selected on Dulbecco's modified Eagle's medium containing mg / ml hygromycin and 10% FBS. Among these cell lines, those expressing LacI mRNA were selected and used in the following experiments.

POPI3BP2 DNA was introduced into the Lac repressor-expressing HeLa S3 cells prepared as described above by electroporation, and 0.3 mg / ml hygromycin, 0.3 mg / ml G418 and 10%
Surviving cell lines were obtained in Dulbecco's modified Eagle's medium containing FBS. IPTG on the cell line obtained here
SREBP-2 (1-481) inducibility, SREBP-2 mRNA before and after IPTG addition
Levels were quantified to confirm inducibility.

Example 2 Investigation of Involvement of SREBP-2 in Regulation of Expression Using the cell line constructed in Example 1, the regulation of transcription of various genes (LDL receptor, fatty acid synthase) upon addition of IPTG was examined. S
The involvement of REBP-2 and the effect of SREBP-2 on metabolic activity were investigated.

The transcriptional activity was measured by measuring the cells with 0.3 mM IPTG, 10 mM
After culturing for 4 hours in Dulbecco's modified Eagle's medium containing ug / ml cholesterol, 1 ug / ml 25-hydroxycholesterol, and 10% FBS, total RNA was prepared from the cells, and (Kawab
e et al., BBRC 219: 515-20, 1996).
Was measured. In the measurement of metabolic activity, for LDL receptor, cells were treated with 0.3 mM IPTG, 10 ug / ml cholesterol, 1 ug
After culturing for 9 hours in Dulbecco's modified Eagle's medium containing 10% FBS / ml 25-hydroxycholesterol, (Goldstei
n, JL et al., Methods Enzymol. 98: 241-260, 198
The amount of ¹²⁵ I-LDL bound to the cell surface was measured by the method of 3). For fatty acid synthesis activity, cells were treated with 0.3 mM IPT.
G, after culturing in Dulbecco's modified Eagle's medium containing 10 ug / ml cholesterol, 1 ug / ml 25-hydroxycholesterol, and 10% FBS for 8 hours, adding 1 mM ¹⁴ C-sodium acetate to the medium, and culturing for 2 hours. , (Brown, MS et a
l., J. Biol. Chem. 253: 1121-1128, 1978). In each case, measurement was performed on cells similarly cultured in a medium from which only IPTG had been removed, and the values were taken as the values of the IPTG untreated control group.

The results obtained are shown in Table 1 below.Table 1 Transcription activity (increase rate over IPTG untreated control group) LDL receptor 2.9 Fatty acid synthase 1.1 Metabolic activity (increase rate over IPTG untreated control group) LDL binding activity 2.6Fatty acid synthesis activity 1.4  As is clear from the table, the LDL receptor mRN was added by adding IPTG.
A and LDL receptor activity increased 2.9 and 2.6 fold
However, fatty acid synthase mRNA does not change and fatty acid synthesis activity
Did not increase.

Example 3 Screening Method for Drug that Selectively Enhances the Action of SREBP-2 1 A test drug was added to a culture of human cultured cells (HeLa S3), incubated, and the cells were recovered. -1a,-
1b, -1c and SREBP-2 mRNA amount or SREBP-1a, -1b, -1c
And the amount of SREBP-2 protein is measured. SREBP-1 mRNA or SR
SREBP-2 mRNA or SREB without increasing EBP-1 protein
A test drug that selectively increases only the P-2 protein is regarded as positive.

Example 4: Screening method 2 for a drug that selectively enhances the action of SREBP-2 Two types of cells (cells 1 and 2) as shown in FIG. 4 were constructed, and the luciferase activity of the test drug was measured. As a result of the measurement, a drug which does not increase the activity in the cell 2 and increases the activity only in the cell 1 is regarded as positive.

The cells 1 and 2 plasmid with the UAS _G luciferase gene upstream a binding sequence of the transcriptional activator GAL4 from yeast has been introduced are both reporter genes. In addition, in cell 1, GAL4 DNA binding region and human influenza virus-derived transcriptional activator VP
A gene that expresses a protein that fuses 16 transcriptional activation regions to the N-terminal side of human SREBP-2 (463-1141 amino acids), which lacks the N-terminal nuclear translocation region and has two endoplasmic reticulum transmembrane regions. Introduce.

On the other hand, in the cell 2, instead of the gene introduced into the cell 1, a protein in which the DNA binding region of GAL4 and the transcription activation region of VP16 are fused to the N-terminal of human SREBP-1a (455-1147 amino acids) is expressed. Introduce a gene to be used. After adding the drug to the culture of each cell and incubating,
The cells are collected and the luciferase activity is measured.

Example 5: Screening method for drug that selectively enhances the action of SREBP-2 3 Cells as shown in FIG. 5 were constructed, and the firefly luciferase activity and sea pansy luciferase activity of the test drug were measured. ,
Drugs that increase only firefly-derived luciferase activity without increasing sea pansy-derived luciferase activity are considered positive.

The cells are transduced with two reporter genes. That is, UAS _G , a binding sequence of the yeast transcriptional activator GAL4, is located upstream of the firefly-derived luciferase gene.
And a plasmid having LexAop, which is a binding sequence of transcription activation factor LexA derived from Escherichia coli, is introduced upstream of the luciferase gene derived from sea pansy. Furthermore, the DNA binding region of GAL4 and the transcription activation region of VP16 were
The gene that expresses the protein fused to the N-terminal side of REBP-2 (463-1141 amino acids), the DNA binding region of LexA, and the transcriptional activation region of VP16 are replaced with the N-terminal of human SREBP-1a (455-1147 amino acids).
A gene that expresses a protein in which the DNA binding region of GAL4 and the transcription activation region of VP16 are fused is introduced into the terminal side. After the drug is added to the culture of the cells and incubated, the cells are collected and the firefly-derived luciferase activity and the sea pansy-derived luciferase activity are measured.

[0038]

Industrial Applicability According to the present invention, there is provided a drug capable of increasing LDL receptor activity without increasing fatty acid synthesis, and therefore a safe therapeutic agent for hyperlipidemia which does not accompany fatty liver caused by activation of SREBP action. It can be provided, and its industrial utility value is extremely large.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the structure of SREBP isoforms and their nuclear translocation types, and translocation to the nucleus by excision of SREBP from the endoplasmic reticulum membrane.

FIG. 2 shows the construction of a cell line to study changes in cellular lipid metabolism resulting from transcriptional activation by SREBP-2.

FIG. 3 is a schematic diagram showing the control mechanism of LDL receptor expression by SREBP-1 and SREBP-2.

FIG. 4 is a schematic diagram showing one example of the screening method of the present invention.

FIG. 5 is a schematic diagram showing one example of the screening method of the present invention.

Claims (6)

[Claims] 1. A method for screening for an agent that selectively increases the action of SREBP (sterol responsive element binding protein) -2. 2. A method in which a test agent is allowed to act on cells expressing SREBP-1 and SREBP-2,
2. The screening method according to claim 1, wherein the method is performed by identifying a drug which greatly increases the action of BP-2. The measurement of the action of SREBP-2 was carried out by increasing the transcription efficiency of the SREBP-2 gene, increasing the stability of SREBP-2 mRNA, and measuring the SREBP-2 mR
Increased NA translation efficiency, increased SREBP-2 protein stability, SRE
Increased nuclear translocation of BP-2 protein, increased nuclear translocation of SREBP-2 protein, increased transcriptional activity of SREBP-2, S in the nucleus
The screening method according to claim 1 or 2, wherein one or more selected from the enhancement of transcription activator binding to REBP-2 is used as an index and compared with a corresponding index of SREBP-1. 4. The screening method according to claim 1, wherein the activity of increasing SREBP-2 activity is measured using an SREBP-2 fusion protein gene in which the SRE binding region has been replaced with another protein. 5. An agent for treating hyperlipidemia, which comprises an agent that selectively increases the action of SREBP-2. [6] a therapeutic agent for hyperlipidemia comprising the agent screened by the method of [1];