DE4400748A1 - Screening test for inhibitors of renin expression - Google Patents

Abstract

Substances (I) that influence expression of renin (II) are identified by (1) transfecting a host cell with a recombinant vector that contains the regulatory region of the (II) gene functionally coupled to a reporter gene (RG); (2) either (a) isolating cells that carry the vector transiently or (b) generating cell lines that have stably integrated the vector; (3) exposing these cells to test cpds., and (4) measuring expression of RG.

Description

The present invention relates to a test method for identi fication of substances that regulate the expression of the reningen can lieren.

Renin is a protein involved in regulating blood pressure. Renin acts as a prorenin in the juxtaglomerular cells of the kidney synthesized, processed and stored in vesicles. At a Drop in blood pressure or increased sodium ion concentration Renin is made from the storage vesicles of the juxtaglomerular cells distributed and catalyzes the free as aspartyl protease setting of the decapeptide angiotensin I from the syn thetized angiotensinogen of blood plasma. The angiotensin I then turns from angiotensin converting enzyme (ACE) to octapeptide Angiotensin II shortened. About the binding of angiotensin II to the type I angiotensinogen receptor on the smooth muscle cells of the vessels and the subsequent intracellular signals comes it for vascular constriction and thus for increasing blood pressure. Next This endocrine disrupting RAS is also a paracrine or autocrine effective tissue known as RAS. Here the angiote sinogenic and possibly also the renin in organs like that Heart, brain or blood vessels. While Renal RAS is believed to be used in acute blood pressure regulation is important, the tissue RAS is more of a function in the long-term regulation of vascular tone and blood pressure wrote. The strong blood pressure regulating effect of ACE Inhibitors show that the RAS is a clear target for Reduction in hypertensive blood pressure (Lee et al., Am. J. Cardiol 70, 12C-19C, 1992).

To further improve the blood pressure without side effects lowering, the regulation of the vascular tone and especially the Ge Barrel and organ protection are other than the ACE Components of the RAS potential targets for new pharmaceuticals. Here is the conversion of angiotensinogen to angio Renin's tensin I is particularly interesting for two reasons:

• I) The rate-determining enzyme reaction of the RAS is Conversion of angiotensinogen to angiotensin I by renin;  
• II) The plasma concentration of the angiotensinogen is in the Magnitude of the Michaelis-Menten constant of the renin. Minor fluctuations in the angiotensinogen or renin concentration therefore have a significant influence on the amount of angiotensin I synthesized

The object was therefore to provide a method which allows the identification of substances by inhibiting the Renin expression lower the concentration of renin.

The invention relates to a test method for identi Identification of substances that influence renin expression and which comprises the following steps:

• a) Transfection of a host cell with a recombinant vector
  • a₁) the regulatory regions of the race,
  • a₂) carries a reporter gene, functionally linked to the regulatory region described in a₁),
• b) Establishing cells containing the vector described under a) contain transient, or cultivation of a cell line that the The vector described in step a) is stably integrated holds,
• c) Exposure of the cells from step b) to different ones testing substances,
• d) Measurement of the expression of the reporter gene in the cells after Step c).

In the first step (a) of the test method according to the invention produced a recombinant vector that contains the regulatory gene region of the renin gene. Below that is the sequence area understand the promoter, enhancer and, if necessary others that positively or negatively affect the transcription of renin contains flowing control sequences.

For example, the regulatory region of renin can be identified human genomic DNA or a human genomic library isolate (see example 1). To this regulatory gene region a reporter gene is linked functionally. Are reporter genes genes whose expressed gene product is an easily measured or provides quantifiable signal.

As a rule, suitable reporter genes are genes for such professionals stones that can be detected using common methods. Prefers are used as reporter genes for enzymes that are easily identified catalyze detectable reaction.  

The genes for chlorine are particularly suitable reporter genes amphenicol acetyl transferase (CAT), β-galactosidase alkaline phosphatase or luciferase.

The functional linkage of the regulatory gene region of Renin and reporter gene means that the transcription of the Reporter gene is carried out under the promoter control of Renin.

In addition to these two gene areas, the recombinant vector carry even more gene segments. In particular, the Ver using a selection marker such as neomycin resistance (neo®) advantageous.

The transformation of a host cell by the recombinant Vek tor can be transformed using all common transformation processes such as elec troporation, calcium phosphate or liposome mediated transfec tion. It is particularly well suited for the transformation transfection mediated by liposomes.

Eukaryotic cell lines are well suited as host cells. Ins special SK-LMS-1 cells (ATCC No. HTB 88).

After the host cell is transformed with the recombinant vector in a further step (b), such cells established that the vector is either transient or stable inte free included. Transient expression means one Expression of limited duration. With stable integration one understands the inclusion of the recombinant vector in the genome the host cell.

In a third step (c), the cells isolated in step (b) are exposed to the substances to be tested for renin reduction. This is done by adding the substances to be tested to the cell culture medium in a concentration of 10 ^-3 M to 10 ^-6 M. The substances to be tested usually remain in contact with the transformed host cells for 2 to 24 hours.

The cells are then lysed and used to evaluate the Reporter gene expression prepared as indicated in Example 3 give is.

Expression of the reporter gene in one with one to be tested Substance treated host cell is measured and compared to a control, d. H. expression in the absence of one testing substance. By comparing these two measured values  immediately make a statement as to whether the sub punch reporter gene expression repressed.

The test method according to the invention has the advantage that it is easy and can be evaluated quickly and thus a large sample sentence allowed. This makes it possible to have an effective mass screening for substances with renin-lowering effects to lead.

The invention is further illustrated by the following examples clear.

example 1 = Construction of the reporter plasmid

From a genomic clone of human reningen (Hardiman et al., DNA, 3, 457-468, 1984) the KpnI / XbaI fragment (approx. 480 bp), which is the ATG start code for the translation of prorenin contains, cut out and into the blues script vector (Strata gene) subcloned. The area around the ATG start codon was then mutagenized so that it subsequently an NcoI interface which the ATG start codon according to the rules Translation initiation for eukaryotic transcripts (Kozak, Nu cleic Acids Res., 15, 8125-8148, 1987) (ACCATGGAT). In front this genomic fragment mutagenized at the ATG then became the 3kb BglII / KpnI Frag cloned (Hardman et al., 1984, supra). This construct was called pRen 3kb + 0.5kb (NcoI). In the commercially available pMaMneo-luc vector (Clontech) was the DNA responsible for the Reporter gene luciferase encoded by xbaI and BamHI cut and provided with linkers so that at the 3 ′ end of the question the ATG start codon of luciferase in pMaMneo-luc (AAAATG- GAA) now also contained an NcoI interface (ACCATGGAA). At the 5'End became an NcoI interface to the BamHI interface added. The luciferase gene was then transferred into sense orien via NcoI tation behind the promoter of the reningen in the mutagenized NcoI interface of the Prorenin-ATG start codon in the pRen 3kb + 0.5 kb (NcoI) ligated. The new construct was pRen 3kb + 0.5kb - called luc. This construct then became XhoI and NotI (both located in the bluescript polylinker) consisting of the fragment from the approx. 3kb renin promoter followed by the reporter gene Luzife lawn cut out and the interfaces by Klenow enzyme replenished.  

The commercially available eukaryotic reporter construct plasmid pMaMneo-luc (Clontech) was cut open by Sal I and there removed by the luciferase gene. In by Klenow enzyme filled Sal I restriction sites of the pMaMneo-luc 3 now the XhoI / MotI renin promoter luciferase fragment from the pRen 3kb + 0.5kb - luc blunt cloned in.

Example 2 Transformation and selection of a stable cell line

SK-LMS-1 cells (ATCC HTB 88) were in DMEM / F12 (1: 1) medium 10% FCS cultivated. The transfection was carried out using the in Game 1 described reporter construct by liposomes Communicate DNA. For this purpose, 1 × 10⁶ cells in 10 cm cell culture Seeded trays. After overnight incubation in FCS-containing medium the cells were 6 hours in 2 ul DNA, 10 ul Transfectam (Fa. Promega No. E 1231) transfected in 2 ml serum-free medium. There after a medium was changed to FCS-containing medium. To 24 hours was on selection medium (DMEM / F12, 10% FCS, 600 ul / ml G418 sulfate). Stable G418-resistant cell clones were found isolated after 10 to 20 days.

Example 3 Measurement of gene activity

Cells according to Example 2 were sown in microtiter plates (1 × 10⁵ cells / well) and after 16 h in DMEM / HamF12 (1: 1) medium without FCS convicted. After two hours, the dilutions become too test substance added. Untreated cells served as Negative control. After 2 to 24 hours of incubation, the Cells by adding 30 µl / well lysis buffer (25 mM Tris-Phos phat, pH 7.8; 2mM DTT; 2 mM 1,2-diaminocyclohexane-N, N.N ′, N′-th traacetic acid, 10% glycerol, 1% Triton X-100®) digested (15 min at room temperature). 50 μl were added to this cell extract Luciferase assay substrates (270 µM coenzyme A, 470 µM luciferin, 530 µM ATP) added. Measurement of luciferase activity can be carried out in conventional luminometers; preferably the measurement is carried out in a 2-D luminometer from Hamamatsu.  

Example 4 Use of the test system in "random screening"

The assay system from Example 3 is used for a "random screening" for substances that regulate the renin influence promotors.

Substances to be used in the screening are in Concentrations of 1 to 5 mM dissolved in DMSO and in suitable Dilutions according to Example 3 used in tests.

Claims (2)

1. A test method for identifying substances that influence re-nine expression, which comprises the following steps:

• a) Transfection of a host cell with a recombinant vector
  • a₁) the regulatory regions of the race,
  • a₂) carries a reporter gene, functionally linked to the regulatory region described in a₁),
• b) establishing cells which transiently contain the vector described under a), or culturing a cell line which stably integrates the vector described under step a),
• c) exposure of the cells from step b) to various substances to be tested,
• d) Measurement of the expression of the reporter gene in the cells after step c).

2. Test method according to claim 1, in which as reporter gene in Step a₂) luciferase and as host cell SK-LMS-1 cells be used.