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Method for screening compounds inhibiting signal transduction through inflammatory cytokines

a technology of inflammatory cytokines and compounds, which is applied in the direction of transferases, peptide sources, instruments, etc., can solve the problems of inhibiting the production of inflammatory cytokines, and achieve the effects of inhibiting signal transduction, inhibiting or suppressing the production of inflammatory cytokines, and inhibiting the production of other inflammatory cytokines

Inactive Publication Date: 2006-02-23
CHUGAI PHARMA CO LTD
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Benefits of technology

[0143] Further, in the case of quantitative measurement, the sample can be quantified based on a standard curve prepared by using values for a positive control group containing known amounts of a compound that is clarified to be capable of inhibiting the binding between TAK1 and TAB1. When the protein bound is larger in quantity, then it can be estimated that the compound has only lower activity for inhibition of the protein binding. On the other hand, when the amount of protein bound is smaller, the binding-inhibitory activity of the compound to inhibit the binding of the protein is presumed to be stronger.
[0194] More specifically, the above-mentioned cell fusion may be conducted, for example, in the presence of cell fusion-enhancing agent in a commonly used nutrient culture medium. The cell fusion-enhancing agent is, for example, polyethylene glycol (PEG), Sendai virus (HVJ), or the like. An adjuvant, such as dimethyl sulfoxide or the like, may optionally be used together to enhance the efficiency of fusion.
[0225] In the above-described method, proteins to be used are expressed in cells used for the assay. Examples described later have revealed that the inhibition of binding between TAK1 and TAB1 inhibits the signal transduction through inflammatory cytokines, which further inhibits the production of other inflammatory cytokines involved in the cytokine network. Accordingly, the detection and / or measurement of the TAK1-, or TAK1- and TAB1-mediated transduction of biological activity makes it possible to inhibit or suppress the signal transduction through inflammatory cytokine of interest, and based on the inhibition or suppression, makes it possible to inhibit or suppress the production of the inflammatory cytokine, to inhibit or suppress physiological activity of the inflammatory cytokine, or possible to screen a compound inhibiting or suppressing the inflammatory stimulation-induced signal transduction.
[0238] When no test sample is added to the cells, the addition of IL-1 increases the luciferase activity. On the other hand, in the presence of a test sample, the luciferase activity is not increased in the cells. In other words, a target compound in the test sample inhibits the IL-1-induced elevation of reporter gene expression. Thus, increases in expression level of the reporter gene can be measured, and thereby successfully screening the target compound.

Problems solved by technology

However, it is not at all known that the inhibition of signal transduction through TAK1 actually results in the inhibition of cellular responses to the inflammatory stimulation such as LPS or cytokine stimulation, for example, the inhibition of signal transduction through an inflammatory cytokine as an inflammatory mediator as well as results in the inhibition of inflammatory cytokine actions and further the inhibition of inflammatory cytokine production.

Method used

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  • Method for screening compounds inhibiting signal transduction through inflammatory cytokines
  • Method for screening compounds inhibiting signal transduction through inflammatory cytokines
  • Method for screening compounds inhibiting signal transduction through inflammatory cytokines

Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of Tak1-DN Expression Vector and Creation Of Transgenic Mice

[0271] A TAK1-DN expression vector that acts as a dominant negative inhibitor was prepared to demonstrate that the signal transduction through LPS or inflammatory cytokines can be inhibited by inhibiting the specific binding between human TAK1 and human TAB1.

[0272] TAK1-DN has the amino acid sequence, corresponding to the TAB1-binding region of TAK1, which covers a stretch from amino acid 77 Glu to amino acid 303 Gln in the amino acid sequence of SEQ ID NO: 2. The TAK1-DN-encoding gene fragment was amplified by using ph-TAK1 (refer to Unexamined Published Japanese Patent Application (JP-A) No. Hei 9-163990) as a template DNA by PCR. Specifically, a sense primer TAK1S (SEQ ID NO: 5) containing a recognition site for restriction enzyme EcoRI and the initiation codon ATG and an antisense primer TAK1AS (SEQ ID NO: 6) containing a recognition site for restriction enzyme EcoRI and the stop codon were used to amplif...

example 2

Cytokine Production Capacity of Peritoneal Macrophages Expressing TAK1-DN

[0282] Peritoneal macrophages were prepared from TAK1-DN-expressing transgenic mice, and the responses of the cells to IL-1 and lipopolysaccharide (LPS; Sigma) were examined.

[0283] Specifically, 10 ml of ice-cold PBS solution containing 0.36% sodium citrate was injected into the peritoneal cavity of a TAK1-DN-expressing transgenic mouse (TAK1-DN Tgm) or a C57BL / 6 mouse (wild-type mouse) of the same weeks of age. The mouse was given a massage for 30 seconds, and then the solution was recovered from the mouse. The solution recovered was centrifuged, and the precipitated cells were resuspended in RPMI1640 (GIBCO-BRL) containing 10% FBS (GIBCO-BRL). The cells were plated in wells at a cell density of 5×104 / well. The cells were cultured at 37° C. for 2 hours, and floating cells were removed by washing with media. Cells adsorbed on the culture plate were used as peritoneal macrophages.

[0284] LPS, at a final concen...

example 3

IκBα Degradation in TAK1-DN Expressing Peritoneal Macrophages Stimulated by LPS and IL-1α

[0286] The macrophages derived from wild-type mice and from TAK1-DN Tgm, which were prepared as described above, were stimulated by LPS or IL-1, and the time course of degradation of IκBα by proteasome was monitored (0, 30, and 60 minutes). Specifically, the peritoneal macrophages were cultured in wells of a 96-well plate at a cell density of 1×106 / well. LPS (1 μg / ml) or IL-1α (10 ng / ml) was added to the culture in the presence or absence of proteasome inhibitor MG132 (40 μM). The cells were harvested after 30 or 60 minutes.

[0287] The cells were washed with PBS (−), and then a lysis buffer was added thereto for the preparation of cell lysate. An aliquot of each lysate corresponding to about 2×105 cells was subjected to gel electrophoresis by 9% SDS-PAGE. The fractionated proteins were blotted onto a nitrocellulose membrane and then incubated with anti-IκBα antibody (SantaCruz) as a primary anti...

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Abstract

Revealed are that the actions of inflammatory cytokine and the production of inflammatory cytokines such as IL-1 and TNF induced by an inflammatory stimulus as well as the production of other inflammatory cytokines such as IL-6 induced by the former class of inflammatory cytokines are all suppressed by inhibiting the signal transduction through TAK1.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for screening compounds inhibiting signal transduction through inflammatory cytokines. The present invention also relates to the compound that can be isolated by the screening method and the use thereof. The present invention further relates to an inhibitor of the signal transduction through inflammatory cytokines, for example, inhibitors of the action and production of inflammatory cytokines, and anti-inflammatory agent, which contain as an active ingredient a compound inhibiting the signal transduction through TAK1. BACKGROUND ART [0002] The system of mitogen-activated protein kinase (MAPK) is known as a system involved in intracellular signal transduction. [0003] MAPK system is a conserved eukaryotic signal transduction system, by which the receptor-mediated signals are converted to a variety of actions. MAPK system contains three types of protein kinases, namely, mitogen-activated protein kinase kinase kinase (MAPK...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68G01N33/542G01N33/53A61K38/00C07K14/47C12N9/12C12Q1/48G01N33/573
CPCA61K38/00C07K14/47G01N2500/02C12Q1/48G01N33/573C12N9/1205
Inventor TSUCHIYA, MASAYUKIOHTOMO, TOSHIHIKOSUGAMATA, YASUHIROMATSUMOTO, KUNIHIRO
Owner CHUGAI PHARMA CO LTD
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