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Screening method, a composition comprising substances chosen in the method thereof, and a binding substance

a screening method and composition technology, applied in the direction of drug compositions, immunological disorders, metabolism disorders, etc., can solve the problems of (inflammation-associated) disorders, no report has shown a relationship between mif and thioredoxin, etc., to strengthen the bond and enhance the activity inhibition of macrophage migration inhibition factor

Inactive Publication Date: 2009-07-02
REDOX BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0032]The present invention effectively works for screening a test substance which strengthens a bond between thioredoxin and macrophage migration inhibition factor. The screened test substance further enhances the activity inhibition of macrophage migration inhibition factor resulting from the binding substance (polypeptide of thioredoxin family etc.).
[0033]The molecular interaction analysis allows to minitor the intermolecular bond state in real time.
[0034]Nitrosoglutathione andprostaglandin I2 enable to certainly strengthen the bond between the polypeptide of the thioredoxin family and macrophage migration inhibitory factor.

Problems solved by technology

However, excessive inflammatory response resulting from excessive production of MIF, which exhibits inflammatory effect, causes many kinds of (inflammation-associated) disorders.
However, no report has shown a relationship between MIF and thioredoxin and a method for screening substances related to MIF so far.

Method used

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  • Screening method, a composition comprising substances chosen in the method thereof, and a binding substance
  • Screening method, a composition comprising substances chosen in the method thereof, and a binding substance
  • Screening method, a composition comprising substances chosen in the method thereof, and a binding substance

Examples

Experimental program
Comparison scheme
Effect test

example 1

Confirmatory Experiment of the Bond Between TRX and MIF in Cells

[0082]The bonds in ATL2 cells and HL60 cells between TRX and MIF were confirmed with an immunoprecipitation method (to be described in detail below)

[Materials]

[0083]ATL2 cells; human adult T-cell leukemia cell strain (See Publication JP62-19532, Tagaya, Y., Y. Maeda, A. Mitsui, N. Kondo, H. Matsui, J. Hamuro, N. Brown, K. Arai, T. Yokota, H. Wakasugi, et al 1989. EMBO J. 8:75)

HL60 cells; human acutepromyelocytic leukemia cell strain (provided by Dr. YODOI Junji, Institute for Virus Research in Kyoto University)

Protein G Sepharose 4 Fast Flow; (GE Healthcare Bio-Sciences Ltd.)

[0084]Anti TRX antibody (ADF 11 antibody); (Redox Bioscience Inc.)

Anti MIF antibody; (Santa Cruz Biotechnology, Inc.)

Anti Rabbit antibody; (Upstate, Inc.)

[Method]

[0085]The cells were cultured with RPMI 1640 (SIGMA inc.) medium containing 10% FCS, 100 U / ml penicillin and 100 μg / ml streptomycin in a 175 cm2 flask.

[0086]1×107-8 / cell of the cells were c...

example 2

Experiment of the Bond between TRX and MIF Outside Cells

[0093]The bond between TRX and MIF outside ATL2 cells was confirmed with an immunoprecipitation method.

[Materials]

[0094]ATL2 cells; human adult T-cell leukemia cell strain

(See Publication JP62-19532, Tagaya, Y., Y. Maeda, A. Mitsui, N. Kondo, H. Matsui, J. Hamuro, N. Brown, K. Arai, T. Yokota, H. Wakasugi, et al 1989. EMBO J. 8:75)

Protein G Sepharose 4 Fast Flow; (GE Healthcare Bio-Sciences Ltd.)

[0095]Anti TRX antibody (ADF 11 antibody); (Redox Bioscience Inc.)

Anti MIF antibody; (Santa Cruz Biotechnology, Inc.)

Anti Rabbit antibody; (Upstate, Inc.)

Amicon ultra-4; (Millipore, Inc.)

[Method]

[0096]1×105 / cell of the ATL2 cells were cultured with RPMI 1640 (SIGMA inc.) medium containing 100 U / ml penicillin and 100 μg / ml streptomycin in a 175 cm2 flask for 3 days. Then, the medium was collected and centrifugated at 10,000 rpm and 4° C., for 15 minutes. Then, FCS was added to the obtained supernatant, and the solution was concentrated w...

example 3

Confirmatory Experiment of Anti Trx Antibody Inhibiting the Cell Internalization of MIF

[0101]MIF is internalized (incorporated) into cells by the autocrine or paracrine action to induce the production of inflammatory cytokine TNF-alpha(α) or IL-1. Thus, the effects of TRX on the cell internalization of MIF were examined to clarify the inflammation response control mechanism.

[Materials]

[0102]ATL2 cells; human adult T-cell leukemia cell strain

Recombinant MIF (rMIF); His-tagged recombinant MIF was expressed in Escherichia coli by using expression vector pQE30 (QIAGEN Inc.), and column-purified with MagneHis™ Protein Purification System (Promega Corp.).

Recombinant TRX (rTRX) (Ajinomoto Co., Inc.)

Anti His-tagged antibody (Promega Corp.).

[Method]

[0103]ATL2 cells were cultured with RPMI 1640 medium containing 10% FCS, 100 U / ml penicillin and 100 μg / ml streptomycin in a 75 cm2 flask.

[0104]1×106 / cell of ATL2 cells were added to a 24 well plate. rTRX having the final concentration of 0 to 250...

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Abstract

(Problems) The main object of the present invention is to provide a screening method for selecting a substance affecting a bond between thioredoxin and MIF.(Means for Solving Problems) The present invention provides a screening method for selecting a test substance which strengthens a bond between a polypeptide of a thioredoxin family and a macrophage migration inhibition factor, comprising: mixing a test substance with at least one binding substance selected from following (1) to (4), (1) the polypeptide belonging to the thioredoxin family, (2) a protein having an amino acid sequence of the polypeptide belonging to the thioredoxin family in which one or more amino acid is deleted, replaced or added, and having an equivalent activity to the polypeptide of the thioredoxin family, (3) a gene coding (1), (4) a gene coding (2); bonding the binding substance to the macrophage migration inhibition factor; and monitoring the bond state between the binding substance and the macrophage migration inhibition factor.

Description

CROSS REFERENCES TO RELATED APPLICATION[0001]This application is a continuation-in-part application based on International Application No. PCT / JP2007 / 60557 (filed on May 23, 2007), which claims the priority of Japanese Application No. 2006-148844 (filed on May 29, 2006).FIELD OF THE INVENTION[0002]The present invention relates to a screening method for selecting a test substance which strengthens a bond between a polypeptide of a thioredoxin family (hereinafter, referred as “TRXs”) and a macrophage migration inhibition factor (hereinafter, referred as “MIF”). The present invention further relates to a composition comprising the substance chosen in the method thereof, and a binding substance.DESCRIPTION OF THE BACKGROUND ART[0003]A macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that inhibits random migration of macrophages to inflammatory regions, and plays an important role in systemic / local inflammation and immune responses (Nishihira J., J Interferon Cyto...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68G01N33/573C07C203/00C12N9/02C12N15/53C07D307/93
CPCA23L1/305A61K31/26C12Y108/01008C12N9/0036A61K38/44A23L33/17A61P1/04A61P1/16A61P3/10A61P9/10A61P11/00A61P11/06A61P13/12A61P15/00A61P17/00A61P19/02A61P29/00A61P37/02A61P37/08A61P43/00
Inventor YODOI, JUNJIKNODO, NORIHIKOSON, AOIKATO, NORIKOHARA, TORNIJIROHORIBE, TOMOHISA
Owner REDOX BIOSCI
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