Identification and characterization of potential new therapeutic targets in inflammatory and autoimmune diseases

被引:17
作者
Mangold, U
Dax, CI
Saar, K
Schwab, W
Kirschbaum, B
Müllner, S
机构
[1] Henkel KGAA, Corp Res, Enzyme Technol, D-40191 Dusseldorf, Germany
[2] Hoechst Marion Roussel, DG Rheumatol, Frankfurt, Germany
[3] BASF AG, Corp Res ZHV, D-6700 Ludwigshafen, Germany
[4] Aventis Res & Technol, Frankfurt, Germany
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1999年 / 266卷 / 03期
关键词
Leflunomide; affinity chromatography; immune system; mode of action; BIAcore (R);
D O I
10.1046/j.1432-1327.1999.00978.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The isoxazole derivative Leflunomide (HWA 486) is a novel immunoregulatory and anti-inflammatory drug. Affinity chromatography was used to purify and identify Leflunomide binding proteins, which might play a role as potential cellular targets in the molecular mode of action. The Leflunomide derivative A 0273 was covalently coupled to a Fractogel(R) matrix. This column was used to separate a cytosolic protein extract of the macrophage cell line RAW 264.7 by several selected and specific gradient elution steps. Proteins that were specifically eluted through the active metabolite of Leflunomide, A 1726, were identified by subsequent protein sequence analysis. This allowed us to specify 10 cytosolic proteins, which bind with high affinity to this matrix. Three of them, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and phosphoglycerate mutase belong to the second part of the glycolytic pathway. The binding specificity of these protein/drug interactions was further evaluated using BIAcore(R) analysis. K-d values of glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and lactic dehydrogenase were similar to the K-d value of a known Leflunomide target protein, dihydroorotate dehydrogenase. In order to elucidate the features as well as the overall relevance of these results, cytosolic fractions of three additional cell lines MOLT-4, A20.2J, HeLa were compared using the same chromatographic protocol. The elution profiles as well as subsequent Western blot analyses confirmed the data obtained previously for the macrophage cell line RAW 264.7.
引用
收藏
页码:1184 / 1191
页数:8
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