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

摘要: The isoxazole derivative Leflunomide (HWA 486) is a novel immunoregulatory and anti-inflammatory drug. Affinity chromatography was used to purify identify binding proteins, which might play role as potential cellular targets in the molecular mode of action. A 0273 covalently coupled Fractogel(R) matrix. This column separate cytosolic protein extract macrophage cell line RAW 264.7 by several selected specific gradient elution steps. Proteins that were specifically eluted through active metabolite Leflunomide, 1726, identified subsequent sequence analysis. allowed us specify 10 bind with high affinity this Three them, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase phosphoglycerate mutase belong second part glycolytic pathway. specificity these protein/drug interactions further evaluated using BIAcore(R) Kd values lactic dehydrogenase similar value known target protein, dihydroorotate dehydrogenase. In order elucidate features well overall relevance results, fractions three additional lines MOLT-4, A20.2J, HeLa compared same chromatographic protocol. profiles Western blot analyses confirmed data obtained previously for 264.7.