COVALENT MODIFICATION OF THE INTERLEUKIN-5 RECEPTOR BY ISOTHIAZOLONES LEADS TO INHIBITION OF THE BINDING OF INTERLEUKIN-5
COVALENT MODIFICATION OF THE INTERLEUKIN-5 RECEPTOR BY ISOTHIAZOLONES LEADS TO INHIBITION OF THE BINDING OF INTERLEUKIN-5
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DOI：
10.1111/j.1432-1033.1994.00635.x
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发表时间：
1994-10-15
期刊：
影响因子：
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通讯作者：
SCHEFFLER, JE
中科院分区：
文献类型：
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作者：
DEVOS, R;GUISEZ, Y;SCHEFFLER, JE
利用人白细胞介素5受体α链(hIL5Rα)和人免疫球蛋白Cγ3链(hIL5Rα-h Gamma 3)的融合蛋白，我们建立了一种用于高通量筛选一系列合成化合物的固相分析方法。我们报道了异噻唑酮类化合物被鉴定为白细胞介素5(IL5)与hIL5Rα结合的有效抑制剂，这是通过固相分析(可溶性hIL5Rα或hIL5Rα-h伽马3)或在表达hIL5Rα的COS-1细胞上进行的。在类似的检测系统中，hIL4和人粒细胞巨噬细胞集落刺激因子(hGM-CSF)与其各自受体的结合不受异噻唑酮类药物的抑制。Scatchard分析表明，这些化合物导致IL5Rα与IL5的亲和力下降。异噻唑酮类化合物对IL5与其受体结合的抑制作用可被二硫苏糖醇等含自由硫的化合物所消除，表明异噻唑酮类化合物与hIL5Rα中游离半胱氨酸残基的巯基发生反应。Cys66突变导致受体仍与hIL5结合，但对异噻唑酮类药物的抑制不敏感。Cys249和Cys296突变为丝氨酸导致IL-5结合活性完全丧失。放射性标记异噻唑酮的使用证实了存在于受体第一结构域的Cys66是导致亲和力降低的共价修饰的目标。
Using a fusion protein of the human interleukin-5-receptor alpha chain (hIL5R alpha) and the human IgG C gamma 3 chain (hIL5R alpha-h gamma 3), we have developed a solid-phase assay for high-flux screening of a collection of synthetic compounds. We report on the identification of isothiazolone derivatives as potent inhibitors of binding of interleukin-5 (IL5) to the hIL5R alpha, as measured in a solid-phase assay (soluble hIL5R alpha or hIL5R alpha-h gamma 3) or on COS-1 cells expressing the hIL5R alpha on the cell membrane. The binding of hIL4 and human granulocyte macrophage colony-stimulating factor (hGM-CSF) to their respective receptors is not inhibited by the isothiazolones in similar assay systems. Scatchard analysis revealed that these compounds caused a decrease in affinity of the IL5R alpha for IL5. The inhibition of binding IL5 to its receptor by the isothiazolone derivatives is abrogated by free-sulfhydryl-containing compounds such as dithiothreitol, indicating that the isothiazolones react with the sulfhydryl group of free cysteine residues in the hIL5R alpha. Mutation of Cys66 led to a receptor which still binds hIL5, but which was insensitive to the inhibition by isothiazolones. Mutation of Cys249 and Cys296 to serine resulted in complete loss of IL-5-binding activity. The use of radiolabeled isothiazolone confirmed that Cys66, present in the first domain of the receptor, is the target for covalent modification leading to a decrease in affinity.