Covalent modification of the interleukin‐5 receptor by isothiazolones leads to inhibition of the binding of interleukin‐5

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Abstract
Using a fusion protein of the human interleukin-5-receptor α chain (hILSRα) and the human IgG Cγ3 chain (hIL5Rα-hγ3), 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 hIL5Ra, as measured in a solid-phase assay (soluble hIL5Rα or hIL5Rα-hγ3) or on COS-1 cells expressing the hIL5Rα on the cell membrane. The binding of ML4 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 IL5Rα 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 hIL5Rα. 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 radio-labeled isothiazolone confirmed that Cys66, present in the first domain of the receptor, is the target for covalent modification leading to a decrease in affinity.