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

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. Copyright © 1994, Wiley Blackwell. All rights reserved