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

Eur J Biochem. 1994 Oct 15;225(2):635-40. doi: 10.1111/j.1432-1033.1994.00635.x.

Abstract

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 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.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites / drug effects
  • Cell Line
  • Cysteine / drug effects
  • Dithiothreitol / pharmacology
  • Granulocyte-Macrophage Colony-Stimulating Factor / metabolism
  • Interleukin-5 / metabolism*
  • Kidney / drug effects
  • Macaca
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Receptors, Interleukin / drug effects*
  • Receptors, Interleukin / metabolism*
  • Receptors, Interleukin-5
  • Recombinant Proteins
  • Sulfhydryl Compounds
  • Thiazoles / antagonists & inhibitors
  • Thiazoles / pharmacology*

Substances

  • Interleukin-5
  • Receptors, Interleukin
  • Receptors, Interleukin-5
  • Recombinant Proteins
  • Sulfhydryl Compounds
  • Thiazoles
  • Granulocyte-Macrophage Colony-Stimulating Factor
  • Cysteine
  • Dithiothreitol