Covalent modification of the interleukin-5 receptor by isothiazolones leads to inhibition of the binding of interleukin-5.
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.
Roche Research Gent, Belgium.
R
R
Devos
Y
Y
Guisez
G
G
Plaetinck
S
S
Cornelis
J
J
Tavernier
J
J
van der Heyden
L H
LH
Foley
J E
JE
Scheffler
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002460:Cell Line; D003545:Cysteine / Q000187:drug effects; D004229:Dithiothreitol / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D015848:Interleukin-5 / Q000378:metabolism*; D007668:Kidney / Q000187:drug effects; D008251:Macaca; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018123:Receptors, Interleukin / Q000187:drug effects* / Q000378:metabolism*; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D013844:Thiazoles / Q000037:antagonists & inhibitors / Q000494:pharmacology*
D015848:Interleukin-5; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D013844:Thiazoles; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D003545:Cysteine; D004229:Dithiothreitol