A synthetic mimic of a discontinuous binding site on interleukin-10.
We synthetically reconstructed a discontinuous binding site on interleukin-10 (IL-10) that recognizes the neutralizing anti-IL-10 antibody CB/RS/1. To design the 32-mer IL-10 mimic, a discontinuous interaction site on IL-10 was mapped, and binding studies with epitope-derived peptides led to specific replacement of several amino acids. Both parts of the interaction site were combined by addition of a linker molecule. Systematic analoging of the combined molecule then led to introduction of several additional substitutions in both regions and the linker. All possible disulfide bridge-containing variants of the 32-mer were tested by binding studies. Parallel syntheses were performed on continuous cellulose membranes by spot synthesis. As a result, a conformationally stabilized IL-10-derived molecule was obtained that both binds to and neutralizes the biological activity of CB/RS/1 in the low nanomolar range. This synthetic approach is a powerful alternative to phage display methods for the design of protein mimics.
Reineke
U
U
Institut für Medizinische Immunologie, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Germany.
Sabat
R
R
Misselwitz
R
R
Welfle
H
H
Volk
H D
HD
Schneider-Mergener
J
J
D019943:Amino Acid Substitution; D001665:Binding Sites / Q000502:physiology*; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006801:Humans; D016753:Interleukin-10 / Q000737:chemistry*; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D010452:Peptide Biosynthesis; D010449:Peptide Mapping; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000939:Epitopes; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10