Use of a solid-phase random peptide library to identify inhibitors of TNF-alpha mediated cytotoxicity in vitro.

Tumour necrosis factor (TNF) is a pleiotropic cytokine which plays a central role in infection, inflammation and autoimmune diseases. Its functions are mediated through binding to high affinity cell surface receptors. An approach to modulate excessive levels of TNF-alpha in the serum is the use of soluble receptors. In this study the potential of a solid-phase combinatorial peptide library was investigated to identify peptide mimics of the binding site of the TNF-alpha receptor. One of the identified mimotopes was shown to inhibit TNF-alpha-mediated cytotoxicity in mouse L929 and in a human KYM-1D4 cell lines in a dose-dependent fashion. Characterization of the mimotope sequence by high-performance liquid chromatography and mass spectroscopy has shown that the inhibitory effect of the mimotope was dependent upon the presence of a protective MTR group on the side chains of the arginine residues in the mimotope sequence. Furthermore, antibodies to the mimotope were shown to recognize the recombinant human TNF 55-kDa receptor in an ELISA assay. These findings highlight the potential of combinatorial peptide libraries in identifying peptide mimics of the binding site of TNF-alpha receptor, which can be used as competitive inhibitors of ligand-receptor interactions.