Subunit disassembly and inhibition of TNFα by a semi-synthetic bicyclic peptide

Macrocyclic peptides are potentially a source of powerful drugs, but their de novo discovery remains challenging. Here we describe the discovery of a high-affinity (K-d = 10 nM) peptide macrocycle (M21) against human tumor necrosis factor-alpha (hTNF alpha), a key drug target in the treatment of inflammatory disorders, directly from diverse semi-synthetic phage peptide repertoires. The bicyclic peptide M21 (ACPPCLWQVLC) comprises two loops covalently anchored to a 2,4,6-trimethyl-mesitylene core and upon binding induces disassembly of the trimeric TNF alpha cytokine into dimers and monomers. A 2.9 angstrom crystal structure of the M21/hTNF alpha complex reveals the peptide bound to a hTNF alpha dimer at a normally buried epitope in the trimer interface overlapping the binding site of a previously discovered small molecule ligand (SPD304), which also induces TNF trimer dissociation and synergizes with M21 in the inhibition of TNF alpha cytotoxicity. The discovery of M21 underlines the potential of semi-synthetic bicyclic peptides as ligands for the discovery of cryptic epitopes, some of which are poorly accessible to antibodies.