Making peptide-based molecules that mimic functional interaction sites on proteins remains a challenge in biomedical sciences. Here, we present a robust technology for the covalent assembly of highly constrained and discontinuous binding site mimics, the potential of which is exemplified for structurally complex binding sites on the "Cys-knot" proteins hFSH and hCG. Peptidic structures were assembled by Ar(CH2 Br)2-promoted peptide cyclizations, combined with oxime ligation and disulfide formation. The technology allows unprotected side chain groups and is applicable to peptides of different lengths and nature. A tetracyclic FSH mimic was constructed, showing >600-fold improved binding compared to linear or monocyclic controls. Binding of a tricyclic hCG mimic to anti-hCG mAb 8G5 was identical to hCG itself (IC50 =260 vs. 470 pM), whereas this mimic displayed an IC50 value of 149 nM for mAb 3468, an hCG-neutralizing antibody with undetectable binding to either linear or monocyclic controls.

The cover picture shows the X‐ray crystal structure of hCGβ (the “pregnancy hormone”, in blue) in complex with a monoclonal antibody (mAb3468, in green) that binds with sub‐nanomolar affinity (PDB ID: 1QFW). The structure illustrates the molecular complexity of the discontinuous and conformational β1/β3 binding site on hCGβ, and further suggests that the binding surfaces of hCGβ and mAb3468 only interact efficiently when they adopt the correct secondary and tertiary structures. On , P. Timmerman et al. present a new synthetic methodology for manufacturing small (3.5–5 kDa), peptide‐based mimics of the highly discontinuous β1/β3 epitope on hCGβ that retain both the secondary and tertiary structures as well as the strong binding to mAb3468, while neglecting 75 % of the total protein. Synthesis of these mimics involves the use of multiple constraints, including cyclization with a synthetic scaffold and connecting the loops both via oxime linkages and natural disulfide bonds. These constraints were shown to be essential for the binding properties of these mimics.