A novel discovery, maturation, and assay integration approach for the development of ruggedized multi-valent capture receptors exemplified against the chikungunya virus E2 protein

Abstract This work reports on the development of synthetic, thermally stable receptors using a novel discovery and multi-valent maturation strategy exemplified against the Chikungunya Virus (CHIKV) E2 surface protein capable of straightforward bioassay integration. An in situ ‘click’ screen of a propargylated folded protein against a comprehensive library of peptide macrocycles with 5-mer variable regions produced four candidate binders with nanomolar affinities. With the guidance of in silico experiments, the individual macrocycles were matured to produce multi-valent cooperative constructs, resulting in successful binding of CHIKV E2 in up to 50% human serum and an over 200-fold improvement in affinity performance compared to the mono-valent macrocycles. The top performing multi-valent construct also exhibits an insignificant loss in capture performance after being heated up to 90 °C for 1 h. The overall methodology provides a rational approach to the general targeting of folded proteins, providing the ability to rapidly develop drop-in biological assay antibody replacements and potentially “polyclonal” mixtures of reagents.