In search of a novel approach for the Detection of Weak Macrocyclic Inhibitors of Challenging Targets: A Proof-of-Concept Study on the Interleukin-23 Receptor

Macrocyclic peptides have emerged as attractive therapeutic agents against challenging targets by combining properties of small molecules and biologics while balancing flexibility and rigidity. They are particularly suited for modulating protein–protein interactions, which can be difficult to target using conventional small molecules due to their large interaction surfaces and the frequent absence of well-defined binding pockets. The discovery of macrocyclic peptide binders often relies on the screening of large peptide libraries. Advances in automated synthesis have enabled the generation of peptide libraries by high-throughput solid-phase peptide synthesis in microwell plates, facilitating hit identification and deconvolution. These libraries also give the possibility of screening using a range of high-throughput assays, including time-resolved Förster resonance energy transfer. However, the detection of weak binders using these approaches can be challenging. In particular, increasing peptide concentrations, necessary for the detection of weak interactions, can promote aggregation and compromise the assay. Here, we investigated a proximity-based strategy to enhance the detection of weak macrocyclic binders without increasing their concentration. The approach relies on conjugating a peptide binder to a cyclic peptide that binds the crystallizable fragment (Fc) of an immunoglobulin G, while expressing the target receptor as an Fc-fusion protein. The interaction between the Fc-binding peptide and the Fc domain is expected to increase the local concentration of the binder in proximity to the target, thereby enhancing its apparent binding potency in the assay. As a proof of concept, the strategy was evaluated using the interleukin-23 receptor and a previously characterized macrocyclic binder P6. The study outlines the strategies and the optimization steps required first for the cyclization of the P6 binder and subsequently for its linking to the Fc-binding peptide. In particular, an on-resin cyclization strategy using a bis-electrophilic linker was explored as an alternative approach for in solution peptide cyclization. The construct, consisting of P6 linked to the Fc-binding cyclic peptide, was evaluated against an Fc-fused interleukin-23 receptor protein and compared with unconjugated P6 using time-resolved Förster resonance energy transfer assays. The linking of P6 to Fc-III resulted in an approximately nine-fold reduction in the half-maximal inhibitory concentration compared with unconjugated P6 when tested against the Fc-fused receptor. In contrast, comparable values were obtained when the conjugate and P6 were tested against an interleukin-23 receptor construct lacking the Fc domain, supporting the contribution of the Fc/Fc-III interaction to the observed effect. These findings highlight the potential of Fc-mediated proximity to enhance the interaction of the binder with its target. Along with further investigation and optimization, this strategy could find application in the screening of large peptide libraries simplifying the detection of weak binders and increasing the success rate of hit discovery processes.