Novel macrocyclic peptidomimetics targeting the insulin-regulated aminopeptidase (IRAP): design, synthesis and evaluation

Inhibition of the insulin-regulated aminopeptidase (IRAP) is a promising therapeutic strategy for neurodegenerative disorders such as Alzheimer's disease, due to its role in cognitive processes. HA08, a macrocyclic peptidomimetic derived from angiotensin IV, is among the most potent known IRAP inhibitors (IC₅₀ = 18 nM). However, detailed structure-activity relationship (SAR) studies at its C-terminus have been limited by synthetic constraints. Herein, we report the design, synthesis, and biological evaluation of a focused series of HA08 analogues to explore the impact of C-terminal modifications on IRAP inhibition. An improved divergent synthetic route was established <i>via</i> a common macrocyclic intermediate, enabling late-stage diversification through coupling with non-natural amino acids which led to the synthesis of twelve novel peptidomimetic scaffolds. Several analogues retained high potency, with one-carbon elongation next to the carboxylic acid moiety or secondary amine being well tolerated. In contrast, aliphatic analogues exhibited markedly reduced potency, highlighting the importance of π-π interactions, while the low activity of phenoxyacetic acid derivatives likely reflects altered geometry within the binding pocket. The most potent inhibitor in the series featured a C-terminal benzyl alcohol (IC₅₀ = 59 nM), approaching the activity of HA08. To rationalise these SAR trends, molecular dynamics simulations were performed based on the IRAP-HA08 co-crystal structure. Partial least squares analysis of protein-ligand contact patterns revealed that sustained interactions between the C-terminal carboxylate and Arg929 correlated with lower potency, whereas interaction with Arg439 was associated with enhanced activity. These findings suggest that subtle shifts in C-terminal positioning influence binding mode and potency and provides valuable insights for the design of future IRAP inhibitors.