Macrocyclic Peptides Containing an Imidazopyridinium (IP⁺) Unit Display Enhanced Passive Cell Permeability

Macrocyclic peptides (MPs) have emerged as interesting therapeutic candidates due to their ability to engage difficult protein targets with high affinity and selectivity. However, their application to intracellular targets is limited by the poor passive membrane permeability of most MPs. We previously showed that incorporation of an imidazopyridinium (IP⁺) moiety into an MP significantly boosted passive membrane permeability, as measured by the parallel artificial membrane permeability assay (PAMPA)(Li, et al. (2024) J. Amer. Chem. Soc. 146, 14633-14644). In this study we report a detailed analysis of the entry of IP⁺-containing MPs into living cells. Chloroalkane penetration assay (CAPA) data show that IP⁺ MPs access the cytoplasm rapidly, often at rates approaching those of drug-like small molecules. Mechanistic studies, including live-cell imaging, ATP-depletion experiments, and organelle co-localization analyses, indicate that IP⁺ MPs traverse the plasma membrane primarily via passive diffusion, avoiding endosomal entrapment. IP⁺ MPs do not localize to mitochondria, as is the case for many positively charged molecules. We show that incorporation of an IP⁺ unit transforms a previously described membrane impermeable macrocyclic antagonist of the p53-MDM2 interaction into a bioactive inhibitor of MCF-7 proliferation. Collectively, these results establish that IP⁺ incorporation is an effective strategy for the development of bioactive MPs targeting intracellular proteins.