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 boosted passive membrane permeability significantly, as measured by the parallel artificial membrane permeability assay (PAMPA) (Li et al. (2024) J. Am. 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 colocalization analyses, indicate that IP + MPs traverse the plasma membrane primarily via passive diffusion, avoiding endosomal entrapment. IP + MPs do not localize to the mitochondria, as is the case for many positively charged molecules. We show that the 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. Enhanced permeability is observed when the IP + unit is positioned either within the macrocyclic backbone or on a side chain. Collectively, these results establish that IP + incorporation is an effective strategy for the development of bioactive MPs targeting intracellular proteins.