Flexible fluorine-thiol displacement stapled peptides with enhanced membrane penetration for the estrogen receptor/coactivator interaction

Understanding how natural and engineered peptides enter cells would facilitate the elucidation of biochemical mechanisms underlying cell biology and is pivotal for developing effective intracellular targeting strategies. In this study, we demonstrate that our peptide stapling technique, fluorine-thiol displacement reaction (FTDR), can produce flexibly constrained peptides with significantly improved cellular uptake, particularly into the nucleus. This platform confers enhanced flexibility, which is further amplified by the inclusion of a D-amino acid, while maintaining environment-dependent α helicity, resulting in highly permeable peptides without the need for additional cell-penetrating motifs. Targeting the estrogen receptor α (ERα)-coactivator interaction prevalent in estrogen receptor-positive (ER+) breast cancers, we showcased that FTDR-stapled peptides, notably SRC2-LD, achieved superior internalization, including cytoplasmic and enriched nuclear uptake, compared to peptides stapled by ring-closing metathesis. These FTDR-stapled peptides use different mechanisms of cellular uptake, including energy-dependent transport such as actin-mediated endocytosis and macropinocytosis. As a result, FTDR peptides exhibit enhanced antiproliferative effects despite their slightly decreased target affinity. Our findings challenge existing perceptions of cell permeability, emphasizing the possibly incomplete understanding of the structural determinants vital for cellular uptake of peptide-like macromolecules. Notably, while α helicity and lipophilicity are positive indicators, they alone are insufficient to determine high-cell permeability, as evidenced by our less helical, more flexible, and less lipophilic FTDR-stapled peptides.