Linker Engineering in Stapled Peptides for Enhanced Membrane Permeability: Screening and Optimization Strategies

The optimization of membrane permeability is a decisive strategy for mitigating late-stage failures in peptide drug development. By leveraging linker chemical diver-sity, stapled peptides utilize linker engineering to precisely modulate key physico-chemical parameters—such as lipophilicity and conformational constraints—to over-come the desolvation energy penalty. This review systematically evaluates link-er-based strategies for enhancing the permeability of stapled peptides, categorized into two primary dimensions: (1) High-throughput screening (HTS) compatibility, focusing on the integration of functionalized linkers into mRNA display, phage display, and DNA-encoded libraries (DELs) to identify lead scaffolds with inherent permeability potential during early discovery ; and (2) Post-screening structural refinement, cover-ing rational design strategies including intramolecular hydrogen bond (IMHB) shield-ing, "chameleonic" adaptations, and stimuli-responsive reversible stapling . Further-more, we analyze the paradigm shift in assessment methodologies from qualitative imaging to quantitative cytosolic delivery assays, which have deepened our under-standing of mechanisms such as the charge/lipophilicity threshold balance and meta-bolic-driven trapping. Overall, linker engineering provides a robust technical roadmap for developing the next generation of cell-permeable stapled peptide therapeutics.