Optimization of stapled peptide inhibitors reveals design principles for targeting talin-induced integrin activation

Talin-induced integrin activation is a central regulator of cell adhesion and signaling. Intracellular Targeting of this pathway remains to be challenging. Here, we report the structure-guided development of next-generation stapled peptidomimetics derived from the RIAM talin-binding site (TBS). Using biochemical, structural, and cellular analyses, we show that the second-generation S2-TBS enhances talin binding and inhibitory potency but exhibits reduced cell uptake due to an extended linker. Guided by these insights, we designed a minimized third-generation peptide, S3-TBS, which restores structural order, improves thermal stability and cell uptake, and retains strong talin binding. NMR and crystallographic analyses confirm conserved binding to specific interfaces in talin head and talin rod domains. Functionally, optimized peptides potently inhibit talin-mediated integrin interactions and suppress invadopodia-driven matrix degradation in cancer cells. These findings establish key principles for balancing affinity, conformational stability, and molecular size in the design of intracellular stapled peptide inhibitors. HIGHLIGHTSO_LIStructure-guided optimization converts a flexible stapled peptide into a highly ordered, high-affinity talin inhibitor. C_LIO_LIS2-TBS increases binding affinity but introduces conformational heterogeneity due to a longer linker. C_LIO_LIS3-TBS achieves improved stability, reduced size, and a well-defined stapled conformation. C_LIO_LIOptimized peptides inhibit talin-mediated integrin function and suppress cancer cell matrix degradation. C_LI