Diversity-Oriented Stapling Yields Intrinsically Cell-Penetrant Inducers of Autophagy

Autophagy is an essential pathway by-which cellular and foreign material are degraded and recycled in eukaryotic cells. Induction of autophagy is a promising approach for treating diverse human diseases, including neurodegenerative disorders and infectious,diseases.. Here, we report the use of a diversity-oriented stapling approach to, produce autophagy-inducing peptides that are intrinsically cell-penetrant. These peptides, induce autophagy at micromolar concentrations in vitro, have aggregate-clearing activity in a cellular model of Huntington's disease, and induce autophagy in vivo. Unexpectedly, the solution structure of the most potentstapled peptide, DD5-o, revealed an alpha-helical conformation in methanol, stabilized by an unusual (i,i+3) staple which cross-links two D-amino acids. We also developed a novel assay for eel-penetration that reports exclusively on cytosolic access and used it to quantitatively compare the cell penetration of DD5-o and other autophagy-inducing peptides. These new, cell-penetrant autophagy inducers and their molecular details are critical advances in the effort to understand and control autophagy: More broadly, diversity-oriented stapling may provide a promising alternative to polycationic sequences as a means for tendering, peptides more cell-penetrant.