In-Cell Penetration Selection–Mass Spectrometry Produces Noncanonical Peptides for Antisense Delivery

• DOI: 10.1021/acschembio.2c00920
• Publication Date: 2023-03-01T19:45:36Z
• Authors: Carly K. Schissel; Charlotte E. Farq...; Andrei Loas; Annika B. Malmberg; Bradley L. Pentelute
• Keywords: Cell-penetrating peptide; Morpholino; Cell fractionation

Peptide-mediated delivery of macromolecules in cells has significant potential therapeutic benefits, but no therapy employing cell-penetrating peptides (CPPs) reached the market after 30 years investigation due to challenges discovery new, more efficient sequences. Here, we demonstrate a method for in-cell penetration selection-mass spectrometry (in-cell PS-MS) discover from synthetic library capable delivering macromolecule cargo cytosol. This was inspired by recent vivo selection approaches cell-surface screening, with an added spatial dimension resulting subcellular fractionation. A representative peptide discovered cytosolic extract, Cyto1a, is nearly 100-fold active toward antisense phosphorodiamidate morpholino oligomer (PMO) compared sequence identified whole cell which includes endosomes. Cyto1a composed d-residues and two non-α-amino acids, stable than its all-l isoform, less toxic known CPPs comparable activity. Pulse-chase microscopy experiments revealed that while PMO-Cyto1a conjugate likely taken up endosomes, it can escape localize nucleus without nonspecifically releasing other endosomal components. In-cell PS-MS introduces means empirically unnatural therapeutically relevant cargo.