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

Abstract

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

Advances in Ultrahigh Throughput Hit Discovery with Tandem Mass Spectrometry Encoded Libraries

“…Schissel et al developed a method for in-cell penetration selection mass spectrometry (in-cell PS−MS) that opens the possibility for delivery of therapeutically relevant cargo. 65 Cell-penetration peptides (CPPs) are able to penetrate the cellular membrane and deliver therapeutic agents such as nucleic acids and proteins, which could not enter the cells otherwise. Schissel et al designed a library including non-α backbones to promote endosomal escape, hydrophobic and aromatic residues for increased membrane penetration and charged residues for enhanced membrane penetration.…”

“…The synthesis of customized building blocks tuning library properties is straightforward, and even building blocks with complex side chain modification can be detected, and sequenced with the automated procedures. 16 We have shown the importance of these unnatural building blocks for improving target recognition, 1,5,16,69 proteolytic stability, 67 cell penetration, 82,83 and in vivo behavior. 4 In the future, a synergistic pairing between display and AS−MS/MS workflows would be a powerful strategy.…”

“…A significant development of the AS–MS/MS technology is the transition to in cell and even in vivo selections. Schissel et al developed a method for in-cell penetration selection mass spectrometry (in-cell PS–MS) that opens the possibility for delivery of therapeutically relevant cargo . Cell-penetration peptides (CPPs) are able to penetrate the cellular membrane and deliver therapeutic agents such as nucleic acids and proteins, which could not enter the cells otherwise.…”

“…The ability to correctly identifying the sequences from complex mixtures with hundreds of synthetic peptide based compounds was further 16 In vivo selections resulted in the identification of erythrocyte binders. 4 Functional screenings enabled the identification of potent cell penetrating peptides 65 and bioconjugation tags. confirmed in further studies by Pomplun et al 16,62 These observations indicated that the approach could be utilized to decode structurally diverse samples of polypeptides and peptidomimetics resulting from affinity selection workflows.…”

“…In vivo selections resulted in the identification of erythrocyte binders 4. Functional screenings enabled the identification of potent cell penetrating peptides65 and bioconjugation tags 66.…”