An l- to d-Amino Acid Conversion in an Endosomolytic Analog of the Cell-penetrating Peptide TAT Influences Proteolytic Stability, Endocytic Uptake, and Endosomal Escape

Abstract

Edited by F. Peter GuengerichCell-penetrating peptides (CPPs) are well established as delivery agents for otherwise cell-impermeable cargos. CPPs can also theoretically be used to modulate intracellular processes. However, their susceptibility to proteolytic degradation often limits their utility in these applications. Previous studies have explored the consequences for cellular uptake of converting the residues in CPPs from L-to D-stereochemistry, but conflicting results have been reported and specific steps en route to intracellular activity have not been explored. Here we use dimeric fluorescence TAT as a model CPP to explore the broader consequences of L-to D-stereochemical conversion. We show that inversion of chirality provides protease resistance without altering the overall mode of cellular entry, a process involving endocytic uptake followed by endosomal escape and cytosolic access. However, whereas inversion of chirality reduces endocytic uptake, the D-peptide, once in the endosome, is significantly more prone to escape than its L-counterpart. Moreover, the D-peptide is retained in the cytosol of cells for several days, whereas the L-peptide is degraded within hours. Notably, while the L-peptide is relatively innocuous to cells, the D-peptide exerts a prolonged antiproliferative activity. Together, our results establish connections between chirality, protease resistance, cellular penetration, and intracellular activity that may be useful for the development of future delivery agents with improved properties. Cell-penetrating peptides (CPPs)2 have become important tools for the delivery of macromolecular cargoes inside cells (1-3). These delivery agents show promise in therapeutic applications and are useful reagents for cell biology assays (4 -6). For instance, CPPs (TAT, penetratin, etc.) are currently being tested in several preclinical and clinical trials (7,8). However, CPPs exposed to cells or serum are rapidly degraded, and this can consequently render these compounds less effective in vivo or in vitro (9 -13). To protect CPPs from degradation, a common strategy has been to employ D-amino acids instead of their L-amino acid counterparts. D-Peptides are protease-resistant, and this approach has been applied to CPPs such as TAT, R9, penetratin, hLF, pVEC, and sweet arrow peptide (10, 14 -17). In addition, the extended in vivo half-lives of D-peptides over L-peptides have contributed to the successful development of D-polyarginine CPPs as cancer contrast agents (18,19). How inversion of chirality impacts the multifaceted functions of CPPs, however, remains unclear.Several reports have indicated that cellular uptake of CPPs is independent of peptide backbone chirality (16,20). Uptake of the CPPs studied was thought to involve direct plasma membrane translocation. This is because uptake persisted at 4°C, a condition that typically abolishes endocytosis (16). In many cases, however, the penetration of CPPs into cells involves different routes of cellular entry (21). Instead of crossing the plasma membr...

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“…Recent examples of delivery methods of proteins include the use of endosomolytic peptides, [12,13] poly(disulfide)s, [14] microinjection, [15] electroporation, [16] small-molecule additives, [17] bioreversible esterification, [18] and modifying the target protein with cell-penetrating peptides (CPPs). Recent examples of delivery methods of proteins include the use of endosomolytic peptides, [12,13] poly(disulfide)s, [14] microinjection, [15] electroporation, [16] small-molecule additives, [17] bioreversible esterification, [18] and modifying the target protein with cell-penetrating peptides (CPPs).…”

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“…Specifically, D-dfTAT, a dfTAT analog made of D-amino acids instead of L residues, is equivalent to dfTAT xin its cell penetration activity. [25] However, while dfTAT is degraded within minutes upon entry into cells, D-dfTAT is resistant to proteolytic degradation and remains intact inside cells for several days. Notably, unlike dfTAT, D-dfTAT inhibits cell proliferation and leads to the dysregulation of hundreds of genes upon cell delivery.…”

“…A possible answer to this question may reside in the fact that dfTAT is readily degraded by cells. Specifically, d ‐dfTAT, a dfTAT analog made of d ‐amino acids instead of l residues, is equivalent to dfTAT xin its cell penetration activity . However, while dfTAT is degraded within minutes upon entry into cells, d ‐dfTAT is resistant to proteolytic degradation and remains intact inside cells for several days.…”