C-end rule peptides mediate neuropilin-1-dependent cell, vascular, and tissue penetration

Abstract

Screening of phage libraries expressing random peptides for binding to prostate cancer cells primarily yielded peptides that had a Cterminal arginine (or rarely lysine) residue, usually in a consensus context R/KXXR/K. Phage expressing these sequences and synthetic nanoparticles coated with them bound to and were internalized into cells. The C-terminal arginine (or lysine) was essential to the activity; adding another amino acid, or even blocking the free carboxyl group of this arginine residue by amidation, eliminated the binding and internalizing activity. An internal R/KXXR/K can be exposed and switched on by a cleavage by a protease. The strict requirement for C-terminal exposure of the motif prompted us to term the phenomenon the C-end rule (CendR). Affinity chromatography showed that the CendR peptides bind to neuropilin-1 (NRP-1) on the target cells. NRP-1 is a cell-surface receptor that plays an essential role in angiogenesis, regulation of vascular permeability, and the development of the nervous system. VEGF-A165 and other ligands of NRP-1 possess a C-terminal CendR sequence that interacts with the b1 domain of NRP-1 and causes cellular internalization and vascular leakage. Our CendR peptides have similar effects, particularly when made multivalent through coupling to a particle. We also noted a unique and important activity of these peptides: penetration and transportation through tissues. The peptides were able to take payloads up to the nanoparticle size scale deep into extravascular tissue. Our observations have implications in drug delivery and penetration of tissue barriers and tumors.homing peptides ͉ vascular permeability ͉ VEGF ͉ cell penetrating peptides

Selection of phage-displayed peptides on live adherent cells in microfluidic channels

“…Previous studies have shown that peptides with a C-terminal (R/K)XX(R/K) sequence exhibit significantly higher affinity for NRP-1-expressing cells than peptides with a C-terminal arginine alone (XXXR) (29). We found that the MiPS system enables more efficient enrichment of phage displaying this higher-affinity motif (Fig.…”

“…We then seeded the chamber with ∼10 4 PPC-1 human prostate carcinoma cells in 2 mL of culture medium. PPC-1 cells were selected as a model because they express high levels of NRP-1, a well characterized receptor that binds and internalizes peptides with C-terminal arginine residues, typically within a consensus context of R/KXXR/K (the C-end Rule or CendR motif) (29)(30)(31). As a negative control, we used phage expressing hepta-glycine (G7), which exhibits negligible binding to PPC-1 cells (28).…”

“…In order to compare the binding affinities between the RPARPAR phage and the R3 phage pool selected by the MiPS chip, we performed a series of inhibitor binding experiments to determine whether the R3 phage pool has sufficient affinity to displace PPC-1-bound RPARPAR phage, and vice versa. We used UV-inactivated noninfectious phage particles expressing about 200 peptides per particle as multivalent inhibitors (29,30). We incubated PPC-1 cell suspensions (2 × 10 4 cells∕sample) with solutions containing a 100-, 300-, or 1,000-fold excess of noninfectious R3 or RPAR-PAR phage (inhibitor) at 4°C for 20 min, after which we added live RPARPAR or R3 phage solutions (1-fold, 3 × 10 8 pfu).…”

“…In both cases, we performed three rounds of selection with a relatively small number of PPC-1 cells (2 × 10 4 ). After each round of selection, the recovered phage were amplified in Escherichia coli BLT5403 cells at 37°C for 2 h, followed by phage precipitation with a polyethylene glycol (PEG)/NaCl solution and purification by CsCl gradient ultracentrifugation (29). We analyzed a small aliquot (100 μL) of the selected phage pool from each round, and found that the enrichment of high-affinity phage was significantly more efficient in the MiPS system; after three rounds of selection in the MiPS chip, the phage from the round 3 pool (R3) demonstrated ∼700-fold higher binding to PPC-1 cells on-chip in comparison to the initial random library (Fig.…”

VEGF-A121a binding to Neuropilins – A concept revisited

“…18 All NRP1-binding proteins and peptides are found to possess a C-terminal arginine. 17 , 19 - 21 The authors further showed that NRP1 has a C-terminal arginine-binding pocket in the b1 domain. Mutating VEGF's C-terminal exon 8a-encoded arginine (R164) resulted in up to 97% loss in retention of mouse VEGF-A164a by NRP1, thus this residue plays a critical role in VEGF-A/NRP1 interactions (Fig.…”