Protease-activated CendR peptides targeting tenascin-C: mitigating off-target tissue accumulation

Area of Science:

• Biotechnology and Pharmaceutical Sciences
• Molecular and Cellular Biology
• Oncology

Background:

• Targeting anticancer compounds and nanoparticles (NPs) to tumors requires precision ligands.
• C-end Rule (CendR) peptides offer tumor-penetrating capabilities but can bind non-target tissues expressing neuropilin-1 (NRP-1).
• The PL3 peptide, a CendR peptide, binds NRP-1 promiscuously via its C-terminal RLVR element.

Purpose of the Study:

• To engineer PL3 peptide derivatives that bind NRP-1 selectively after proteolytic activation by urokinase-type plasminogen activator (uPA).
• To maintain binding affinity to tenascin-C C-domain (TNC-C) while achieving uPA-dependent NRP-1 engagement.
• To develop a screening technology for proteolytically activated tumor-penetrating peptides.

Main Methods:

• Rational design and screening of a uPA-treated peptide-phage library based on the PL3 peptide.
• Identification of peptide derivatives that bind recombinant NRP-1 only after uPA cleavage.
• In vitro assays (cleavage, binding, internalization) and in vivo biodistribution studies in glioblastoma mouse models.

Main Results:

• Two novel peptides, PL3uCendR and SKLG, were identified, exhibiting uPA-dependent binding to NRP-1.
• These peptides maintain binding to TNC-C while demonstrating reduced off-target binding to healthy NRP-1-expressing tissues.
• In vivo studies confirmed the targeted delivery and efficacy of the developed peptides.

Conclusions:

• Novel uPA-dependent CendR peptides targeting TNC-C and NRP-1 have been successfully developed.
• This study establishes a technology for screening proteolytically activated tumor-penetrating peptides.
• The developed peptides offer a promising strategy for precise tumor targeting and reduced systemic toxicity in cancer therapy.