From In Silico to Experimental Validation: Tailoring Peptide Substrates for a Serine Protease
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Updated: Dec 26, 2025
The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes
Published on: May 25, 2018
Philip Maximilian Knaff1,2, Christian Kersten3, Ramona Willbold4
1Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany.
Abstract:
Smart nanocarriers for the transport of drugs to tumor cells are nowadays of great interest for treating cancer. The use of enzymatic stimuli to cleave peptide-based drug nanocapsules for the selective release of nanocapsule cargo in close proximity to tumor cells opens new possibilities in cancer research. In the present work, we demonstrate a methodology for finding and optimizing cleavable substrate sequences by the type II transmembrane serine protease hepsin, which is highly overexpressed in prostate cancer. The design and screening of combinatorial libraries in silico against the binding cavity of hepsin allow the identification of a panel of promising substrates with high-calculated docking scores. In vitro screening verifies the predictions and showed that all substrates are cleaved by hepsin with higher efficiency than the literature known hepsin substrate RQLR↓VVGG. The introduction of d-amino acids on a selected peptide with the highest catalytic efficiency (kcat/Km) renders it resistant to cleavage by plasma or serum while maintaining their susceptibility to hepsin.
Insights
Researchers optimized peptide substrates for hepsin, a protease overexpressed in prostate cancer. These novel substrates enable targeted drug release from smart nanocarriers, enhancing cancer therapy while resisting degradation in plasma.
Area of Science:
- Biochemistry
- Nanotechnology
- Oncology
Background:
- Smart nanocarriers offer targeted drug delivery for cancer treatment.
- Enzymatic cleavage of nanocapsules enables selective drug release near tumor cells.
- Hepsin, a type II transmembrane serine protease, is overexpressed in prostate cancer.
Purpose of the Study:
- To develop and optimize peptide substrates cleavable by hepsin for targeted drug delivery.
- To identify novel hepsin substrates with enhanced cleavage efficiency.
- To create hepsin-cleavable nanocarriers resistant to plasma degradation.
Main Methods:
- In silico screening of combinatorial peptide libraries against hepsin's binding cavity.
- In vitro enzymatic assays to verify substrate cleavage efficiency.
- Introduction of d-amino acids to enhance peptide stability.
Main Results:
- Identification of multiple hepsin substrates with high in silico docking scores.
- In vitro validation confirmed hepsin cleavage of designed substrates, surpassing known substrates.
- Optimized peptides with d-amino acids showed hepsin specificity and resistance to plasma/serum degradation.
Conclusions:
- A methodology for designing and optimizing hepsin-specific peptide substrates was established.
- These substrates are effective for developing smart nanocarriers for targeted prostate cancer therapy.
- The d-amino acid modification provides stability, crucial for in vivo drug delivery applications.
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