Related Experiment Video
Updated: Jan 11, 2026
05:20
A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl
Published on: November 28, 2014
13.8K
Development of Granzyme A Turn-ON Fluorescent Activity-Based Probes
Sebastian M Malespini1, Muhammad Kazim1, Euna Yoo1
1Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA.
Chembiochem : a European Journal of Chemical Biology
|November 19, 2025
Summary
Researchers developed novel probes to detect Granzyme A (GzmA) activity, crucial for understanding its dual role in cancer immunity and inflammation. These tools enable precise visualization of GzmA in biological systems.
Area of Science:
- Biochemistry
- Immunology
- Molecular Biology
Background:
- Granzyme A (GzmA) is a serine protease in cytotoxic immune cells with complex roles in antitumor immunity and inflammation.
- Elevated GzmA correlates with better cancer outcomes, yet extracellular GzmA can promote tumorigenesis via inflammation.
Purpose of the Study:
- To develop selective tools for detecting GzmA activity with high spatiotemporal resolution in native biological contexts.
- To enable precise visualization and dissection of GzmA's multifaceted roles in the tumor immune microenvironment.
Main Methods:
- Design and synthesis of quenched activity-based probes (ABPs) that fluoresce upon binding to active GzmA.
- Utilizing an Ile-Gly-Asn-Arg recognition sequence, phenyl phosphinate ester warhead, and a near-infrared sulfo-Cy5/QSY21 fluorophore/quencher pair.
- In vitro and cellular assays to evaluate probe reactivity, selectivity, and cell permeability.
Main Results:
- Developed a lead ABP with high reactivity, selectivity, and cell permeability.
- Demonstrated robust detection of GzmA in vitro and in cells with an excellent signal-to-noise ratio.
- The probe enables visualization of GzmA activity in the tumor immune microenvironment.
Conclusions:
- Activatable probes provide a powerful means to study GzmA biology.
- These probes support activity-based protein profiling and noninvasive imaging of GzmA activity.
- Facilitates a deeper understanding of GzmA's complex functions in cancer and inflammation.
