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A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

Published on: November 28, 2014

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

This summary is machine-generated.

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.

Keywords:

activity‐based probes granzyme A immunity optical imaging protease

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Published on: September 13, 2022

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.