Developing a toolbox of chemical probes for the detection of Granzyme A activity in inflammatory diseases
Creators: name: Cheng, Zhi Ming
Publisher: name: The University of Edinburgh
Year: 2024
Subjects: Granzyme A activity
inflammatory diseases
natural killer (NK) cells
cytotoxic T-lymphocytes (CTLs)
cytotoxic cell involve
immunemediated cell death
Granzyme A (GzmA)
Inflammatory Bowel Diseases (IBD)
near-infrared (NIR) fluorogenic GzmA activitybased probes
human GzmA activity chemiluminescence probes
NIR photodynamic therapy (PDT) agents
NIR photosensitizer
non-toxic NIR-I light

description: Granzymes are a family of serine proteases expressed in cytolytic granules within natural killer (NK) cells and cytotoxic T-lymphocytes (CTLs). These enzymes play a pivotal role in the immune system, where NK and CTLs release granzymes to eliminate target cells, such as cancerous or pathogen-infected cells. Therefore, granzymes are often considered markers of cytotoxic cell involvement in immune-mediated cell death. Granzyme A (GzmA), the most abundant of this family, has been reported as a potential biomarker in various inflammatory diseases, including Inflammatory Bowel Diseases (IBD). However, its exact role in these conditions remains a subject of debate, as conflicting studies propose both pro-inflammatory and pro-apoptotic functions. This uncertainty is partly due to the inadequacy of existing tools to assess GzmA's role effectively. Conventional methods, such as enzyme level measurements using antibodies, often fall short in providing insights into its proteolytic events, including the precise location and timing of enzyme activation, as well as its functional context. To address the need for enhanced tools to investigate GzmA's role in diseases, we have developed a diverse chemical toolbox of GzmA-responsive probes, including a GzmA inhibitor, human and mouse near-infrared (NIR) fluorogenic GzmA activitybased probes, and human GzmA activity chemiluminescence probes. We demonstrated that these probes were able to perform real-time imaging of immune cell responses to antigen-driven recognition of cancer cells, and detect GzmA activity in biosamples of infected mice. Additionally, they also facilitated the exploration of GzmA's role in inflammatory diseases and potentially aiding in the stratification of patients with inflammatory bowel disease (IBD). At the same time, recognizing the limitations for current NIR photodynamic therapy (PDT) agents, we have developed novel NIR photosensitizers for biomedical applications, with the potential to be activated only when exposed to specific overexpressed cancer biomarkers. We demonstrated that the chemical modification of the phenol moiety in Se-bridged hemicyanines leads to a significant reduction of their photodynamic activity and can be exploited to prepare targeted PDT agents for enzymatic decaging in cancer cells. We proved the versatility of this platform by synthesizing some of the first NIR-I organic PS with selective reactivity against human cathepsin B as a relevant biomarker in cancer cells. With the new photosensitizers, we were able to safely ablate U87 microtumors in vivo in a zebrafish preclinical model of cancer after illumination with non-toxic NIR-I light.