Periodic Reporting for period 1 - MULTIGLOW (Multi-modal chemical probes as diagnostic tools)
Berichtszeitraum: 2023-10-01 bis 2025-09-30
Early detection of cancer remains one of the most urgent challenges facing European healthcare systems. Across the EU, cancer causes 1.3 million deaths annually, and prostate and colorectal cancers remain among the most prevalent and difficult to diagnose at early stages. Current diagnostic tools rely predominantly on protein expression levels or genetic markers, which often fail to reflect the functional biochemical activity driving disease progression. One such activity is that of serine proteases- enzymes whose dysregulation contributes to cancer invasion, metastasis, and altered cell–microenvironment interactions. Among these, TMPRSS2 has emerged as a clinically relevant but poorly understood protease, implicated in prostate cancer biology and several epithelial cancers. Despite its relevance, there is a lack of sensitive and selective tools to detect its active form in cells, tissues, or patient-derived samples.
This gap- between the biological importance of TMPRSS2 and the limited availability of chemical tools to study it- formed the central motivation for the MULTIGLOW project. The project set out to design, synthesize, and validate a new generation of multi-modal, chemiluminescent and activity-based probes capable of selectively reporting TMPRSS2 activity in vitro, in cell systems, and in clinically relevant samples. By focusing on enzyme activity rather than expression alone, the project aligns strongly with the EU’s scientific and societal priorities, including Europe’s Beating Cancer Plan, the mission for cancer prevention and early detection, and the objective of building a more resilient healthcare system under the “Economy That Works for People” policy area.
The project pursued four interconnected scientific goals:
1) Development of innovative chemical probes – including chemiluminescent substrates, classical activity-based probes, and multi-modal (luminescent + fluorescent/inhibitory) tools selective for TMPRSS2 and other P1-arginine serine proteases.
2) Biochemical characterization and selectivity profiling – validating probe performance and identifying potent, selective lead candidates for biological applications.
3) Application of probes in biological systems – using the most promising tools to detect TMPRSS2 activity in cancer cell lines, 3D spheroids, and in complex samples, demonstrating their diagnostic and imaging potential.
4) Initial assessment of clinical relevance – examining whether TMPRSS2 activity can be detected in patient-derived biological fluids, evaluating feasibility, limitations, and future diagnostic pathways.
Together, these objectives form a pipeline from molecular design to real-world applicability, aiming not only to generate new scientific knowledge but also to build early-stage diagnostic strategies and foundational technologies with long-term translational potential.
This gap- between the biological importance of TMPRSS2 and the limited availability of chemical tools to study it- formed the central motivation for the MULTIGLOW project. The project set out to design, synthesize, and validate a new generation of multi-modal, chemiluminescent and activity-based probes capable of selectively reporting TMPRSS2 activity in vitro, in cell systems, and in clinically relevant samples. By focusing on enzyme activity rather than expression alone, the project aligns strongly with the EU’s scientific and societal priorities, including Europe’s Beating Cancer Plan, the mission for cancer prevention and early detection, and the objective of building a more resilient healthcare system under the “Economy That Works for People” policy area.
The project pursued four interconnected scientific goals:
1) Development of innovative chemical probes – including chemiluminescent substrates, classical activity-based probes, and multi-modal (luminescent + fluorescent/inhibitory) tools selective for TMPRSS2 and other P1-arginine serine proteases.
2) Biochemical characterization and selectivity profiling – validating probe performance and identifying potent, selective lead candidates for biological applications.
3) Application of probes in biological systems – using the most promising tools to detect TMPRSS2 activity in cancer cell lines, 3D spheroids, and in complex samples, demonstrating their diagnostic and imaging potential.
4) Initial assessment of clinical relevance – examining whether TMPRSS2 activity can be detected in patient-derived biological fluids, evaluating feasibility, limitations, and future diagnostic pathways.
Together, these objectives form a pipeline from molecular design to real-world applicability, aiming not only to generate new scientific knowledge but also to build early-stage diagnostic strategies and foundational technologies with long-term translational potential.
During the project, substantial scientific and technical progress was achieved across all planned research activities, resulting in the successful development of a comprehensive chemical toolkit for studying TMPRSS2 and related serine proteases.
1) Design and synthesis of chemical probes. The project successfully generated a diverse portfolio of chemical tools, including: a) A library of P1-arginine chemiluminescent substrates designed for sensitive detection of serine protease activity; b) A series of classical covalent activity-based probes (ABPs) targeting TMPRSS2; c) A validated multi-modal ABP, demonstrated with human neutrophil elastase (HNE), confirming the feasibility of combining chemiluminescent, fluorescent, and inhibitory elements in a single molecule.
2) Biochemical characterization and kinetics. Using high-quality recombinant TMPRSS2 obtained through collaborative efforts, all synthesized substrates and covalent probes were subjected to: a)Inhibition assays using a fluorogenic reporter; b) Determination of kinetic parameters (k2/Ki and Ki) for the most potent compounds; c) Selectivity profiling across a panel of related serine proteases (thrombin, trypsin, furin, HAT).
This systematic analysis identified several selective and high-potency candidates suitable for cell- and tissue-based applications. The most promising chemiluminescent substrates were applied in: Prostate cancer cell lines (e.g. LNCaP), revealing dynamic TMPRSS2 activity changes during androgen stimulation. Additionally usage of 3D spheroid models (4T1), demonstrate substrate penetration and robust signal in complex environments. Comparative substrate screening in cancer cells to identify the most sensitive reagents for biological imaging. These studies confirmed that the developed tools offer high sensitivity, excellent signal-to-noise ratio, and compatibility with live-cell and spheroid imaging.
Preliminary analysis of TMPRSS2 in patient-derived samples- Chemiluminescent assays were conducted on serum from prostate cancer patients. Although TMPRSS2-associated signals were detected, variability and sample-related matrix effects limited diagnostic conclusiveness. These results indicate that:
Sample preparation optimization is needed (e.g. protein depletion strategies). Urine or tissue-conditioned media may represent more suitable biofluids for future diagnostic development.
Main technical achievements:
1) Creation of a novel and versatile chemiluminescent substrate platform.
2) First demonstration of a multi-modal ABP combining luminescence, fluorescence, and covalent inhibition.
3) Identification of selective molecular tools for TMPRSS2 detection.
4) Validation of substrate performance in 2D and 3D cancer models.
5) Establishment of feasibility for functional TMPRSS2 detection in clinically relevant matrices.
Overall, the project successfully delivered its core scientific objectives, significantly advancing chemical and biological understanding of TMPRSS2 activity.
1) Design and synthesis of chemical probes. The project successfully generated a diverse portfolio of chemical tools, including: a) A library of P1-arginine chemiluminescent substrates designed for sensitive detection of serine protease activity; b) A series of classical covalent activity-based probes (ABPs) targeting TMPRSS2; c) A validated multi-modal ABP, demonstrated with human neutrophil elastase (HNE), confirming the feasibility of combining chemiluminescent, fluorescent, and inhibitory elements in a single molecule.
2) Biochemical characterization and kinetics. Using high-quality recombinant TMPRSS2 obtained through collaborative efforts, all synthesized substrates and covalent probes were subjected to: a)Inhibition assays using a fluorogenic reporter; b) Determination of kinetic parameters (k2/Ki and Ki) for the most potent compounds; c) Selectivity profiling across a panel of related serine proteases (thrombin, trypsin, furin, HAT).
This systematic analysis identified several selective and high-potency candidates suitable for cell- and tissue-based applications. The most promising chemiluminescent substrates were applied in: Prostate cancer cell lines (e.g. LNCaP), revealing dynamic TMPRSS2 activity changes during androgen stimulation. Additionally usage of 3D spheroid models (4T1), demonstrate substrate penetration and robust signal in complex environments. Comparative substrate screening in cancer cells to identify the most sensitive reagents for biological imaging. These studies confirmed that the developed tools offer high sensitivity, excellent signal-to-noise ratio, and compatibility with live-cell and spheroid imaging.
Preliminary analysis of TMPRSS2 in patient-derived samples- Chemiluminescent assays were conducted on serum from prostate cancer patients. Although TMPRSS2-associated signals were detected, variability and sample-related matrix effects limited diagnostic conclusiveness. These results indicate that:
Sample preparation optimization is needed (e.g. protein depletion strategies). Urine or tissue-conditioned media may represent more suitable biofluids for future diagnostic development.
Main technical achievements:
1) Creation of a novel and versatile chemiluminescent substrate platform.
2) First demonstration of a multi-modal ABP combining luminescence, fluorescence, and covalent inhibition.
3) Identification of selective molecular tools for TMPRSS2 detection.
4) Validation of substrate performance in 2D and 3D cancer models.
5) Establishment of feasibility for functional TMPRSS2 detection in clinically relevant matrices.
Overall, the project successfully delivered its core scientific objectives, significantly advancing chemical and biological understanding of TMPRSS2 activity.
The project achieved several results that substantially advance the state of the art in chemical biology, protease imaging, and diagnostic tool development:
1) Novel chemiluminescent substrate library- A broad, modular panel of substrates for P1-arginine serine proteases was developed. These substrates demonstrate high sensitivity, tunable selectivity, and compatibility with living cells and complex matrices. Such a substrate library did not previously exist and now provides a powerful resource for protease research and diagnostics.
2) Validation of a new multi-modal ABP strategy- The integration of chemiluminescent, fluorescent, and covalent detection modalities into a single molecule represents a major conceptual innovation. This approach enables multi-scale readouts (biochemical, cellular, imaging) from a single probe — a capability not previously demonstrated for serine proteases. Although validated on HNE, the modular design allows adaptation to TMPRSS2 and other proteases.
3) Functional detection of TMPRSS2 activity- The project provides the first demonstration that TMPRSS2 activity can be sensitively detected in cell lines and spheroids using turn-on chemiluminescent substrates. This enables functional studies of TMPRSS2 under physiologically relevant conditions, going beyond expression-level biomarkers.
4) Early exploration of TMPRSS2 as a diagnostic biomarker. Preliminary detection of TMPRSS2 activity in serum suggests feasibility, while also highlighting technical challenges and opportunities for improvement.
1) Novel chemiluminescent substrate library- A broad, modular panel of substrates for P1-arginine serine proteases was developed. These substrates demonstrate high sensitivity, tunable selectivity, and compatibility with living cells and complex matrices. Such a substrate library did not previously exist and now provides a powerful resource for protease research and diagnostics.
2) Validation of a new multi-modal ABP strategy- The integration of chemiluminescent, fluorescent, and covalent detection modalities into a single molecule represents a major conceptual innovation. This approach enables multi-scale readouts (biochemical, cellular, imaging) from a single probe — a capability not previously demonstrated for serine proteases. Although validated on HNE, the modular design allows adaptation to TMPRSS2 and other proteases.
3) Functional detection of TMPRSS2 activity- The project provides the first demonstration that TMPRSS2 activity can be sensitively detected in cell lines and spheroids using turn-on chemiluminescent substrates. This enables functional studies of TMPRSS2 under physiologically relevant conditions, going beyond expression-level biomarkers.
4) Early exploration of TMPRSS2 as a diagnostic biomarker. Preliminary detection of TMPRSS2 activity in serum suggests feasibility, while also highlighting technical challenges and opportunities for improvement.