Farmaceutische, Biomedische en Diergeneeskundige Wetenschappen

Public defense Joni De Loose 11/03/2025 - On dipeptidyl peptidase 9 and the road towards understanding its role in human cells - Department Pharmaceutical Sciences

Promotors: prof. dr. Ingrid De Meester - prof. dr. ir. Yann Sterckx

Location defense: Aula Q002, gebouw Q, Campus Drie Eiken

Abstract:

This thesis focuses on human dipeptidyl peptidase 9 (DPP9), an intracellular serine protease that cleaves off the N-terminal dipeptide of its substrates. DPP9 is involved in cellular homeostasis and programmed cell death but remains less characterized than its family members DPP4 and fibroblast activation protein (FAP). Unlike these family members, DPP9 is essential for cell survival and neonatal development, highlighting the importance of this protease in the human body.
A significant challenge at the outset of this research was the absence of selective DPP9 compounds. To address this gap, we evaluated a small molecule-based toolset for studying DPP9. Novel inhibitors synthesized at the University of Antwerp’s Laboratory of Medicinal Chemistry (UAMC) were biochemically characterized. Among these, cpd-42 exhibited in vitro nanomolar affinity for DPP9 and high selectivity towards homologue DPP8. Cellular assays with cpd-42 and a second novel DPP8/9 inhibitor, cpd-6a, confirmed that DPP8/9 inhibition induces caspase-1-dependent lytic cell death in myeloid cells. Notably, gasdermin D was not essential for cell death in myeloid cells, suggesting the involvement of alternative cell death pathways beyond the expected gasdermin D-driven pyroptosis. The inhibitors also induced lytic cell death in primary human CD4+ T cells, monocytes, monocyte-derived macrophages and dendritic cells. However, further analyses will be necessary for a comprehensive interpretation of their intracellular effects.
We also investigated DPP9 expression and activity in peripheral blood mononuclear cells (PBMCs) and endothelial cells. DPP8/9 activity increased as monocytes differentiated into macrophages and dendritic cells. Intriguingly, DPP9 localized to the cytoplasm and the nucleus in PBMCs, displaying variable expression patterns across different cells and cell types, while in endothelial cells, DPP9 was primarily detected in the cytoplasm. The significance of DPP9’s nuclear localization and the related regulatory mechanisms remain unresolved.
We further characterized fluorescently labeled inhibitors that were synthesized at UAMC. Among these, an NBD-linked DPP8/9 inhibitor showed promise as the first intracellular DPP9 fluorescent probe. However, challenges such as photobleaching and compatibility issues with antibodies limited its applicability.
In the final experimental section, we evaluated proteolysis-targeting chimeras (PROTACs) for their ability to degrade DPP9 intracellularly. While these PROTACs inhibited DPP9 activity in vitro, intracellular DPP9 degradation was inconsistent in THP-1 cells, highlighting the need for further optimization of these molecules.
This thesis underscores the necessity for improved selective inhibitors, probes, and PROTACs to study DPP9’s functions. Future research should focus on fundamental research questions concerning DPP9’s homeostatic regulation, the nuclear functions, and the functional redundancy with DPP8. Advancing our understanding of DPP9’s role in cellular processes will be crucial in determining if and how DPP9 could ever become a druggable target.

Public defense Yentl Van Rymenant 18/02/2025 - Exploring Fibroblast Activation Protein (FAP) in the tumor microenvironment and Crohn’s disease using novel FAP-selective tools and methods - Department Pharmaceutical Sciences

Promotors: Prof. Ingrid De Meester - Prof. Pieter Van der Veken

Location defence: Aula Q002, gebouw Q, Campus Drie Eiken

Abstract:

Fibroblast activation protein (FAP) is a post-prolyl proteolytic enzyme that is prominently expressed in cancer-associated fibroblasts (CAFs) within the tumor microenvironment (TME) and in activated fibroblasts involved in tissue remodeling in chronic inflammatory diseases, such as Crohn’s disease (CD). Interestingly, FAP exhibits low expression and activity in healthy tissues, making it a promising diagnostic and therapeutic target for cancer and chronic inflammatory diseases. Unfortunately, FAP’s functional role in these pathological conditions remains poorly understood, and current tools and methods for studying FAP are often characterized by a lack of selectivity. Additionally, research has primarily focused on FAP mRNA and protein expression, often overlooking its enzymatic activity, which is critical for understanding its biological functions. These limitations highlight the urgent need for innovative, highly selective tools and methods to detect FAP and elucidate its role in disease mechanisms.
Hence, the goal of this doctoral research was to develop and validate novel FAP-selective tools and methods to enhance our understanding of FAP’s biological function in cancer and Crohn’s disease.
To achieve this, we established an in-house recombinant human FAP (rhFAP) production system to ensure a robust supply of high-quality active enzyme for downstream evaluation and validation of novel FAP-targeting tools and compounds. We produced and purified a highly specific antibody against FAP and developed fluorescent FAP-targeting activity-based probes (ABPs) that enable selective, high-affinity visualization of FAP’s enzymatic activity. Furthermore, we evaluated novel [18F]-labeled FAP inhibitors ([18F]-FAPIs) for PET/CT imaging as a non-invasive diagnostic approach for tumors and fibrotic diseases in which FAP plays a role.
With these tools in hand, we gained new insights into the expression and function of FAP in the TME, with a particular focus on its activity and role in natural killer (NK) cells. Our findings suggest that FAP is involved in NK cell activity and immune evasion. In addition, we used these FAP-specific tools to investigate its function in CD, revealing its involvement in chronic inflammation and intestinal fibrosis.
Overall, this research not only offers new insights into FAP’s role in the TME and CD, but also provides the scientific community with a comprehensive toolkit for the selective detection and functional analysis of FAP. By filling critical knowledge gaps in FAP’s biological function, our findings pave the way for novel diagnostic and therapeutic strategies targeting FAP in oncology, Crohn’s disease and beyond.

Public defence Agnese Compagnone 30/01/2025 - Implementation of in vivo models to identify potential candidates for DED treatment - Department Pharmaceutical Sciences

Promotors: Prof. Paul Cos - Prof. Peter Delputte

Location: Q002

Abstract:

This thesis explores the development of an evaporative dry eye (EDE) animal model based on Meibomian gland cauterisation, aiming to replicate the tear film alteration caused by Meibomian gland dysfunction (MGD), a major driver of EDE. Dry eye disease (DED), which causes ocular surface inflammation, pain, and discomfort, is a multifactorial condition with significant impact on quality of life. EDE is the most common subtype, linked to MGD, which impairs lipid secretion from Meibomian glands, increasing the tear evaporation rate. The cauterisation method, as suggested by existing literature, faced challenges: technique variability and unclear guidelines on how many glands to cauterise or whether both eyelids should be involved. These inconsistencies can compromise the model’s reliability, indicating the need for further optimisation to ensure reproducibility and consistency in experimental outcomes.
This thesis also presents a novel multiplex electrochemiluminescence immunoassay (ECLIA) for detecting key biomarkers in rat tear samples, critical for DED research. Biomarkers such as interleukin-17 (IL-17), matrix metalloproteinase-9 (MMP-9), and intercellular adhesion molecule-1 (ICAM-1) were targeted. The assay was validated with promising results, showing that the detection ranges for these biomarkers met the required parameters, though precision was identified as a limiting factor. Variations were mainly due to human error, such as pipetting inconsistencies and evaporation during incubation. Suggestions for improving precision include standardising operator expertise, minimising reagent variability, and controlling incubation conditions. These improvements would increase reliability and broaden the assay's detection range.
Additionally, the thesis investigates the role of oxidative stress and ferroptosis in DED. Ferroptosis, a form of iron-dependent cell death linked to lipid peroxidation, is thought to contribute to DED pathogenesis. In vitro studies with the ferroptosis inhibitor UAMC-3203 suggested potential in reducing oxidative stress. However, clear evidence for ferroptosis in DED models was not found. In vivo tests showed that UAMC-3203 could restore GPX4 gene expression closer to the control group, hinting at potential benefits of ferroptosis inhibition.
Finally, the thesis proposes optimising the EDE model with inflammation-triggering agents like LFA-1 and ICAM-1 inhibitors, or toll-like receptor agonists, to enhance chronicity and inflammation. The use of advanced 3D cell culture models for better simulation of the ocular surface is also discussed, highlighting their potential for testing new therapies. Further research is needed to address the multifactorial causes of DED and improve treatments for this widespread condition