Public defence Ellen Wuyts 24/03/2026 - Structural and functional insights into the Leishmania donovani dynamin-1-like protein as a promising drug target - Department Pharmaceutical Sciences
Public defence Ellen Wuyts 24/03/2026 - Structural and functional insights into the Leishmania donovani dynamin-1-like protein as a promising drug target - Department Pharmaceutical Sciences
Promotors:
Prof. dr. Yann Sterckx (promotor)
Prof. dr. Guy Caljon (promotor)
Prof. dr. Malgorzata Domagalska (promotor, ITG)
Location: Aula O7, gebouw O, Campus Drie Eiken
Abstract:
Leishmaniasis remains a major neglected tropical disease requiring a multifaceted control strategy that integrates vaccination, vector control, and chemotherapy. Among these pillars, chemotherapy is indispensable for clinical management and disease control. However, increasing reports of treatment failure and the limited arsenal of effective drugs underscore the urgent need for novel antileishmanial compounds with innovative modes of action. This thesis focuses on the characterization of TCMDC-143345, a promising compound identified from the GlaxoSmithKline Leishbox, and its molecular target, the Leishmania donovani dynamin-1–like protein (LdoDLP1). Previous work established LdoDLP1 as a resistance marker for TCMDC-143345, strongly suggesting that it constitutes the compound’s molecular target. The central objective of this thesis was to deepen our understanding of the structure-function relationship of LdoDLP1 and to unravel the molecular basis underlying the antileishmanial activity of TCMDC-143345 through biochemical, structural, biophysical, and parasitological approaches.
A comprehensive biophysical characterization of LdoDLP1 demonstrated its intrinsic propensity to self-assemble into higher-order oligomers, a defining feature of dynamin-related proteins (DRPs). Structure-guided mutagenesis led to the identification of a G354D/R357S double mutant with markedly altered oligomerization behavior. Analytical gel filtration, small-angle X-ray scattering, and negative-stain electron microscopy confirmed that LdoDLP1 is a bona fide DRP, lacking a canonical PH domain but containing a B-insert, thereby providing important insights into its structural architecture. To elucidate the interaction between TCMDC-143345 and LdoDLP1, extensive efforts were undertaken to obtain a high-resolution structure of the protein and its complex with the compound. Although structural determination was not yet achieved, grating-coupled interferometry provided preliminary evidence supporting a direct interaction. In parallel, efforts were initiated to establish a GTPase activity assay to assess the functional impact of compound binding.
As complementary tools to probe LdoDLP1 function, camelid single-domain antibodies (sdAbs) were generated. Immunization yielded 33 unique LdoDLP1-specific sdAbs, which were recombinantly produced and characterized, offering innovative reagents to further dissect LdoDLP1 self-assembly and enzymatic activity.
Finally, the potential of TCMDC-143345 to induce parasite quiescence was investigated. High-dose exposure experiments followed by fluorescence-activated cell sorting demonstrated quiescent behavior, after which susceptibility was reassessed to evaluate potential shifts in drug response.
Overall, LdoDLP1 appears a more challenging target than anticipated but nevertheless valuable insights into its structure, function and potential as a drug target were obtained with the potential of invigorating future research.
Public defence Emile Verhulst 18/03/2026 - Development of biochemical assays to enable FAP-targeted inhibitor discovery and cancer biomarker applications - Department Pharmaceutical Sciences
Public defence Emile Verhulst 18/03/2026 - Development of biochemical assays to enable FAP-targeted inhibitor discovery and cancer biomarker applications - Department Pharmaceutical Sciences
Promotors: Prof. dr. Ingrid De Meester - Prof. dr. Pieter Van der Veken
Location: Aula O2, building O, Campus Drie Eiken, Universiteitsplein 1, 2610 Antwerpen (Wilrijk)
Abstract:
Fibroblast activation protein α (FAP) is highly expressed on cancer-associated fibroblasts and has emerged as a promising target for tumor molecular imaging and therapy. Although many FAP-targeted ligands have entered clinical evaluation, important aspects of FAP biochemistry, enzyme kinetics, and target engagement are still not well understood, limiting rational ligand design and therapeutic optimization. The overarching aim of this thesis was to establish robust recombinant production of highly active FAP, and to develop an in vitro screening framework for FAP-targeted theranostic ligands that enables quantitative kinetic characterization of target engagement and residence time.
First, this study reviews the historical development of FAP-targeting strategies in oncology (Chapter 3). Subsequently, recombinant production of human FAP (Chapter 4) and related proteases (Chapter 5) was optimized, focusing on yield, specific activity and stability. The enhanced quality of the recombinant human FAP facilitated an initial structural characterization, and the development of more elaborate enzymatic assay formats. Building on this foundation, medium-throughput kinetic assays were established to accurately determine association and dissociation rate constants of FAP-inhibitors, allowing target-residence time to be quantified as a key parameter for ligand selection (Chapter 6). Application of this framework revealed that structurally related FAP-inhibitors can display markedly distinct kinetic profiles despite similar equilibrium potency, highlighting the limitations of conventional IC50-based screening.
In addition, this work also describes the development of a FAP-targeted gold nanoparticle conjugate as an exploratory translational tool for FAP detection (Chapter 7). Multiple targeting strategies were evaluated, including monoclonal antibodies, small-molecule FAP inhibitors, and single-domain antibodies, to assess their compatibility with gold nanoparticle conjugation and retention of functional FAP binding. The final conjugate was evaluated in a proof-of-principle anti-FAP lateral flow assay. Beyond this diagnostic application, the conjugate represents a versatile platform with broader potential, for example as a tracking reagent to study FAP localization and behavior in vitro, including live-cell imaging applications.
Collectively, this work provides a mechanistic basis for the rational design and evaluation of FAP-targeted radioligands and diagnostic tools. The findings contribute to ongoing efforts to improve the specificity, efficacy, and clinical robustness of FAP-based theranostic strategies.
Public defence Sam Corthaut 11/03/2026 - Insights in the structure-function relationship of DPP9 binding interactions and DPP9-selective inhibition - Department Pharmaceutical Sciences
Public defence Sam Corthaut 11/03/2026 - Insights in the structure-function relationship of DPP9 binding interactions and DPP9-selective inhibition - Department Pharmaceutical Sciences
Promotors: Prof. dr. Ingrid De Meester - Prof. dr. Yann Sterckx
Location defence: Aula O4, building O, Campus Drie Eiken, Universiteitsplein 1, 2610 Antwerpen (Wilrijk)
Abstract:
This thesis explores the molecular biology of two human serine proteases dipeptidyl peptidase 8 (DPP8) and dipeptidyl peptidase 9 (DPP9). Both are intracellular members of the DPP4 subfamily of serine proteases and cleave off dipeptides at the N-terminus of their substrates when a Pro or, in lesser extent, an Ala residue is at penultimate (P1) position. DPP9 was shown to be crucial in several cellular pathways and processes. Well-studied is the role of DPP9 as a regulator of pyroptosis – an inflammatory type of cell death which occurs upon infectious triggers. The physiological roles of DPP8 are more obscure. As both proteases have been related with various cancers and infectious diseases, they are currently investigated as possible targets for drug development.
DPP8 and DPP9 are highly similar proteins with a protein sequence identity of approximately 60%. This significantly impedes the research of both proteases as methods for the individual study of DPP8 or DPP9 are limited. Currently, a major research objective is the development of inhibitors which are highly selective for DPP8 or DPP9 with no affinity towards the other. Earlier collaboration with the University of Antwerp research group of Medicinal Chemistry, resulted in the development of compound 42, which is, up to now, the most selective DPP9 inhibitor. However, the molecular mechanisms behind this selectivity remains unclear.
Here, we aimed to elucidate these mechanisms. First, we obtained structural insights of binding of compound 42 to both DPP8 and DPP9 by small-angle X-ray scattering experiments and cryogenic transmission electron microscopy. Employing these techniques, we showed that there are no major conformational changes in the protein structure of DPP8 and DPP9 upon inhibitor binding. Moreover, the binding pose of compound 42 is identical in DPP8 as in DPP9. However, DPP9 undergoes a small compaction of its overall protein fold upon inhibitor binding, which could not be observed for DPP8. Second, we used enzymatic assays in combination with DPP9 mutants to study the effects of various regions in the DPP8/9 protein structures on compound 42 inhibition. With this approach, we revealed that the Tyr280/Phe253 residue (DPP8/DPP9) and two regions in the putative entry/exit path of the inhibitor, are important for a selective binding of compound 42 to DPP9. In addition, we developed camelid single-domain antibodies targeting DPP8 and DPP9, as potential research tools.
In conclusion, this thesis contributes in further unentangling the physiological roles of DPP8 and DPP9 by providing molecular insights into these proteases and their inhibition, and by the development of research tools.