Research Yann Sterckx

Abstract

Fibroblast activation protein (FAP) is a protease biomarker that is selectively expressed on activated fibroblasts. Strongly FAP+ fibroblasts are found in >90% of all tumors, in fibrotic tissue and in tissue remodelling. At UAntwerp, UAMC1110 was earlier discovered: a very potent and selective FAP inhibitor. Radiolabeled derivatives of UAMC1110, called FAPIs, can be used as diagnostics or as therapeutics ('theranostics'). Nowadays, a steeply increasing number of FAPIs is being synthesised. However, there are no good predictions for the in vivo behaviour of novel FAPIs and the understanding of the interactions between FAP and its inhibitors is still limited. Within this FWO-SB application, we will bridge the gap between the present in vitro biochemical evaluation and the in vivo preclinical experiments. In addition, we will provide an expansion of our knowledge about FAP-FAPI interactions. This increase in insights will be realised by: 1) FAPI-PLA, a highly interdisciplinary in vitro platform to accelerate the preclinical development of FAP-targeted theranostics. FAPI-PLA will enable a thorough evaluation of new FAPIs through a combination of biophysical characterization and assessment of their behaviour in a cellular context such that they may eventually be used as theranostics. 2) the elucidation of FAP-FAPI interactions by nanoscale structure determination to expand our knowledge on the interactions.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Elvas Filipe
• Co-promoter: Sterckx Yann
• Co-promoter: Van Der Veken Pieter
• Fellow: de Groot Anke

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Malaria, caused by Plasmodium parasites, is one of the 'Big Three' infectious diseases. Each year more than 200 million cases are documented, including more than half a million deaths (>76% of the deceased are children under the age of five). P. vivax is the most widespread human-infective malaria parasite and severe cases are increasingly reported. Despite having a severe socio-economic impact on large parts of the world, the progress in battling P. vivax is slow. Problems are worsened due to low-efficacy vaccines, drug-resistant parasites and global disease (re-)emergence. This calls for active research into P. vivax biology. Invasion of a host reticulocyte (retic) by the merozoite (MRZ) is an essential event in the parasite's life cycle. Yet, our understanding of interactions at the MRZ-retic interface is limited. The PvTRAgs are MRZ surface antigens mediating retic binding. PvTRAg35.2 and PvTRAg38 are known to interact with basigin. Many aspects of these basigin binding PvTRAgs are yet to be investigated: i) the structural basis for basigin recognition is unknown, ii) the molecular determinants underlying the versatility displayed by PvTRAg-basigin interactions remain enigmatic, and iii) how these events relate to retic invasion is unclear. Given the knowledge gap in P. vivax biology and the importance of PvTRAgs in MRZ biology, tackling these issues is expected to generate many novel findings that may support P. vivax specific vaccine design efforts.

Researcher(s)

• Promoter: Sterckx Yann
• Fellow: De Cleene Witse

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Human African trypanosomiasis (HAT), caused by Trypanosoma brucei gambiense parasites, is a neglected tropical disease prevalent in Sub-Saharan Africa. While significant progress has been made in reducing HAT cases, enhanced diagnosis is crucial for the upcoming post-elimination phase. Current diagnostic methods rely on serological tests detecting antibodies (Abs) against variant surface glycoproteins (VSGs), specifically LiTat 1.3, LiTat 1.5 and (to a lesser extent) LiTat 1.6. To date, it is unclear why these VSGs are such robust diagnostic antigens for gambiense-HAT (gHAT). Their universal use is speculated to be due to their predominant character, meaning that they occur in nearly all gHAT cases during the early stages of infection and induce a strong and specific Ab response. However, substantial evidence for this hypothesis is currently lacking. The general lack of structures for Ab-VSG complexes hampers our understanding of immune recognition. This study aims to bridge this gap by elucidating the structural features and epitopes involved in Ab-VSG interactions, focusing on the predominant gHAT VSGs. Furthermore, it will investigate early VSG expression patterns and validate whether predominant VSGs indeed occur early after natural transmission. The outcomes of this project will not only contribute to a fundamental understanding of trypanosome immunobiology but also provide a molecular basis for improving gHAT diagnosis, supporting the global effort to eliminate HAT.

Researcher(s)

• Promoter: Sterckx Yann
• Fellow: Danel Niki

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Thymus specific serine protease (TSSP) is a serine protease first identified in the late nineties. It is highly expressed in the thymus and barely detectable in other organs. TSSP is the third member of the S28 group of serine proteases, along with prolyl carboxypeptidase (PRCP) and dipeptidyl peptidase II (DPPII). It is named a serine protease because of its predicted enzymatic activity due to the presence of three amino acids (serine, aspartate and histidine) at positions highly similar to the ones forming the catalytic site of PRCP. PRCP cleaves off C-terminal amino acids adjacent to a proline, while DPPII only cleaves short peptides after proline at the second position starting from the N-terminus, hence the name 'dipeptidyl'-peptidase. This difference in substrate specificity makes it hard to predict the cleavage specificity of the third member, TSSP. Why should TSSP be studied further? In the thymus, TSSP is highly expressed by the cortical epithelial cells (cTEC's) and at lower levels by thymic dendritic cells. Since the functional T-cell repertoire is shaped in the thymus by positive and negative selection through interactions with Major Histocompatibility Complex (MHC)-peptide complexes expressed by cTECs and bone marrow derived antigen-presenting cells, TSSP could possibly be involved in this process. Peptides for loading on MHC class II molecules are generated by sequential proteolysis of endosomal proteins. Furthermore, during T-cell maturation in the thymus, massive cell death occurs in the cortical region as thymocytes that recognize self-antigens have to be deleted. The molecular mechanisms underlying this 'thymocyte cleanup campaign' and the high expression of TSSP in this region remain outstanding questions in the field. On the long term, a better knowledge on the structure-function relationship of TSSP may contribute to a better insight in the T-cell selection processes in the thymus. It is hypothesized that a primary function of TSSP is to somehow limit central tolerance to increase the diversity of the functional CD4+ T cell repertoire. Clearly, further characterization of TSSP's enzymatic activity is an essential next step in the search for its function in the shaping of the immune repertoire. In this basic science project we want to start the biochemical study of this hitherto uncharacterized protein by 1) structure-guided recombinant production, purification and quality control of recombinant mouse TSSP (rmTSSP) and human TSSP (rhTSSP); 2) characterization of TSSP substrate specificity and comparison with DPPII and PRCP and 3) selection and development of antibodies and inhibitors as further tools to study TSSP biology. To increase the chance of obtaining valuable antibodies we will use two approaches: i) selection of camelid single domain antibodies (sdAbs) from a library and ii) generation of classical monoclonal antibodies (mAbs). As a first step in the development of potent and selective inhibitors, we will screen a protease inhibitor library of small molecules to select a lead compound that can be used as a starting point for follow-up research. It is astonishing that this thymus specific enzyme has not yet been studied in more detail. One reason may be that the recombinant production of well-folded active TSSP could not be achieved to date. The Laboratory of Medical Biochemistry (LMB) has a longstanding expertise in studying the other members of the serine protease family S28. Therefore, there is a momentum to build on recent AlphaFold 2-based structural knowledge and experimental expertise on related proteins to definitely unravel the catalytic activity of TSS.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Sterckx Yann
• Fellow: Van Meel Kim

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

(Bio)chemists think about molecules in terms of connectivity and spatial structure. These concepts match well with the actual structure of molecules on the nanoscale. Based on irradiation with a wavelength in the order of magnitude of the interatomic sizes (x-rays) in a periodically ordered structure (a crystal), from the diffraction pattern, the underlying structure can be calculated. Since the '80s this is a standard technique for experimentally visualizing molecules. The importance of it is impossible to overestimate – a majority of the 3D information about atoms and molecules, from molecules consisting of a few atoms to proteins and even complete cell organs like ribosomes, stems from x-ray diffraction measurements. The technique is of incredible importance both for the unambiguous characterization of newly synthesized small molecules, including their stereochemistry, as well as for macromolecules like proteins, and their complexes with pharmacologically active compounds. This allows to elucidate drug and disease mechanisms. This project concerns the purchase of a modern x-ray diffractometer, which will allow to obtain this information faster, with better quality, close to the research involved, and in-house. This will lead to a substantial acceleration within these research topics, to new cooperations both within and outside UAntwerp, and to the initiation of new research, by making this technique broadly available and easily accessible.

Researcher(s)

• Promoter: Vande Velde Christophe
• Co-promoter: Maes Bert
• Co-promoter: Meynen Vera
• Co-promoter: Sterckx Yann
• Co-promoter: Van Der Veken Pieter
• Co-promoter: Van Doorslaer Sabine

Research team(s)

• Intelligence in PRocesses, Advanced Catalysts and Solvents (iPRACS)

Project type(s)

• Research Project

Abstract

Visceral leishmaniasis is a major neglected lethal parasitic disease for which treatment options are scarce and toxicity, resistance and post-treatment relapse are common. No human vaccine is currently available and parasite quiescence is completely overlooked in the development of novel vaccination strategies. Our cutting-edge research has recently identified stem cells in the bone marrow as a sanctuary site where parasites can hide and survive drug treatment by transitioning to a quiescent state. Transcriptional profiling of quiescent and non-quiescent parasites provided differential genes, uniquely expressed during quiescence, that constitute attractive therapeutic vaccine antigen candidates. This project will provide unprecedented information about host-pathogen interactions and explore vaccination strategies to prevent relapse by: (i) obtaining essential data on antigenic presentation properties of Leishmania-infected stem cells, (ii) editing parasitic quiescence genes and selecting single domain antibodies (sdAbs) against quiescence gene products, and (iii) exploring immunity to quiescence genes during infection and following immunization. Taken together, it is expected that in-depth understanding of parasitic quiescence and corresponding antigenic/immunogenic properties will be revolutionary for the development of novel therapeutic vaccination strategies that can be incorporated with drug treatment to prevent relapse.

Researcher(s)

• Promoter: Caljon Guy
• Co-promoter: Sterckx Yann
• Fellow: Present Cassandra

Research team(s)

• Laboratory for Microbiology, Parasitology and Hygiene (LMPH)

Project type(s)

• Research Project

Abstract

Malaria, caused by Plasmodium parasites, is one of the 'Big Three' infectious diseases, together with HIV and TB. Each year more than 200 million cases of the disease are documented, including more than half a million deaths (>76% of the deceased are children under the age of five). Problems are worsened due to low-efficacy vaccines, drug-resistant parasites and the (re-)emergence of the disease around the globe. This clearly indicates that novel intervention strategies are still direly needed. Antibodies (Abs) are potent tools for parasite neutralisation. Besides conventional Abs, the natural immune repertoire of mammals contains so-called unconventional diversification strategies, which extend the coverage of antigen space. Interestingly, unconventional Abs appear to excel in neutralising highly sophisticated pathogens. Camelid single-domain Abs (sdAbs) are prime examples of such unconventional Ab fragments. Extensive knowledge on the camelid sdAb structure-function relationship enables the construction of highly diverse synthetic libraries that offer several advantages over immune libraries obtained through immunisation. This project aims to harness the potential of synthetic sdAb libraries with unconventional diversification strategies to tackle the malaria sporozoite through an interdisciplinary research approach combining molecular, structural, and parasitological methods.

Researcher(s)

• Promoter: Sterckx Yann
• Co-promoter: Caljon Guy
• Fellow: De Vocht Line

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Plasmodium vivax is the most widespread species causing malaria in humans, but the lack of a long-term culture system has limited knowledge about the biology of this parasite. A key step in the infection of P. vivax is the reticulocyte (young red blood cells) invasion process which involves several host receptor and parasite ligand interactions. Description of P. vivax invasion ligands entails relevant information for the development of vaccines, essential to design targeted control and prevention strategies. In vitro studies and transcriptomic profiles of P. vivax isolates highlighted the potential invasion role of some PvTRAg proteins. In addition, their high immunogenicity and conserved sequence among isolates makes them promising vaccine targets. As the function of the PvTRAgs remains undescribed, this project aims to characterize the involvement of five PvTRAg proteins during the process of erythrocyte invasion. We will carry out in vitro studies using recombinant PVTRAg proteins to evaluate their binding capacity to erythrocytes, and will create transgenic P. knowlesi lines to elucidate the role of the selected PvTRAgs and their P. knowlesi orthologs during invasion. Finally, the PvTRAg proteins that show strong indications of being invasion ligands, will be confirmed in ex vivo invasion assays using P. vivax isolates.

Researcher(s)

• Promoter: Sterckx Yann
• Fellow: Díaz Delgado Dalia

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Infectious disease research (including diagnostic, preventative and therapeutic development) has been a longstanding spearhead initiative of the University of Antwerp. This is driven by a vibrant research community, which is embedded in a larger "infectious disease ecosystem" in Flanders. A significant portion of these efforts is specifically devoted towards tackling protozoan parasites, a group of unicellular eukaryotes that affect the livelihoods of billions of people and their livestock around the world. Protozoan parasites cause some of the most daunting infectious diseases to have burdened humankind in past and present times (e.g., malaria, leishmaniasis, trypanosomiasis). These diseases are hallmarked by a significant mortality and a high morbidity, thereby severely impacting the quality of life and socio-economic status of those affected. Protozoan parasites are currently endemic in large parts of the world (over 100 countries ranging from the Americas to Southeast Asia) and pose a global risk due to human migration, climate change and an expanded distribution of the insect vectors that enable parasite transmission. Consequently, even currently unaffected areas (including the Western world) are confronted with disease (re-)emergence. Hence, the current burden and pandemic potential of protozoan parasites advocate the urgency and necessity to invest in tools that enable swift parasite detection and control. Some of the most potent and promising tools employed by humans in the battle against their pathogens are obtained from other animal species. A striking example is provided by the Belgian discovery of a peculiar antibody subset that naturally occurs in camelids (e.g., alpacas, llamas, camels, and dromedaries). In these antibodies, antigen recognition is mediated by a single domain, which is why it is often referred to as a "single-domain antibody" (sdAb). During the past decades it has been recognised that sdAbs possess many remarkable properties that render them highly suitable for discovery, application, and valorisation in life sciences (including diagnostics and therapeutics). These very same properties also make them unique and potent tools for pandemic preparedness and responsiveness. Literature and market analyses reveal that sdAbs remain largely under-utilised in the battle against protozoan parasites. Consequently, the application of sdAbs in the field of human and veterinary parasitology represents uncharted territory. This project aims to harness the highly complementary expertise at UAntwerp with regards to the generation and application of sdAbs in the field of parasitology to establish PREPARAS, a hybrid platform for the generation and identification of anti-parasite sdAbs via both immune and synthetic libraries. This will generate a fruitful synergy between research, application development, and valorisation given i) the veterinary expertise and strong research focus of the participating laboratories on protozoan parasite biology, ii) the unique opportunity of exploiting a hybrid platform for sdAb generation, and iii) the potential of sdAbs to address scientifical, medical and market-driven needs. Hence, PREPARAS will provide in-house access to unique research and development tools to remain at the forefront in the global battle against protozoan parasites of human and veterinary importance.

Researcher(s)

• Promoter: Sterckx Yann
• Co-promoter: Caljon Guy
• Co-promoter: Leroy Jo

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Fibroblast activation protein (FAP) is a cell surface marker of Cancer- Associated Fibroblasts (CAFs) in most sarcomas and in > 90% of carcinomas. Together with its negligible expression in most other tissues, this makes FAP a nearly-universal biomarker of tumors. During the past years, diagnostic and therapeutic targeting of FAP with so-called 'FAPIs' has attracted strong attention from nuclear medicine/oncology specialists. Noteworthy, all FAPIs owe their remarkable tumor homing potential to a potent and selective FAP-binding subunit: UAMC1110, reported by the applicants of this proposal. Because FAPIs require further optimization of tumor residence time, we aim to link multiple FAPIs or FAPI subunits to gold nanoparticles (AuNPs). In this way, we hope to obtain FAP-targeting AuNPs with unprecedented FAP affinity and tumor residence, due to the 'multivalency effect'. The nanoparticles will be investigated as cancer theranostics in a mouse model of colorectal cancer and as diagnostics in a lateral flow assay.

Researcher(s)

• Promoter: Van Der Veken Pieter
• Co-promoter: De Meester Ingrid
• Co-promoter: Elvas Filipe
• Co-promoter: Sterckx Yann
• Co-promoter: Stroobants Sigrid

Research team(s)

• Medicinal Chemistry (UAMC)

Project type(s)

• Research Project

Abstract

Inflammation is an immune response in which the cytosolic multiprotein complexes, inflammasomes, are crucial signaling platforms. Only recently, a master inflammasome regulator has been uncovered: the widely distributed intracellular serine protease dipeptidyl peptidase 9 (DPP9). More specifically, DPP9 is a binding partner and a negative regulator of two related Pathogen Recognition Receptors (PRRs), named 'NLRP1' and 'CARD8'. Precise insight into the mechanism is lacking as no selective DPP9 inhibitors have been reported to date. Interestingly, our preliminary data indicate that there are some differences in DPP9 (co)localization/complex formation with these PRRs among different human blood cell types. UAntwerp developed promising DPP9 inhibitors (Benramdane S, submitted) and PROTACs (heterobifunctional molecules that target a protein to degradation), creating a momentum to characterize and validate them for use in a cellular context. This PhD's overarching goal is to apply the two best DPP9 inhibitors to visualize [DPP9-CARD8] and [DPP9-NLRP1] interactions in situ in primary human blood cells and, at the molecular level, to determine the binding parameters of these interactions, both in the absence and presence of the best DPP9 inhibitors. To check whether effects are 'on-target', control experiments using PROTACs and a DPP9-/- cell line will be included. Understanding DPP9-inflammasome-related protein interactions is required to evaluate their potential as drug-targets.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Sterckx Yann
• Co-promoter: Van Der Veken Pieter
• Fellow: Mertens Kathleen

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Malaria, caused by Plasmodium parasites, is one of the 'Big Three' infectious diseases, together with HIV and TB. Each year more than 200 million cases of the disease are documented, including more than half a million deaths (>65% of the deceased are children under the age of five). P. falciparum and P. vivax are the most widespread and notorious members infective to humans. Although P. vivax induces a milder form of the disease, severe cases are increasingly reported. In addition, P. vivax has a much larger geographic range compared to P. falciparum; while falciparum malaria predominantly burdens Sub-Saharan Africa, vivax malaria affects the lives of millions across Latin America and South-East Asia. Despite having a severe socio-economic impact on large parts of the world, the progress in battling P. vivax is slow. Problems are worsened due to low-efficacy vaccines, drug-resistant parasites and the (re-)emergence of the disease around the globe. This calls for active research into the biology of the malaria parasite. Productive invasion of a host liver cell by a form of the parasite called the sporozoite (SPZ) represents an essential event in the parasite's life cycle. Infectious SPZs express several 6Cys proteins on their surface (P36, P52, B9, P38 and P12p) and P36, P52 and B9 have been shown to be essential for productive SPZ invasion. Many aspects of the structure-function relationship of SPZ 6Cys proteins are unknown: i) studies on P. vivax P36, P52, and B9 are very limited, ii) the existence of a P. vivax P36-P52-B9 complex remains to be validated, iii) the molecular determinants and affinities of interactions within this complex are yet to be revealed, and iv) the molecular basis for SR-BI receptor recognition is enigmatic.This research project will aim to shed light on these relevant questions through an interdisciplinary research approach combining biophysical, structural, and functional assays. Given the knowledge gap in P. vivax biology and the general importance of SPZ 6Cys proteins in SPZ biology, tackling the above-mentioned questions is expected to generate many novel, relevant findings that may be used in the battle against malaria.

Researcher(s)

• Promoter: Sterckx Yann
• Fellow: De Vocht Line

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Chemotherapy is a cornerstone in the battle against leishmaniasis, a neglected tropical disease caused by Leishmania parasites that affects millions worldwide. In addition, currently unaffected areas are confronted with the (re-)emergence of the disease. Unfortunately, an alarming number of reports are describing treatment failure with currently available drugs, which can be traced back to three main mechanisms employed by the parasite to cope with the exposure to chemotherapy: drug resistance, hiding in so-called "sanctuary sites" and parasite quiescence. Given that the current number of anti-leishmanial treatment options is limited and that those available are unsatisfactory, there is a dire need for the discovery of novel compounds, preferably with yet unexplored modes of action. In this quest, DNDI-6690 has been identified as a promising lead. While the molecular target of this compound has been identified, many aspects for the molecular basis of the anti-leishmanial activity of DNDI-6690 remain enigmatic. First, a biophysical and structural characterisation of the target – DNDI-6690 complex is still lacking. Second, the breadth of the compound's activity within the Leishmania genus has not been fully explored. Finally, the link between the action of DNDI-6690 and parasite quiescence remains to be investigated. Given the promising nature of DNDI-6690 and the dire need for novel tools to combat leishmaniasis, this warrants further investigation.

Researcher(s)

• Promoter: Sterckx Yann
• Co-promoter: Caljon Guy
• Fellow: Wuyts Ellen

Research team(s)

• Medical Biochemistry

Project website

Project type(s)

• Research Project

Abstract

Neglected tropical diseases (NTDs) comprise a wide variety of communicable diseases that are prevalent in (sub)tropical regions and affect more than 1 billion people worldwide. NTDs are hallmarked by a significant mortality and a high morbidity, thereby severely impacting the quality of life and socio-economic status of those affected. The WHO has listed 20 NTDs that should be tackled in the interest of global health and well-being. Three of these are caused by kinetoplastids, a group of flagellated, single-celled eukaryotic organisms comprising parasites of the Trypanosoma and Leishmania genera. Trypanosomes are the causative agents of animal and human trypanosomiasis (AT and HT, respectively) and are of the most cunning pathogens to have burdened humankind in past and present times. While anthroponotic HT (T. brucei gambiense) is perceived as a minor threat, zoonotic HT (T. b. rhodesiense) remains a worrisome health problem. Likewise, AT (T. b. brucei, T. congolense, T. vivax, T. evansi) still has a devastating socio-economic impact (annual losses of ~$5 billion). The battle against trypanosomes requires a concerted approach involving vaccination and drug treatment. However, the development of an effective vaccine against trypanosomes is thwarted by sophisticated immune-evasion strategies and the current drug treatment schemes are largely unsatisfactory. Hence, there is a dire need for alternative control strategies, which advocates the need for active research into trypanosome (immuno)biology. The life cycle of salivarian trypanosomes requires passages through two host organisms: the tsetse fly and mammals. As part of their obligate bipartite life cycle, these parasites have evolved to adapt, mediate immune evasion, and undergo developmental transitions within changing host environments. While trypanosomes are notorious for their ability to masterfully manipulate host-parasite interactions, many of the underlying molecular mechanisms remain poorly characterised. Trypanosomal receptor-like adenylate cyclases (TrypRACs) have been identified as important operators in these processes. The TrypRACs represent a large polymorphic family displaying a conserved architecture in which a single transmembrane helix separates an N-terminal extracellular receptor domain from a cytosolic enzymatic domain with cyclase activity. Specific TrypRACs are expressed in insect vector and mammalian host stages. In the mammalian host, it has been shown that the activation of the bloodstream-specific TrypRAC ESAG4 via mild acid stress results in massive cAMP production, thereby inhibiting TNF-α synthesis by host myeloid cells and contributing to innate immune evasion at the onset of infection. In the tsetse fly, several insect-specific TrypRACs coordinate so-called "social motility (SoMo)" of the parasite population, which is crucial for vector infection. SoMo is regulated by a cAMP signaling complex containing specific TrypRAC isoforms (especially the TrypRAC ACP5 is essential) and other trypanosome factors. While our preliminary data indicate that the TrypRAC ectodomain is pivotal for the control of cyclase activity, the molecular mechanisms that underlie ectodomain-mediated TrypRAC activation are poorly understood. Especially the effect of putative natural ligands on the TrypRAC structure-function relationship remains unknown. Therefore, this project aims to study the molecular aspects of TrypRAC ectodomain-ligand interactions using a combination of functional, biophysical, and structural methods (the research stay at UAntwerp will be critical to support the structural work). We propose that the TrypRAC extracellular sensor domains are promising targets for the development of novel anti-trypanosomal therapies and that a thorough characterisation of their structure-function relationship will yield highly valuable insights.

Researcher(s)

• Promoter: Sterckx Yann
• Fellow: Oliveira Alves Desirée

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Host-parasite interactions involved in the process of P. vivax reticulocyte invasion are poorly understood due to the lack of long-term culture methods (in contrast to P. falciparum). Surface antigens of the P. vivax tryptophan-rich antigen (PvTRAg) family are expressed in the early ring or the late schizont stages and have been shown to bind erythrocytes in vitro. Each PvTRAg appears to recognize at least two different host receptors, among them basigin and Band3. By using ex vivo invasion assays coupled to transcriptomic analysis of P. vivax isolates, we have recently demonstrated that Band3 is a P. vivax invasion receptor that binds to PvTRAg38 (and potentially other PvTRAgs). However, the molecular determinants underlying host receptor recognition by PvTRAgs, the functional redundancy in these interactions, and how this relates to subsequent reticulocyte invasion by P. vivax remains unknown. With this "RecTRAgs" jPPP we aim to establish the molecular basis of PvTRAg38-basigin interaction and its role in invasion. By using transgenic P. knowlesi as a model for P. vivax and producing recombinant PvTRAg38 and basigin, we will thoroughly characterize the interaction of Pv/PkTRAg38 and human basigin and its function during invasion. Results of "RecTRAgs" will guide future projects on broader PvTRAg-receptor interactions and its biological function.

Researcher(s)

• Promoter: Sterckx Yann

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Various proteases play an important role in the tumor microenvironment (TME). Hence, tackling tumors by targeting these TME proteases is a very promising approach in the fight against cancer. FAPIs, highly potent and selective probes based on UAMC1110, an inhibitor developed at the UAntwerp, are currently evaluated in clinical studies. In contrast, the targeting of other highly relevant TME proteases is lagging behind. Granzyme B (GRZB) is the most abundant protease present in the granules of cytotoxic immune cells present in the TME and plays a role in the targeted tumor cell destruction. Despite decades of research, many aspects of GRZB immunobiology remain enigmatic. It is currently unknown which percentage of GRZB is active in the TME. To study whether imaging or measuring active GRZB levels has advantages over visualizing total GRZB, there is a need for selective and high affinity GRZB probes. Given the potential of GRZB in cancer diagnosis and treatment, this postdoc challenge aims to reinvigorate the quest for the generation of highly selective GRZB inhibitors starting from a literature-based lead compound. The postdoc will be challenged to determine the high-resolution structure of this inhibitor – GRZB complex to fuel rational ligand design. The labs participating in this call are involved in the recently funded OncoProTools (Protease-guided tumor targeting tools to revolutionize cancer diagnostics and treatment) HE-MSCA-Doctoral Network (granted upon first submission, UAntwerp as the lead applicant). UAntwerp will host two PhD students (PhD1 and PhD2) from January 2023 onwards. Since this international project will be the start of a new GRZB-research line within the 'Tumor Micro-environment Proteases' theme, support by a postdoc is highly desirable. The project will be supported by docking studies for in silico design of new inhibitors (UAMC, Hans De Winter). We will offer in-house access to granzyme activity assays, recombinant protein production and purification infrastructure, protein-ligand interaction assays and a lab fully equipped for structural biology (LMB, Y. Sterckx). The Postdoc candidate is expected to bring own experimental expertise with protein expression and structural biology into the GRZB theme and he/she will benefit from a dynamic international network of academic and industrial partners in the field of oncology.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: De Winter Hans
• Co-promoter: Elvas Filipe
• Co-promoter: Sterckx Yann
• Co-promoter: Van Der Veken Pieter
• Fellow: Pimenta Fernandes Andreia

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

The current application envisages extending the infectious disease and inflammation research and drug discovery platforms at the University of Antwerp to accommodate highly flexible and versatile live cell imaging and biochemical read-outs with a possibility to upgrade from median to high throughput. The apparatus will be embedded in a high-end immunoprofiling platform and BSL-2 environment at the Laboratory of Microbiology, Parasitology and Hygiene (LMPH) with biosafety approvals for basic research on infection with microbial pathogens. The TECAN SPARK Cyto 600 is a highly versatile multimodal plate reader that enables cellular and in situ molecular assays in controlled O2/CO2, humidity cassette and temperature regulation environments with real-time absorbance, fluorescence and luminescence measurements. Three different optical measurement options exist by using filters, monochromators or fusion optics which eliminates the compromise between sensitivity and flexibility. The unique lid-lifting function enables substrate addition or immune cell priming through the included 2-channel injector. SPARK Cyto 600 is equipped with 2×, 4× and 10× objective lenses and a CMOS camera to enable live cell imaging. In addition to bright field imaging, fluorescence imaging is possible in 4 optical channels with capability of Time-Resolved Fluorescence (TRF) and Fluorescence Resonance Energy Transfer (FRET). An additional asset is the compatibility with bead-based proximity assays using Alpha Technology with optimized integrated filters and laser-based excitation. While all known competitors limit live cell imaging systems to bright field and fluorescence measurements, the SPARK Cyto 600 also allows for real-time detection of glow and flash luminescence signals and Bioluminescence Resonance Energy Transfer (BRET) applications to enable sensitive real-time follow-up of protein-protein interactions in cells. Given the high versatility and pressing need for such equipment for infection and inflammatory disease research, this unique apparatus will allow real-time imaging of cellular as well as molecular events in controlled conditions. This new infrastructure will therefore boost research of many research groups at the University of Antwerp and will contribute to fundamental insights at the cellular and molecular level as well as to the development of novel therapeutics and diagnostics.

Researcher(s)

• Promoter: Caljon Guy
• Co-promoter: Augustyns Koen
• Co-promoter: Sterckx Yann
• Co-promoter: Vanden Berghe Wim
• Co-promoter: Wullaert Andy

Research team(s)

• Laboratory for Microbiology, Parasitology and Hygiene (LMPH)

Project type(s)

• Research Project

Abstract

This proposal is situated in the field of drug discovery and has the overall objective of further validating and implementing a proof-of-concept high-throughput screening (HTS) assay we have developed in-house. The acronym EVZYM refers to our findings that extracellular vesicles (EVs) represent a novel source for native, active target enzymes against which small-molecule compounds can be screened for their potential to inhibit target enzyme activity. This poses a significant advantage in the quest for novel drug candidates as the availability of active enzyme preparations is essential for successful HTS assay outcome; i.e., large compound libraries are screened for their inhibitory potential and the identified "hits" form the starting point for further optimization. A first goal of the project encompasses upscaling EV isolation, and determining optimal storage conditions that guarantee EV stability such to maximally enhance their application in industrial settings. The second goal consists of further validating and implementing our in-house EVZYM-based HTS assay for a target protease which is naturally present and enriched in EVs. This target protease is TMPRSS2, a human serine-type protease present on the cell surface of which the activity enhances corona- and influenzavirus infections, thereby making it an attractive target for the development of anti-viral chemotherapeutics. Within this second objective, we also aim to obtain a recombinant version of TMPRSS2, which represents an added value because i) this represents an alternative source of target protease for HTS assay validation and ii) no documented highquality preparations of recombinant TMPRSS2 are currently available on the market.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Sterckx Yann

Research team(s)

• Medical Biochemistry

Project website

Project type(s)

• Research Project

Abstract

Cryo-electron microscopy (cryo-EM) has evolved tremendously over the last five years, thereby becoming a promising method to gain high-resolution structural information on proteins with a relevance in human (patho)physiology (e.g., cancer, host-pathogen interactions, and neuropathologies). This rapid evolution has sparked the interest of pharmaceutical companies in cryo-EM, since obtaining detailed structural information on proteins yields better insights into their function, which can be used to develop novel and/or better pharmaceuticals. However, as a result of its success, several inefficiencies within the cryo-EM workflow have emerged, especially related to sample preparation. Novel technologies have been proposed to optimize these, but these new techniques (i) often address only a single step within the overall workflow, (ii) are incompatible with other novel protocol/procedures or (iii) are difficult to implement by non-expert users. In a previous PoC study we developed a novel type of affinity grid that can be used for on-grid protein purification. Furthermore, market interviews have revealed that the introduction of this technology is best achieved through a service for protein structure determination (including a workflow from protein sample to protein structure) rather than simply providing the technology as such. The aim of this follow-up PoC is to validate the technology by resolving different protein structures using this grid technology and meanwhile establishing a service pipeline for high-resolution protein structure determination. This will illustrate the value of the grids towards potential customers (Pharma, Biotech) and investors.

Researcher(s)

• Promoter: Timmermans Jean-Pierre
• Co-promoter: Sterckx Yann

Research team(s)

• Laboratory of cell biology and histology

Project type(s)

• Research Project

Abstract

The coronavirus SARS-CoV-2, causative of COVID-19, currently causes an unprecedented pandemic. Human SARS-CoV-2 infections are enabled by two events that occur at the host-virus interface. First, viral attachment to host cells is mediated by an interaction between the SARS-CoV-2 'spike' protein and its host receptor angiotensin converting enzyme 2 (ACE2).Next, the virus is "primed" for host cell entry through proteolytic cleavage of SARS-CoV-2-spike protein by other surface-exposed host proteases such as TMPRSS2. Inhibition of TMPRSS2-enabled "priming" negatively impacts SARS-CoV-2 infectivity. Unfortunately, the currently available TMPRSS2 inhibitors (such as camostat) are nonspecific. For the development of inhibitors with an increased specificity and high potency, a better knowledge of the characteristics of the protease are urgently needed. This project aims to lay the indispensable foundation for the rational design of specific TMPRSS2 inhibitors in the battle against SARS-CoV-2 and COVID-19. This will be realized in two work packages (WPs) and 6 interrelated and measurable deliverables (D). The project will focus on following research questions: (1) What is the extended substrate specificity of TMPRSS2? and (2) What is the correlation between TMPRSS2 inhibition and neutralization of SARSCoV- 2 infectivity in vitro? The deliverables of the project include the availability of active recombinant human TMPRSS2, methods to quantify its activity, data on the extended substrate specificity and on the inhibitory potency of a set of 100 compounds from the library of protease inhibitors of the UAntwerp research group on Medicinal Chemistry (UAMC). The correlation of the inhibitory potency of these compounds with their effect on in vitro infectivity of SARS-CoV-2, together with data on extended TMPRSS2 substrate specificity, are an indispensable prerequisite for optimal planning of larger collaborative projects on host protease targeting as a therapeutic approach in the fight against COVID-19. Moreover, given the recently acquired expertise in structural biology in our lab, this project will lay a solid foundation for future structural studies of hit compounds in complex with TMPRSS2, which can in turn fuel rational drug design to generate more potent and specific compounds.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Bracke An
• Co-promoter: Sterckx Yann

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Despite the presence of a sound expertise, structural biology is currently not well-embedded within the University of Antwerp. Hence, UAntwerp researchers are dependent on collaborations with external partners to be productive and competitive in this field. Structural biology at UAntwerp will only successfully come of age by investing in the acquisition of basic infrastructure that will adequately support the existing expertise. In this project proposal, funding is requested for the purchase of the Mosquito Xtal3, a state-of-the-art crystallization robot that has become an indispensable workhorse in any structural biology laboratory. The Mosquito Xtal3 allows fast, robust and high-throughput crystallization of biological macromolecules, which is a basic requirement for structure determination through macromolecular X-ray crystallography.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Cappoen Davie
• Co-promoter: De Winter Hans
• Co-promoter: Sterckx Yann
• Co-promoter: Van Ostade Xaveer

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

There is growing conviction that certain parasites successfully initiate infection in the skin by specifically targeting and co-opting immune cells present or recruited to the dermis following inoculation by their arthropod vector. One such pathogen is the protozoan parasite Trypanosoma brucei which causes sleeping sickness and is inoculated by the tsetse fly. These inoculated parasites are peculiarly infective despite the rapid recruitment of activated innate immune cells at the inoculation site, revealing that the parasite has evolved powerful mechanisms to either evade or overcome the host's vigorous innate immune response. Extracellular vesicles (EV) are believed to play a major role in this parasite-host interplay. Novel cutting-edge technologies are required to gain fundamental insights in the role of parasitic EV proteins because current gene editing and silencing methodologies happen to be inappropriate. Using Nanobodies, this project will develop a strategy to selectively deplete proteins from the EV cargo to allow detailed scrutiny of the molecular players involved in the parasite-immune cell interaction. The impact of EV proteins on kinase activity fingerprints of innate immune cells and their role in a vector-based parasite transmission cycle will be assessed. Collectively, this project will significantly progress our understanding of fundamental aspects of the trypanosome-host interaction.

Researcher(s)

• Promoter: Caljon Guy
• Co-promoter: Sterckx Yann
• Fellow: Claes Mathieu

Research team(s)

• Laboratory for Microbiology, Parasitology and Hygiene (LMPH)

Project type(s)

• Research Project

Abstract

There is compelling evidence that the enzyme dipeptidyl peptidase (DPP) 9 is involved in inflammation and cell death in macrophages. However, large gaps in our understanding of the exact underlying mechanisms remain. Research has mainly been limited to macrophage cell lines and murine primary macrophages. Therefore, our first objective is to study the effect of the DPP8/9 inhibitor 1G244, currently the most selective inhibitor available, on the production and secretion of cytokines and chemokines by human peripheral blood mononuclear cells, monocyte-derived macrophages, M1, M2 and M4 macrophages. The effect on cell viability will also be evaluated in these primary cells. Our second objective includes the identification of DPP9 interaction partners in the monocytic cell line THP-1 and human primary macrophages. Pull-down experiments using recombinant human DPP9 as a bait, followed by LC-MS/MS identification, as well as proximity ligation assays will be applied. We foresee to identify at least one additional interaction partner apart from the FIIND domain in NLRP1/CARD8. The third objective is to characterize the interaction between DPP9 and the bindings partner(s) identified in objective 2 at the molecular level, using isothermal titration calorimetry and grating-coupled interferometry. After the initial characterization of the interactions, we will use anti-DPP9 antibodies with known epitopes in order to identify the regions in DPP9 that are involved in the interaction.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Sterckx Yann
• Fellow: De Loose Joni

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Malaria is one of the 'Big Three' infectious diseases, together with HIV and tuberculosis. According to the World Health Organisation, malaria is endemic in 104 countries thereby endangering the health and lives of 3.4 billion people. Each year around 200 million cases of the disease are documented, including more than half a million deaths. More than 70% of the deceased are children under the age of five. The etiological agents of malaria are parasites from the Plasmodium genus, of which P. falciparum is the most virulent. Malaria parasites are transmitted by mosquitoes, which inject the parasites into the human body during a blood meal. This initiates the infection, which is characterized by two stages. The first stage (known as liver stage malaria) is caused by a form of the parasite called the sporozoite and is typically asymptomatic. The sporozoite infects the liver and develops into the next form of the parasite called the merozoite. This marks the start of the second stage of the malaria known as the blood stage. This phase, during which merozoites infect red blood cells, causes the infamous malaria pathology. Sporozoites are ideal targets for anti-malarial therapies as their elimination from the human host would prevent the onset of disease. Therefore, the sporozoite surface proteins are interesting candidates for the development of novel anti-malarial drugs and vaccine strategies. The presented research project aims at unraveling the mechanistic principles behind several processes that are crucial in the establishment of liver stage malaria. The first is the invasion of hepatocytes by the parasite. While it is known that the parasite's main surface antigen, the circumsporozoite protein (CSP), plays a pivotal part in successful hepatocyte invasion, the structural and functional aspects of this event remain unchartered territory. A thorough structural and biophysical study of the molecular aspects of CSP-mediated hepatocyte invasion will provide relevant insights into the biology of the malaria parasite. Once the parasite has invaded a hepatocyte, it forms a vacuole from within which it exports CSP to the host cell cytoplasm. There, CSP competes with NFkB for binding with the importin proteins in order to dampen NFkB-driven inflammatory responses. This increases the odds of parasite survival inside the infected hepatocyte and, hence, ensures continuation of the life cycle. Although it is known that CSP and importin proteins interact, the structural and biophysical aspects of this encounter have not yet been investigated. Obtaining a detailed picture of this interaction will allow a better understanding of immune evasion strategies adopted by the malaria parasite during the liver stage of the infection. Finally, the fundamental mechanism of CSP export from the parasite to the host hepatocyte cytoplasm will also be investigated. As investigating sporozoite antigens has produced significant scientific breakthroughs in the battle against malaria, it is anticipated that tackling the above-mentioned issues will not only yield insights into the parasite's immunobiology, but also generate a molecular basis to contribute to the design of novel anti-malarial therapies.

Researcher(s)

• Promoter: Sterckx Yann
• Fellow: Geens Rob

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Trypanosoma evansi is a widely spread parasite that causes a debilitating disease called animal trypanosomosis in all types of ungulates (cattle, buffaloes, horses, pigs and deer). Animal trypanosomosis is characterised by weight loss, drastic reductions of draft power, diminished meat and milk production, and, often, death of the infected animals. This severely challenges rearing livestock in the affected areas and heavily weighs on their socio-economic development. The presented research project aims at contributing to the development of novel tools for T. evansi detection and control. First, a new DNA-based assay for the diagnosis of active T. evansi infections has been successfully developed. Second, the use of the antigen-binding fragments of camelid heavy-chain only antibodies (so-called Nanobodies) has allowed the identification of the glycolytic enzyme T. evansi enolase (TevENO) as a potential novel specific biomarker for infection. In addition, because of the central importance of glycolysis for trypanosome survival within the host, TevENO might also have a therapeutic value. Nanobodies will again be employed as research tools to facilitate the discovery of novel diagnostic and therapeutic tools to achieve parasite detection and control by targeting TevENO. Given the heavy socio-economic burden imposed by T. evansi in large regions of the world, it is anticipated that the proposed work will contribute significantly to the battle against animal trypanosomosis caused by this parasite.

Researcher(s)

• Promoter: Sterckx Yann
• Fellow: Li Zeng

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project