Projects

Abstract

Antibodies (Abs) have a proven track record in biotechnology and -medicine. Most applications are based on conventional Abs (mostly IgGs), which have constituted a highly profitable market for decades. However, despite their track record, conventional Abs have their drawbacks and are ill-suited for certain applications. These shortcomings can usually be overcome by unconventional Abs found in other mammals. A prime example is provided by the Belgian discovery of a peculiar Ab subset that naturally occurs in camelids (e.g., camels, dromedaries, and llamas). In these Abs, antigen recognition is mediated by a single domain, which is why this domain is often referred to as a "single-domain antibody" (sdAb aka nanobody®). Camelid sdAbs possess unique features that are not usually found in conventional Abs: a small size (~15 kDa), an increased solubility, robust folding properties, a high intrinsic stability, poor immunogenicity, and the relative ease to tailor them (modifications according to a "plug-and-play" principle). These remarkable properties render them highly suitable for discovery, application, and valorisation in life sciences (including diagnostics and therapeutics). Importantly, the number of sdAbs in clinical trials and approved by the relevant regulatory agencies is on the rise (sdAbs are catching up with conventional Abs): in the past five years, four sdAbs have been approved for clinical use, ~50 others are currently in clinical trials, and many patents have been submitted/granted around the world. sdAbs are readily obtained through camelid immunisation or in silico designed synthetic sdAb libraries that have been shown to perform equally well. Immune and synthetic sdAb libraries each have their strengths and drawbacks and therefore complement each other. In most cases, interested parties have access to either immune or synthetic libraries but very rarely to both. Clearly, access to both library types through a hybrid platform will create a powerful synergy that can fuel discovery, innovation, and valorisation. The unique selling proposition of this project is the establishment of HyCAbs, an in-house hybrid sdAb platform based on the combined strengths of immune and synthetic libraries that can be employed to swiftly identify sdAbs against a myriad of target antigens. HyCAbs represents a continuation of the previously awarded PREPARAS project (Antigoon ID 49344). With this IOF PoC CREATE proposal, we aspire to consolidate and open this initiative up to i) UAntwerp researchers active in other life science domains and ii) interested external parties (both academic and industrial). In addition, we aim to unleash the potential of machine learning on deep sequencing data obtained from camelids to design and construct next-generation synthetic sdAb libraries by marrying our in-house sdAb expertise with the know-how of the BIOMINA core facility. The hybrid nature of HyCAbs is unique. One of its features would be to offer the interested party full flexibility in acquiring sdAbs through camelid immunisation, screening against synthetic libraries, or both. This flexibility enables the simultaneous consideration of sample amounts, time from antigen provision to binder identification, and budgetary constraints. For UAntwerp researchers, HyCAbs offers relatively cheap sdAb access with in-house IP from the start, which will add value for the university. For external parties, service agreements will be negotiated. Hence, we expect HyCAbs to provide various valorisation routes. HyCAbs presents a unique opportunity to establish a robust sdAb platform that enables discovery, innovation, and valorisation in its current form and supports low risk expansion and implementation of innovative elements in the field of sdAb technology in the future.

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 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

Granzyme B (GzmB) is a serine protease involved in the targeted destruction of tumor cells and is stored in granules of cytotoxic T (CD8+) and natural killer (NK) cells. After perforin-mediated delivery to the tumor cell cytoplasm, GzmB is responsible for initiating caspase-mediated apoptosis. As such, GzmB is a strong candidate for monitoring tumor cell-directed killing, which renders the cytotoxic activity of CD8+ and NK cells even promising for revolutionizing cell-based anti-cancer therapies. Detecting both the presence and activity of GzmB in the tumor microenvironment (TME) can be achieved by the use of enzyme-specific activity-based probes (ABPs). However, only two such probes have been developed to date and their selectivity has not been validated. In addition, details on the structural basis of GzmB inhibition by small molecules are scarce, and this crucial to accelerate the rational design of novel inhibitors and ABPs. Thus, we aim to obtain crystals of GzmB in complex with a reference inhibitor and to determine the high resolution structure of the complex for rational drug design, and consequently to generate and characterize a library of novel GzmB inhibitors. Our findings may deliver a moiety for the future development of a novel fluorescent ABP for detection of active GzmB in the microenvironment of different solid tumors.

Researcher(s)

• Promoter: Pimenta Fernandes Andreia

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 (>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

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

Preliminary evidence shows that the enzyme dipeptidyl peptidase 9 (DPP9), which is present in macrophages, plays a role in controlling the inflammatory response and associated cell death. Inflammation is a normal response of tissues against infections, chemical insults and physical injuries. However, in some cases, it becomes chronic and causes harm. A better understanding of the inflammatory process provides new therapeutic opportunities. In this project, we will focus on the role of the intracellular protease DPP9 in macrophages derived from human white blood cells. The crystal structure of DPP9 was recently solved, and can now be used to design DPP9 inhibitors. We will develop selective and potent DPP9 inhibitors and substrates. We will also engineer the human THP-1 cell line to eliminate DPP9. Using the DPP9 inhibitors and engineered cell lines, we will determine under which conditions DPP9 plays a key role in macrophage function. Finally, the molecular binding partners of DPP9 will be identified. We will look for natural substrates as well as non-substrate interaction partners of DPP9 in macrophages. The interactions will be characterized at the molecular level. Our fundamental biochemical findings and novel chemical tools will significantly advance the development of future therapies for diseases where DPP9 plays a role.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Van Der Veken Pieter

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

This project represents a research contract awarded by the University of Antwerp. The supervisor provides the Antwerp University research mentioned in the title of the project under the conditions stipulated by the university.

Researcher(s)

• Promoter: De Meester Ingrid
• Promoter: Hendriks Dirk

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Fibroblast activation protein (FAP) is a protease biomarker that is selectively expressed on activated fibroblasts. Strongly FAP+ fibroblast are found in >90 of all tumors, in fibrotic tissue and in tissue remodeling. Researchers at UAntwerp earlier discovered UAMC1110: today the most potent and selective FAP inhibitor available. Radiolabeled derivatives of UAMC1110, called FAPIs, can be used as diagnostics or as therapeutics ('theranostics'). Nowadays, a steeply increasing number of FAPIs is being synthesized. This research proposal aims to bridge the current in vitro gap between FAPI design, synthesis and initial biochemical characterization on the one hand and the preclinical in vivo evaluation on the other. This will be realized by the development of FAPi-PLA, a test platform comprising: (1) a validated method for structure elucidation of FAP-FAPI complexes, (2) highly sensitive biosensor-based FAP-FAPI-binding assays for kinetic analyses of FAP-FAPI interactions and (3) a relevant cell-based assay to predict the FAPI-target residence time in a biological context. The project aims to obtain data for three existing FAPIs as reference compounds and at least two new FAPIs that are in the pipeline. To accelerate the development of FAPIs, there is an urgent need for such an efficient centralized platform. Within this project, we will develop the technology needed to create 'FAPi-PLA', a complete and modular in vitro FAPI test platform to accelerate FAPI development.

Researcher(s)

• Promoter: De Meester Ingrid
• Fellow: de Groot Anke

Research team(s)

• Medical Biochemistry

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

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

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 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

Immunotherapy shows great potential in the treatment of cancer. Unfortunately, primary resistance is frequently occurring, hence the urgent need for biomarkers. Fibroblast activation protein (FAP), an enzyme that is selectively expressed in cancer-associated fibroblasts in 90% of all human epithelial tumors, may become a promising predictive biomarker for immunotherapy resistance. However, to date, there are no validated, specific tools to determine FAP expression and/or activity within the tumor microenvironment (TME). Therefore, the first goal of this project, is the development of specific tools and accurate methods to detect FAP within the TME. We will develop 2 fluorescent FAP-assays, one based on a fluorescently labelled inhibitor and the other on a fluorescently labelled anti-FAP antibody. The third assay is based on a FAP-specific substrate for histochemistry that allows in situ amplification of the signal. Secondly, early detection of metastasis remains a major hurdle in diagnosis and there is an urgent need to explore new biomarkers of early metastasis. Recently, it is suggested that circulating FAP +cCAFs within the blood of metastatic patients could be interesting for that purpose. Nonetheless, no specific, accurate tools and techniques are available for identification and enrichment of FAP +cCAFs. Consequently, the second goal of this project is to develop new tools and methods to identify and enrich FAP+cCAFs in the peripheral blood of metastatic cancer patients

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Van Der Veken Pieter
• Fellow: Van Rymenant Yentl

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

Immunotherapy is revolutionizing the clinical management of multiple cancer types including DNA mismatch repair-deficient tumors (Galluzi et al., 2018). However, even in this preselected group of patients, good and complete response are achieved in only one fifth of the patients (Le et al., 2016; Le et al., 2017). Thus, there are still enormous opportunities for improvement. The presence of an immunosuppressive tumor environment may be key to further optimization of immunotherapy (Jiang et al., 2016). Extracellular matrix accumulation and rigidity, and neo-expression of matrix proteins may be involved in T cell exclusion from the proximity of cancer cells or impaired T cell functionality. Cancer-associated fibroblasts (CAF) create an imbalanced biomechanical force in the tumor by the deposition of extracellular matrix, and by their contractility and proteolytic activity. CAF are abundant in mismatch repair-deficient tumors and their presence is associated with poor disease outcome (Kalluri 2016). Clinical targeting of CAF has been challenging due to its heterogeneous nature. We propose to target CAF making use of commonly expressed fibroblast growth factor receptor (FGFR) and platelet-derived growth factor receptor (PDGFR) or protease activity by fibroblast-activation protein (FAP). The advantage of FGFR/PDGFR inhibitors and FAP inhibitors is their complementary targeting of CAF biology either growth/differentiation vs proteolysis. Thus, we will evaluate whether impeding immune cell infiltration/functionality by CAF could explain non-responsiveness to immune checkpoint inhibitors in patients with DNA mismatch repair-deficient tumors. To test this hypothesis we will make use of three work packages: Workpackage 1: Human DNA mismatch repair-deficient tumors will be analyzed for the spatial localization and organization of collagen fibers, CAF and T-cells and data will be correlated with clinicopathological parameters. Workpackage 2: Patient-derived CAF will be treated with FGFR/PDGFR inhibitors or FAP inhibitors and outcomes such as (neo)matrix deposition, collagen rearrangement, and contractility will be evaluated and the consequent functional impact on T-cell chemotaxis, proliferation and differentiation will be tested. Workpackage 3: Syngeneic mouse tumor models will be used to evaluate if CAF targeting may improve efficacy of immune checkpoint inhibitors (such as anti-PD-1). We anticipate that CAF targeting combined with immunotherapy will synergistically induce and sustain the anti-tumor immune response, resulting in durable tumor regression in a larger population. Encouraging data in the preclinical study will enable fast translation into a clinical phase I/II trial.

Researcher(s)

• Promoter: De Meester Ingrid

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

(This research project aims to acquire dedicated knowledge on the protein 'Fibroblast activation protein alpha', further called 'FAP', in the context of personalized immune therapy. We will investigate whether FAP can be a biomarker for resistance to immunotherapy in urothelial carcinomas. In addition, we explore the presence of FAP in urine (as a non-invasive biological biomatrix) to monitor or predict the response to therapy for urothelial carcinomas.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Van Der Veken Pieter

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

Abstract

Analysis of off-target effects of experimental compounds following the agreement between the Antwerp University and the university of Helsinki. Further details are confidential------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Researcher(s)

• Promoter: De Meester Ingrid

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Thrombolysis with tissue plasminogen activator remains the only approved pharmacological treatment for acute ischemic stroke, AIS. Besides the narrow therapeutic time window, its use is limited by its efficacy: in up to 50% of the treated patients, timely recanalization is not achieved. Moreover, administration involves serious side effects such as intracranial hemorrhage and neurotoxicity. Consequently, the search for new agents for improvement of AIS treatment is urgently needed. Research has demonstrated that the enzyme carboxypeptidase U (CPU, TAFIa) is an important player in thrombus lysis. After activation from its precursor proCPU, the released CPU is able to potently attenuate fibrinolysis. Consequently, inhibition of CPU activity is a novel approach to enhance fibrinolysis. We want to explore the involvement of this enzyme in AIS in more detail. The usefulness of CPU as a diagnostic marker to discriminate ischemic from hemorrhagic stroke and the relationship of CPU with clinical outcome and thrombolytic treatment efficacy will be investigated. We plan to optimize the Thrombodynamics assay in order to assess the effect of CPU-inhibition on clot lysis during thrombolysis. Furthermore, in a preclinical setting, we will evaluate the effect of CPU inhibition in an experimental stroke model in rats. This research will provide essential information on the role of the CPU system and the usefulness of CPU inhibitors as potentially efficient and safer treatment of AIS.

Researcher(s)

• Promoter: Hendriks Dirk
• Co-promoter: Lambeir Anne-Marie
• Fellow: Mertens Joachim

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Physical and functional interactions between biomolecules play pivotal roles in all aspects of human health and disease. Gaining a greater understanding of these biomolecular interactions will further expand our understanding of diseases such as cancer, metabolic diseases and neurodegeneration. At UAntwerp, 7 research groups have joined forces to obtain the absolutely necessary equipment to measure these interactions with a Biomolecular Interactions Platform (BIP). This will allow to detect interactions and precisely determine binding affinities between any kind of molecule, from ions and small molecules to high-molecular weight and multi-protein complexes. The BIP will also allow to identify collateral off- targets, crucial in the drug discovery field. Access to a BIP will strongly support ongoing research projects and bring research within the BIP-consortium to a higher level. Since biomolecular interactions are highly influenced by the methodology, it is recommended to measure the interaction by several, independent techniques and continue with the most appropriate one. For this reason, the consortium aims at installing a BIP, consisting of several complementary instruments that each measure biomolecular interactions based on different physical principles. They wish to expand the existing Isothermal Titration Calorimetry with two complementary state-of-the- art techniques: MicroScale Thermophoresis and Grating- Coupled waveguide Interferometry.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Delputte Peter
• Co-promoter: De Wael Karolien
• Co-promoter: Dewilde Sylvia
• Co-promoter: De Winter Hans
• Co-promoter: Kooy Frank
• Co-promoter: Lambeir Anne-Marie
• Co-promoter: Maudsley Stuart
• Co-promoter: Van Der Veken Pieter
• Co-promoter: Van Ostade Xaveer

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

The complex mechanism involved in the pathogenesis of these metabolic disorders remains poorly understood. Recent findings indicate that the enzyme prolyl carboxypeptidase (PRCP) plays a role in body weight control and glucose homeostasis by inactivating melanocyt-stimulating hormone MSH, a neuropeptide which causes a loss of appetite. We aim to investigate whether 'peripheral' (outside the central nervous system) actions of PRCP also contribute to this function. Our preliminary experiments have identified apelin, which also regulates feeding behaviour and glucose metabolism, as a novel PRCP substrate. Firstly, we will characterize this cleavage in vitro and investigate whether truncation by PRCP alters the function of apelin and other peptide substrates. Secondly, the role of PRCP on in vivo cleavage of peripheral apelin will be investigated. The effect of pharmacological inhibition of PRCP will be studied with emphasis on apelin activity.

Researcher(s)

• Promoter: De Meester Ingrid
• Fellow: De Hert Emilie

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Thrombolysis with tissue plasminogen activator remains the only approved pharmacological treatment for acute ischemic stroke, AIS. Besides the narrow therapeutic time window, its use is limited by its efficacy: in up to 50% of the treated patients, timely recanalization is not achieved. Moreover, administration involves serious side effects such as intracranial hemorrhage and neurotoxicity. Consequently, the search for new agents for improvement of AIS treatment is urgently needed. Research has demonstrated that the enzyme carboxypeptidase U (CPU, TAFIa) is an important player in thrombus lysis. After activation from its precursor proCPU, the released CPU is able to potently attenuate fibrinolysis. Consequently, inhibition of CPU activity is a novel approach to enhance fibrinolysis. We want to explore the involvement of this enzyme in AIS in more detail. The usefulness of CPU as a diagnostic marker to discriminate ischemic from hemorrhagic stroke and the relationship of CPU with clinical outcome and thrombolytic treatment efficacy will be investigated. We plan to optimize the Thrombodynamics assay in order to assess the effect of CPU-inhibition on clot lysis during thrombolysis. Furthermore, in a preclinical setting, we will evaluate the effect of CPU inhibition in an experimental stroke model in rats. This research will provide essential information on the role of the CPU system and the usefulness of CPU inhibitors as potentially efficient and safer treatment of AIS.

Researcher(s)

• Promoter: Hendriks Dirk
• Co-promoter: Lambeir Anne-Marie
• Fellow: Mertens Joachim

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

The complex mechanism involved in the pathogenesis of these metabolic disorders remains poorly understood. Recent findings indicate that the enzyme prolyl carboxypeptidase (PRCP) plays a role in body weight control and glucose homeostasis by inactivating melanocyt-stimulating hormone MSH, a neuropeptide which causes a loss of appetite. We aim to investigate whether 'peripheral' (outside the central nervous system) actions of PRCP also contribute to this function. Our preliminary experiments have identified apelin, which also regulates feeding behaviour and glucose metabolism, as a novel PRCP substrate. Firstly, we will characterize this cleavage in vitro and investigate whether truncation by PRCP alters the function of apelin and other peptide substrates. Secondly, the role of PRCP on in vivo cleavage of peripheral apelin will be investigated.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Lambeir Anne-Marie
• Fellow: De Hert Emilie

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

This project represents a formal research agreement between UA and on the other hand the client. UA provides the client research results mentioned in the title of the project under the conditions as stipulated in this contract.

Researcher(s)

• Promoter: De Meester Ingrid

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

In the first stage of the project we will extend the knowledge about assays for the measurement of CPU, i.e. the clot lysis assay and an ultrasensitive activity-based method. Next we will use these techniques to investigate the role of CPU in patients with acute myocardial infarction. A third objective is to explore the localization of CPU in atherosclerotic plaques and thrombi. Finally, we will evaluate the role of CPU in a novel plaque rupture model. Investigating the role of CPU in atherosclerosis and its clinical manifestations may open new approaches for plaque stabilising therapies and treatment of acute ischemic syndromes.

Researcher(s)

• Promoter: Hendriks Dirk
• Co-promoter: Lambeir Anne-Marie
• Fellow: Leenaerts Dorien

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

In this project we aim (1) to characterize at the molecular level the role of PREP in hallmark processes of neurodegenerative disease: neuronal death, synapse loss and neuroinflammation; and (2) to investigate whether PREP-inhibitors can be used to modulate these processes.

Researcher(s)

• Promoter: Lambeir Anne-Marie
• Co-promoter: Dedeurwaerdere Stefanie
• Co-promoter: Van Der Veken Pieter

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

In a first part of the project we will investigate the protein expression and enzymatic activity of PRCP in the circulation. Thereafter, we will perform in vitro experiments to study the cleavage of apelin and enterostatin by PRCP and compare cleavage efficiency with other known PRCP substrates. A third objective is to investigate apelin and enterostatin cleavage by PRCP in vivo and to find out whether PRCP influences their effect on body weight and metabolism. Understanding how peptide hormones are degraded by PRCP may open new approaches for therapeutic intervention.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Lambeir Anne-Marie
• Fellow: Kehoe Kaat

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

This project fits in the study of the role and expression of DPP9 in lung pathophysiology with an emphasis on lung fibrosis. However, DPP9 is part of a larger family of DPPs with structural similarities and overlapping substrate specificities. Initially, we want to establish a DPP atlas of the lung to provide context to the study of DPP9 specifically. Our second and main objective is to form a picture of DPP9's role and expression in the lung, starting from our current knowledge of DPP9 in human macrophages, towards a broader take on DPP9 and lung fibrosis.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Lambeir Anne-Marie
• Fellow: Vliegen Gwendolyn

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

The effect of vaccination during pregnancy on bioactive components in breast milk (antibodies against pertussis and influenza, the total amount of SIgA and its subclasses and lactoferrin) will be examined. In addition, we will study the influence of storage conditions on the bioactive components. The differences between breast milk from mothers of preterm and term infants will be investigated in cooperation with the department of Neonatology at UZA.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Van Damme Pierre
• Fellow: De Schutter Sara

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

In the first stage of the project we will extend the knowledge about assays for the measurement of CPU, i.e. the clot lysis assay and an ultrasensitive activity-based method. Next we will use these techniques to investigate the role of CPU in patients with acute myocardial infarction. A third objective is to explore the localization of CPU in atherosclerotic plaques and thrombi. Finally, we will evaluate the role of CPU in a novel plaque rupture model. Investigating the role of CPU in atherosclerosis and its clinical manifestations may open new approaches for plaque stabilising therapies and treatment of acute ischemic syndromes.

Researcher(s)

• Promoter: Hendriks Dirk
• Co-promoter: Lambeir Anne-Marie
• Fellow: Leenaerts Dorien

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

In a first part of the project we will investigate the protein expression and enzymatic activity of PRCP in the circulation. Thereafter, we will perform in vitro experiments to study the cleavage of apelin and enterostatin by PRCP and compare cleavage efficiency with other known PRCP substrates. A third objective is to investigate apelin and enterostatin cleavage by PRCP in vivo and to find out whether PRCP influences their effect on body weight and metabolism. Understanding how peptide hormones are degraded by PRCP may open new approaches for therapeutic intervention.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Lambeir Anne-Marie
• Fellow: Kehoe Kaat

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

This project will focus on the role of dipeptidyl-peptidase 9 (DPP9) in leukocytes. This enzyme is capable of cutting a dipeptide off a peptide after a proline at the penultimate position. However, its physiological role remains elusive. We want to solve the answer to this problem by tackling three key questions. Where exactly do we find DPP9 in leukocytes? It is important to know where in the cell DPP9 resides and if it moves upon cellular activation. Also, do we find equal amounts of DPP9 in different types of leukocytes? Can we identify interaction partners of this large intracellular protein? To determine the function of DPP9, it helps to know with which proteins it interacts. If the function of those proteins is known, it is likely that DPP9 has a function in the same pathway. Can we change cellular functions by changing the expression and/or enzyme activity of DPP9? We can inhibit DPP9 function by either inhibiting its formation or its enzymatic activity. Both methods might lead to (different) effects on the cellular level and indicate in which cellular roles DPP9 is involved. With these three approaches the physiological role of DPP9 in leukocytes might finally be revealed.

Researcher(s)

• Promoter: De Meester Ingrid
• Fellow: Waumans Yannick

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

This project aims to unravel the effects of DPP4 inhibition on collagen and collagen-derived dipeptide metabolism. We will develop and validate methods to quantify, in biological matrices, dipeptides resulting from DPP4 mediated cleavage. Next, the effects of DPP4 inhibition on the collagen metabolism and the dipeptide profile will be studied in vitro in fibroblast and osteoblast cultures. The in vivo relevance of our findings will be evaluated by studying the effects of long-term DPP4 inhibition in vivo in a rat model of type 2 diabetes. The focus will be on cardiac and renal collagen metabolism and dipeptide levels in plasma, urine and tissues.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Behets Geert
• Co-promoter: Covaci Adrian

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

This project represents a formal service agreement between UA and on the other hand Vestre Viken Hospital Trust. UA provides Vestre Viken Hospital Trust research results mentioned in the title of the project under the conditions as stipulated in this contract.

Researcher(s)

• Promoter: Hendriks Dirk

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

This project represents a formal research agreement between UA and on the other hand a private institution. UA provides the private institution research results mentioned in the title of the project under the conditions as stipulated in this contract.

Researcher(s)

• Promoter: Hendriks Dirk

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

It is hypothesized that Prolyl oligopeptidase (PREP) might have a functional role in the development of neurodegenerative diseases such as synucleinopathies. In order to gain a better insight in the nature of this modulating role of PREP and PREP inhibitors on the fibril formation, we here propose to study the interaction between PREP and a-synuclein by using site directed mutagenesis, aggregation assays and a mass spectrometry based approach.

Researcher(s)

• Promoter: Van Elzen Roos

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

This project will focus on the role of dipeptidyl-peptidase 9 (DPP9) in leukocytes. This enzyme is capable of cutting a dipeptide off a peptide after a proline at the penultimate position. However, its physiological role remains elusive. We want to solve the answer to this problem by tackling three key questions. Where exactly do we find DPP9 in leukocytes? It is important to know where in the cell DPP9 resides and if it moves upon cellular activation. Also, do we find equal amounts of DPP9 in different types of leukocytes? Can we identify interaction partners of this large intracellular protein? To determine the function of DPP9, it helps to know with which proteins it interacts. If the function of those proteins is known, it is likely that DPP9 has a function in the same pathway. Can we change cellular functions by changing the expression and/or enzyme activity of DPP9? We can inhibit DPP9 function by either inhibiting its formation or its enzymatic activity. Both methods might lead to (different) effects on the cellular level and indicate in which cellular roles DPP9 is involved. With these three approaches the physiological role of DPP9 in leukocytes might finally be revealed.

Researcher(s)

• Promoter: De Meester Ingrid
• Fellow: Waumans Yannick

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

The aim of the project is to identify interaction partners of human carboxypeptidase M (CPM). The research plan has two components. On the one hand we study the interaction with bioactive peptides that are substrates for the carboxypeptidase enzymatic activity. On the other hand we investigate a potential role of this membrane bound protein in cell-cell interactions, extracellular matrix interactions and cell migration.

Researcher(s)

• Promoter: Lambeir Anne-Marie
• Co-promoter: Hendriks Dirk
• Fellow: Denis Catherine

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

The aim of this research project is to further clarify the interaction between propyl oligopeptidase (POP) and the deposition of misfolded proteins, especially alpha-synuclein aggregation. Another aim in this project is to test the effects of POP inhibitor to the aplha-synuclein overexpressing mouse model that is obtained from prof. V. Baekelandt.

Researcher(s)

• Promoter: Lambeir Anne-Marie

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Dipeptidyl peptidases are enzymes that cleave N-terminal dipeptides from peptides with proline at the penultimate position. Dipeptidyl peptidase IV is by far the most extensively studied member of this family of serine proteases. Recently, Zhai et al [1] described a decrease in ischemia/reperfusion injury after lung transplantation by flushing and storage of the graft in a solution with the irriversible DPPIV inhibitor, AB192. [2] The goal of this doctoral thesis is to investigate this positive effect on cellular level by measuring the expression of dipeptidyl peptidases (activity-, protein- and mRNA-level) in primarily islolated lung microvascular endothelial cells and the effect of reduced oxygen tension on the expression levels. Secondly, other possible targets of AB192 are investigated and the therapeutically available DPPIV inhibitors (vildagliptin and sitagliptin) are tested for the same positive effects during ischemia-reperfusion injury.

Researcher(s)

• Promoter: De Meester Ingrid
• Fellow: Matheeussen Veerle

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Carboxypeptidase U (CPU), also referred to as active Thrombin-Activatable Fibrinolysis Inhibitor (TAFIa), is a recently discovered attenuator of the fibrinolytic rate and is considered to be the molecular link between coagulation and fibrinolysis. The measurement of active CPU in plasma is associated with mayor pitfalls. It is our goal to develop a sensitive and selective assay allowing us to determine limited amounts of CPU in circulation. In a second phase, this assay is being used to identify patient groups (MI, PE, DVT, ischemic stroke, sepsis, ¿) were the CPU system is being activated and thus could affect the outcome or severity of the pathology.

Researcher(s)

• Promoter: Hendriks Dirk
• Fellow: Heylen Evelien

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

The objective of this project is to investigate the interactions between prolyl oligopeptidase and relevant biomolecules, in vitro and in the cell. We envisage three lines of research: (1) the interaction with alpha-synuclein and the connection with Parkinson's disease, (2) the interaction with tubulin, intracellular transport and secretion, (3) co-localisation with other cytosolic peptidases and the connection with the aggresome.

Researcher(s)

• Promoter: Lambeir Anne-Marie

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

This project aims to better understand the effects of chronic dipeptidyl peptidase (DPP) inhibition on pre-defined aspects of cardiovascular, renal and bone (patho)physiology. Inhibitors with defined selectivity profiles will be developed as tools. Expression and inhibition of DPP4 and related peptidases will be studied on the molecular level, in cultured cells and in rat models of ischemia/reperfusion injury of heart and kidney.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Augustyns Koen
• Co-promoter: De Keulenaer Gilles
• Co-promoter: D'Haese Patrick
• Co-promoter: Lambeir Anne-Marie

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

this research project studies proline selective peptidases. It has the following aims : 1. Characterization of inhibitors and selection of potent and specific inhibitors of DPP4 related enzymes DPP8, DPP9 and FAP. 2. Study of the expression of the DPPP mambers in endothelia of different origin and this under normoxia as well as hypoxia. 3. In vitro study of the effect of selective ans not selective inhibition on endothelial cell activation. 4. The in vitro study of the effect of selective and non-selective DPP inhibition on collagen metabolism of fibroblasts.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Lambeir Anne-Marie
• Co-promoter: Scharpe Simon

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

The three main objectives of the consortium are: 1) to unravel the mode of action of PREP and PREP-like enzymes in health and disease; 2) to develop new drugs: 3) to discover new therapeutic targets. In order to achieve this, studies will be performed to identify the physiological substrates PREP and PREP-like enzymes and to characterize the pathways in which they are involved.

Researcher(s)

• Promoter: Lambeir Anne-Marie

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

The aim of the project is to identify interaction partners of human carboxypeptidase M (CPM). The research plan has two components. On the one hand we study the interaction with bioactive peptides that are substrates for the carboxypeptidase enzymatic activity. On the other hand we investigate a potential role of this membrane bound protein in cell-cell interactions, extracellular matrix interactions and cell migration.

Researcher(s)

• Promoter: Lambeir Anne-Marie
• Co-promoter: Hendriks Dirk
• Fellow: Denis Catherine

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

The objective of this project is to investigate the interactions between P0 and relevant biomolecules, in vitro and in the cell, We envisage four lines of research: (1) the interaction with alpha-synuclein and the connection with Parkinsons disease, (2) the interaction with tubulin, intracellular transport and secretion, (3) co-localisation with other cytosolic peptidases and the connection with the aggresome, (4) comparative peptidomics of mouse brain after administration of a P0 inhibitor. The four research lines are interconnected.

Researcher(s)

• Promoter: Lambeir Anne-Marie

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Dipeptidyl peptidases are enzymes that cleave N-terminal dipeptides from peptides with proline at the penultimate position. Dipeptidyl peptidase IV is by far the most extensively studied member of this family of serine proteases. Recently, Zhai et al [1] described a decrease in ischemia/reperfusion injury after lung transplantation by flushing and storage of the graft in a solution with the irriversible DPPIV inhibitor, AB192. [2] The goal of this doctoral thesis is to investigate this positive effect on cellular level by measuring the expression of dipeptidyl peptidases (activity-, protein- and mRNA-level) in primarily islolated lung microvascular endothelial cells and the effect of reduced oxygen tension on the expression levels. Secondly, other possible targets of AB192 are investigated and the therapeutically available DPPIV inhibitors (vildagliptin and sitagliptin) are tested for the same positive effects during ischemia-reperfusion injury.

Researcher(s)

• Promoter: De Meester Ingrid
• Fellow: Matheeussen Veerle

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Carboxypeptidase U (CPU), also referred to as active Thrombin-Activatable Fibrinolysis Inhibitor (TAFIa), is a recently discovered attenuator of the fibrinolytic rate and is considered to be the molecular link between coagulation and fibrinolysis. The measurement of active CPU in plasma is associated with mayor pitfalls. It is our goal to develop a sensitive and selective assay allowing us to determine limited amounts of CPU in circulation. In a second phase, this assay is being used to identify patient groups (MI, PE, DVT, ischemic stroke, sepsis, ¿) were the CPU system is being activated and thus could affect the outcome or severity of the pathology.

Researcher(s)

• Promoter: Hendriks Dirk
• Fellow: Heylen Evelien

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

The aim of the project is to identify interaction partners of human carboxypeptidase M (CPM). The research plan has two components. On the one hand we study the interaction with bioactive peptides that are substrates for the carboxypeptidase enzymatic activity. On the other hand we investigate a potential role of this membrane bound protein in cell-cell interactions, extracellular matrix interactions and cell migration.

Researcher(s)

• Promoter: Lambeir Anne-Marie
• Co-promoter: Hendriks Dirk
• Fellow: Denis Catherine

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

The aims are to study systematically the substrate specificity of the basic carboxypeptidases CPU, CPN, CPM, proCPU and the pancreatic enzyme CPB. This will be done by means of a library of short peptides where 1 or 2 of the amino acids preceding the C-terminal arginine are varied. In addition the degradation of postulated natural substrates will be investigated. The knowledge of the substrate speicificty will be applied for the development of specifici enzymatic methods for CPU, CPN, and CPM in plasma or serum and for CPM on cell surfaces and membrane preparations.

Researcher(s)

• Promoter: Lambeir Anne-Marie

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Carboxypeptidase M (CPM) is a cell-surface bound carboxypeptidase which removes C-terminal basic aminoacids from bioactive peptides. CPM has been reported to be distributed in many organs and fluids. CPM is also present on immunological and inflammatory cells. However, a physiological role of CPM in these tissues has not yet been clearly defined. Commonly, it is accepted that CPM can be a player in the processing or inactivation of peptide hormones, growth factors, inflammatory important peptides, et cetera. As there is very little knowledge of the molecular properties of CPM, we aim to systematically characterise the catalytic mechanisme and substrate specificity of CPM. These investigations must allow to differentiate between the various carboxypeptidases, specifically concerning the substrate specificity. Carboxypeptidase U (CPU) inhibitors are currently under clinical investigation as adjuvans in the fibrinolytic therapy. The importance of studying the selectivity of the CPU inhibitors with regard to other carboxypeptidases is self-evident. In this context, CPM is particulary important because it is constitutionally expressed in many tissues. In the first phase of our project, CPM will be cloned, expressed and purified. A comparative study of the recombinant and natural enzyme will be performed. We wish to study the different elements of the catalytic proces using enzymological methods. The substrate specificity of CPM will be studied using site-directed mutagenesis. In the second phase, we aim to identify naturally occuring substrates of CPM in tissue extracts (proteomics analysis). Extracted polypeptides will be fractionated and analysed for the presence of substrate (LC-MS). The enzyme kinetics of these substrates will be studied in vitro. A last aim is to set up a database of CPM values in normal and clinical samples. The results of our study can be used in the development of new CPU inhibitors. New therapeutic strategies can be proposed if the involvement of CPM in inflammation is confirmed. The possibility of administering therpeutic peptides as aerosol is under research. The presence of CPM on type I alveolar cells can influence the absorption and pharmacokinetics of these drugs.

Researcher(s)

• Promoter: Lambeir Anne-Marie
• Co-promoter: Hendriks Dirk
• Fellow: Deiteren Kathleen

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

The current work will focus on exploring the function of the novel enzyme carboxypeptidase U and its role in the fibrinolysis. We will also investigate if the proCPU/CPU system is involved in the activation or inactivation of other physiological substrates bearing a C-terminal basic amino-acid.

Researcher(s)

• Promoter: Hendriks Dirk
• Fellow: Willemse Johan

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

This project concerns the molecular and biochemical study of new human dipeptidyl peptidases (DPPs). The project aims to (1) determine which dipeptidyl peptidases are present in human leucocytes besides DPPIV, (2) identify and characterize these peptidases physicochemically, (3) study which substrates are cleaved by these peptidases and (4) test known inhibitors of dipeptidyl peptidases for selectivity.

Researcher(s)

• Promoter: De Meester Ingrid

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

This research plan aims to answer the following specific questions concerning FAP, DPPII, DP8 and DP9. The experimental objectives can be grouped around 3 themes: chromogenic and fluorogenic substrates, peptide substrates and inhibitors. The experimental results will be interpreted with the aid of available structural information (crystal structure of DPIV is published recently; DP8 and 9 show primary sequence homology, DPPII does not) in order to obtain a better insight in substrate binding and catalysis by the members of this family of serine type peptidases.

Researcher(s)

• Promoter: De Meester Ingrid
• Co-promoter: Lambeir Anne-Marie
• Co-promoter: Scharpe Simon

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Prolyloligopeptidase (PO) and dipeptidyl peptidase IV (DPP-IV) are proline specific peptidases. Their biological function is connected with the metabolism of biologically active peptides. It is generally assumed that they exert their catalytic function through the classical serine protease mechanism. The specificity and the selectivity are mainly determined by the primary binding site and a few sub-binding sites for adjacent amino acids. The substrate-specificity of DPP-IV for natural peptides could not be predicted based on these assumptions and the data obtained by using small dipeptide-derived substrates. It was not possible to unambiguously identify the rate limiting step in the mechanism from solvent isotope effects. Under certain conditions a conformational change seems to be rate limiting for the hydrolysis of chromogenic substrates by PO. The interpretation of structure-activity-relationships of inhibitors also remains very difficult. The object of this project is to get a better insight in the details of the catalytic mechanism of PO and DPP-IV. Different approaches are being considered: kinetic experiments, site-directed mutagenesis in the active site and the substrate binding sites in PO, kwantitative structure-activity relationships of inhibitors and ligands.

Researcher(s)

• Promoter: Scharpe Simon
• Co-promoter: Lambeir Anne-Marie
• Fellow: Brandt Inger

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Carboxypeptidase M (CPM) is a cell-surface bound carboxypeptidase which removes C-terminal basic aminoacids from bioactive peptides. CPM has been reported to be distributed in many organs and fluids. CPM is also present on immunological and inflammatory cells. However, a physiological role of CPM in these tissues has not yet been clearly defined. Commonly, it is accepted that CPM can be a player in the processing or inactivation of peptide hormones, growth factors, inflammatory important peptides, et cetera. As there is very little knowledge of the molecular properties of CPM, we aim to systematically characterise the catalytic mechanisme and substrate specificity of CPM. These investigations must allow to differentiate between the various carboxypeptidases, specifically concerning the substrate specificity. Carboxypeptidase U (CPU) inhibitors are currently under clinical investigation as adjuvans in the fibrinolytic therapy. The importance of studying the selectivity of the CPU inhibitors with regard to other carboxypeptidases is self-evident. In this context, CPM is particulary important because it is constitutionally expressed in many tissues. In the first phase of our project, CPM will be cloned, expressed and purified. A comparative study of the recombinant and natural enzyme will be performed. We wish to study the different elements of the catalytic proces using enzymological methods. The substrate specificity of CPM will be studied using site-directed mutagenesis. In the second phase, we aim to identify naturally occuring substrates of CPM in tissue extracts (proteomics analysis). Extracted polypeptides will be fractionated and analysed for the presence of substrate (LC-MS). The enzyme kinetics of these substrates will be studied in vitro. A last aim is to set up a database of CPM values in normal and clinical samples. The results of our study can be used in the development of new CPU inhibitors. New therapeutic strategies can be proposed if the involvement of CPM in inflammation is confirmed. The possibility of administering therpeutic peptides as aerosol is under research. The presence of CPM on type I alveolar cells can influence the absorption and pharmacokinetics of these drugs.

Researcher(s)

• Promoter: Lambeir Anne-Marie
• Fellow: Deiteren Kathleen

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Researcher(s)

• Promoter: De Meester Ingrid
• Fellow: Maes Marie-Berthe

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Researcher(s)

• Promoter: Scharpe Simon

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Patients presenting with non thrombocytopenic mucocutaneous haemorrhages (MHC) of hereditary nature are usually difficult to diagnose. It is widely accepted that most of these patients have a mild disorder of primary haemostasis, most frequently, type 1 von Willebrand disease (vWD) and platelet aggregation/ secretion defects (PASD). However, many patients presenting with clinically significant MCH have no demonstrable alterations in plasma von Willebrand factor and ex vivo platelet function. On the other hand, it is common to observe abnormalities in these tests in subjects with no abnormal haemorrhages. These observations suggest that the pathogenesis of the bleeding disorder in many of these patients is still unknown. We hypothesize that the concentration of other haemostatic factors influence or modulate the clinical expression of MCH, the bleeding symptoms resulting from the interactions and final balance among pro- and anticoagulant factors. Specifically, we propose that the overall activity of the fibrinolytic system is enhanced in patients as compared with controls, and that this hyperactivity contributes to bleeding. Among the determinants of fibrinolytic activity, in this project we propose to analyze the influence of plasma procarboxypeptidase U (proCPU, TAFI) levels and activity in patients consulting for MCH. Carboxypeptidase U, a recently discovered exopeptidase, circulates in plasma as an inactive proform, proCPU. Thrombin activates CPU, and the active form attenuates fibrinolysis by cleaving C-terminal lysine residues exposed on partially degraded fibrin by the action of plasmin. Theoretically, the lower the CPU activity, the higher the rate of fibrinolysis and thus the speed of fibrin clot degradation and removal. Thus, severely suppressed proCPU activation or concentration might be associated with a tendency to bleed, and accordingly, we propose that patients with MCH have a lower proCPU concentration and/or activity. In this context, low plasma proCPU would be considered a risk factor for haemorrhages and, as such, would potentiate the bleeding tendency in patients with vWD and PASD and high plasma proCPU would attenuate their bleeding tendency. Moreover, we hypothesize that patients with MCH, but with no specific diagnosis and those with prolonged bleeding time have lower plasma levels of proCPU than non-bleeder controls and patients with normal bleeding time.The prospective, controlled study, will be performed in patients consulting for MCH, which will be classified in different diagnostic categories (vWD, PASD, vWD+PASD, and patients with no specific diagnosis). Age and sex-matched healthy individuals, with no history of personal or familial bleeding will be used as controls. In all of them, plasma proCPU will be measured by antigenic and functional assays.

Researcher(s)

• Promoter: Hendriks Dirk

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

The current work will focus on exploring the function of the novel enzyme carboxypeptidase U and its role in the fibrinolysis. We will also investigate if the proCPU/CPU system is involved in the activation or inactivation of other physiological substrates bearing a C-terminal basic amino-acid.

Researcher(s)

• Promoter: Hendriks Dirk
• Fellow: Willemse Johan

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Carboxypeptidase M (CPM) is a cell-surface bound carboxypeptidase which removes C-terminal basic aminoacids from bioactive peptides. CPM has been reported to be distributed in many organs and fluids. CPM is also present on immunological and inflammatory cells. However, a physiological role of CPM in these tissues has not yet been clearly defined. Commonly, it is accepted that CPM can be a player in the processing or inactivation of peptide hormones, growth factors, inflammatory important peptides, et cetera. As there is very little knowledge of the molecular properties of CPM, we aim to systematically characterise the catalytic mechanisme and substrate specificity of CPM. These investigations must allow to differentiate between the various carboxypeptidases, specifically concerning the substrate specificity. Carboxypeptidase U (CPU) inhibitors are currently under clinical investigation as adjuvans in the fibrinolytic therapy. The importance of studying the selectivity of the CPU inhibitors with regard to other carboxypeptidases is self-evident. In this context, CPM is particulary important because it is constitutionally expressed in many tissues. In the first phase of our project, CPM will be cloned, expressed and purified. A comparative study of the recombinant and natural enzyme will be performed. We wish to study the different elements of the catalytic proces using enzymological methods. The substrate specificity of CPM will be studied using site-directed mutagenesis. In the second phase, we aim to identify naturally occuring substrates of CPM in tissue extracts (proteomics analysis). Extracted polypeptides will be fractionated and analysed for the presence of substrate (LC-MS). The enzyme kinetics of these substrates will be studied in vitro. A last aim is to set up a database of CPM values in normal and clinical samples. The results of our study can be used in the development of new CPU inhibitors. New therapeutic strategies can be proposed if the involvement of CPM in inflammation is confirmed. The possibility of administering therpeutic peptides as aerosol is under research. The presence of CPM on type I alveolar cells can influence the absorption and pharmacokinetics of these drugs.

Researcher(s)

• Promoter: Lambeir Anne-Marie
• Fellow: Deiteren Kathleen

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Researcher(s)

• Promoter: Hendriks Dirk

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Prolyloligopeptidase (PO) and dipeptidyl peptidase IV (DPP-IV) are proline specific peptidases. Their biological function is connected with the metabolism of biologically active peptides. It is generally assumed that they exert their catalytic function through the classical serine protease mechanism. The specificity and the selectivity are mainly determined by the primary binding site and a few sub-binding sites for adjacent amino acids. The substrate-specificity of DPP-IV for natural peptides could not be predicted based on these assumptions and the data obtained by using small dipeptide-derived substrates. It was not possible to unambiguously identify the rate limiting step in the mechanism from solvent isotope effects. Under certain conditions a conformational change seems to be rate limiting for the hydrolysis of chromogenic substrates by PO. The interpretation of structure-activity-relationships of inhibitors also remains very difficult. The object of this project is to get a better insight in the details of the catalytic mechanism of PO and DPP-IV. Different approaches are being considered: kinetic experiments, site-directed mutagenesis in the active site and the substrate binding sites in PO, kwantitative structure-activity relationships of inhibitors and ligands.

Researcher(s)

• Promoter: Scharpe Simon
• Co-promoter: Lambeir Anne-Marie
• Fellow: Brandt Inger

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Carboxypeptidase U (CPU, EC 3.4.17.20) belongs to the group of the basic carboxypeptidases. These enzymes cleave basic amino acid (lysine or arginine) from the C-terminus of the peptide chain. Carboxypeptidase U circulates in blood as an inactive zymogen,procarboxyppetidase U. During the process of coagulation and fibrinolysis it is converted to its active form, carboxypeptidase U. The physiological function of CPU is probably restricted to the coagulation-fibrinolysis system where it is involved in regulating the rate of fibrin lysis. The velocity of fibrinolysis is determining for the balance between coagulation and fibrinolysis. Disturbance of this equilibrium could result either in thrombosis or haemorrhage. Whether the proCPU plasmaconcentration plays a significant role and thus can be regarded as risk factor for cardiovascular diseases, remains to be investigated.

Researcher(s)

• Promoter: Hendriks Dirk
• Fellow: Leurs Judith

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

During the study of CD26/DPPIV in human leucocytes, it appeared that some of the X-Pro-releasing activity wasn't inhibited by DPPIV specific inhibitors. Preliminary experiments showed that at least some of the X-Pro-releasing activity was due to several other dipeptidylpeptidases. Primarily human dipeptidyl peptidase II (DPPII) will be purified, followed by a biochemical and enzymological characterization (synthetical substrates and natural occuring peptides). Human leucocyte cellines and periferal blood mononuclear cells will be screened on the presence of DPPII. Following biological evaluation of new DPPII inhibitors (collaboration with the laboratory of farmaceutical chemistry, Prof. A. Haemers and Prof. K. Augustyns), the function of DPPII in human leucocytes will be studied.

Researcher(s)

• Promoter: De Meester Ingrid
• Fellow: Maes Marie-Berthe

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Researcher(s)

• Promoter: Scharpe Simon

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

The project deals with the search for physiological substrates of dipeptidyl peptidase IV (=CD26), a very specific aminopeptidase that is found in the bloodstream and on differentiated epithelial cells. It aims (I) to investigate which peptides are hydrolysed ; (2) to characterize the cleavage kinetically and (3) to study the biological consequences ofthe hydrolysis. Focus will be put on peptides ofthe chemokine and glucagon superfamily.

Researcher(s)

• Promoter: De Meester Ingrid

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project

Abstract

Researcher(s)

• Promoter: Scharpe Simon
• Co-promoter: De Meester Ingrid

Research team(s)

• Medical Biochemistry

Project type(s)

• Research Project