Ongoing projects

Elucidation of the molecular basis for the activation of trypanosomal receptor adenylate cyclases by stimulus-induced ectodomain conformational change. 09/01/2023 - 08/07/2023

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.

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Intracellular dipeptidyl peptidase 9 (DPP9) interactions in primary human blood cells: how are they influenced by novel DPP9 inhibitors and PROTACs? 01/11/2022 - 31/10/2024

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.

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Investigation of the structure-function relationship of sporozoite 6Cys proteins from the malaria parasite Plasmodium vivax. 01/10/2022 - 30/09/2026

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.

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Molecular basis for the potency and selectivity of DNDI-6690, a promising lead for the development of novel anti-leishmanial drugs 01/11/2021 - 31/10/2023

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.

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EVZYM: A new source of native human targets in high throughput screening (HTS) of enzyme inhibitors – TMPRSS2 as an example. 01/09/2021 - 01/04/2023

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.

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The role of dipeptidyl peptidase 9 (DPP9) in human monocyte-derived macrophages: Discovery of DPP9 binding partners & natural substrates using novel chemical and cellular tools. 01/01/2021 - 31/12/2024

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.

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Dipeptidyl peptidase 9 (DPP9) characterization in primary human cells. 01/11/2019 - 31/10/2023

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.

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Development of tools and methods for the evaluation of FAP as a predictive/prognostic biomarker in cancer. 01/11/2019 - 31/10/2023

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

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Investigation of the structural and functional role of the Plasmodium falciparum circumsporozoite protein in the development of liver stage malaria. 01/10/2019 - 30/09/2023

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.

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Impact of extracellular matrix organization in the tumor environment on efficacy of immunotherapy in DNA mismatch repair deficient tumors. 01/09/2019 - 31/08/2023

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.

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Personalized immune therapy: FAP as a marker for resistance to immune checkpoint therapy in urothelial carcinomas. 01/09/2019 - 31/12/2022

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.

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Support maintenance scientific equipment (Medical Biochemistry). 01/01/2017 - 31/12/2022

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.

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Past projects

Host proteases at the interface between humans and SARS-CoV-2: Focus on TMPRSS2 as a therapeutic target. 01/06/2020 - 31/05/2021

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.

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Towards the realization of a structural biology platform at the University of Antwerp: The Mosquito Xtal3 crystallization robot as the missing link. 01/01/2020 - 31/12/2021

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.

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Investigating the potential of the glycolytic enzyme enolase from Trypanosoma evansi as a target for parasite detection and control. 15/07/2019 - 14/07/2020

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.

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Analyse of possible off-target effects of customer's experimental compounds. 08/07/2019 - 31/05/2020

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------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

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Carboxypeptidase U - a new drug target for the improvement of treatment in acute ischemic stroke. 01/10/2018 - 30/09/2020

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.

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The Biomolecular Interaction Platform (BIP) at UAntwerp. 01/05/2018 - 30/04/2021

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.

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Prolyl carboxypeptidase as a novel therapeutic target in the fight against obesity. 01/01/2017 - 31/12/2020

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.

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Carboxypeptidase U - a new drug target for the improvement of treatment in acute ischemic stroke. 01/10/2016 - 30/09/2018

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.

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Prolyl carboxypeptidase in peripheral body weight control and glucose homeostasis: is cleavage of apelin-13 playing a role? 01/10/2016 - 31/12/2016

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.

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Consulting Services. 01/04/2016 - 31/12/2017

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.

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The role of carboxypeptidase U in atherosclerosis and its thrombotic complications. 01/10/2015 - 30/09/2017

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.

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Project type(s)

  • Research Project

Prolyl oligopeptidase: partners and pathways in neuronal function and neurodegeneration. 01/01/2015 - 31/12/2018

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.

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  • Research Project

Prolylcarboxypeptidase in body weight control: a role for peripheral peptide cleavage? 01/10/2014 - 30/09/2016

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.

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  • Research Project

An in vitro and ex vivo Study on the Expression and Role of Dipeptidyl Peptidase 9 in the Lung. 01/10/2014 - 30/09/2015

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.

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  • Research Project

Bioactive components in human milk. 01/01/2014 - 31/12/2014

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.

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  • Research Project

The role of carboxypeptidase U in atherosclerosis and its thrombotic complications. 01/10/2013 - 30/09/2015

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)

Research team(s)

Project type(s)

  • Research Project

Prolylcarboxypeptidase in body weight control: a role for peripheral peptide cleavage? 01/10/2012 - 30/09/2014

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)

Research team(s)

Project type(s)

  • Research Project

Dipeptidyl-peptidase 9 in human leucocytes. 01/10/2012 - 30/09/2014

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.

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Project type(s)

  • Research Project

Role of dipeptidyl peptidase 4 (DPP4) in collagen metabolism and dipeptide homeostasis. 01/01/2012 - 31/12/2015

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.

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  • Research Project

HPLC analyses in activation of the kynurenine pathway in the acute phase of stroke, and its role in depression and fatigue following stroke. 01/12/2011 - 31/12/2012

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.

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  • Research Project

HPLC analyses in Mood Disorder Project. 01/12/2011 - 31/12/2012

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.

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  • Research Project

A study towards the functional role of prolyl oligopeptidase and prolyl oligopeptidase inhibitors in synucleinopathy 01/01/2011 - 31/12/2012

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.

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    • Research Project

    Dipeptidyl-peptidase 9 in human leukocytes. 01/10/2010 - 30/09/2012

    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)

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    Project type(s)

    • Research Project

    Carboxypeptidase M, substrates and ligands. 01/10/2010 - 31/03/2012

    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)

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    • Research Project

    FWO Visiting Postdoctoral Fellowship (T. MYOHANEN, Finland). Investigation of prolyl oligopeptidase as a therapeutic target for neurodegenerative diseases: inhibitors, substrates and ligands. 15/04/2010 - 14/10/2010

    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.

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    • Research Project

    Dipeptidyl peptidase IV (CD26) in ischemia-reperfusion injury. 01/10/2009 - 30/09/2011

    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)

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    • Research Project

    Carboxypeptidase U: a metallocarboxypeptidase with a distinct role in haemostasis and a possible risk factor for thrombotic disease. 01/10/2009 - 31/03/2011

    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)

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    Project type(s)

    • Research Project

    Prolyl oligopeptidase and prolyl oligopeptidase inhibitors in models of neurodegeneration. 01/10/2009 - 31/03/2010

    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.

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    • Research Project

    Dipeptidyl peptidases beyond glucose homeostasis: from biochemistry to physiological importance. 01/01/2009 - 31/12/2012

    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.

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    • Research Project

    Comparative biochemical and functional study of the different dipeptidyl peptidases. 01/01/2009 - 31/12/2012

    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.

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    • Research Project

    Oligopeptidase inhibitors in brain function and dysfunction: towards new therapeutic strategies for neuroprotection (NEUROPRO). 01/10/2008 - 30/09/2012

    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)

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    • Research Project

    Carboxypeptidase M, substrates and ligands. 01/10/2008 - 30/09/2010

    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)

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    • Research Project

    Investigation of prolyl oligopeptidase as a therapeutic target for neuropathological diseases: inhibitors, substrates and ligands. 01/01/2008 - 31/12/2011

    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)

    Research team(s)

    Project type(s)

    • Research Project

    Dipeptidyl peptidase IV (CD26) in ischemia-reperfusion injury. 01/10/2007 - 30/09/2009

    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)

    Research team(s)

    Project type(s)

    • Research Project

    Carboxypeptidase U: a metallocarboxypeptidase with a distinct role in haemostasis and a possible risk factor for thrombotic disease. 01/10/2007 - 30/09/2009

    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)

    Research team(s)

    Project type(s)

    • Research Project

    Carboxypeptidase M, substrates and ligands. 01/10/2007 - 30/09/2008

    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)

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    • Research Project

    Substrate specificity of basic carboxypeptidases. 01/10/2006 - 31/12/2008

    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.

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    • Research Project

    Molecular characterisation of carboxypeptidase M, an extracellular membrane-bound peptidase involved in the tissue-specific response toward biologically active peptides. 01/10/2006 - 30/09/2008

    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.

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    • Research Project

    Novel mechanisms in the physiological and pathophysiological regulation of the fibrinolytic pathway. 01/10/2005 - 30/09/2007

    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.

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    • Research Project

    New Human Dipeptidyl Peptidases. 01/05/2005 - 30/04/2009

    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.

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    • Research Project

    Biochemical study of recently discovered human dipeptidyl peptidases. 01/01/2005 - 31/12/2008

    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.

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    • Research Project

    Substrate specificity and structure-function relationships in the prolyl oligopeptidase family of serine proteases. 01/10/2004 - 30/09/2006

    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.

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    • Research Project

    Molecular characterisation of carboxypeptidase M, an extracellular membrane-bound peptidase involved in the tissue-specific response toward biologically active peptides. 01/10/2004 - 30/09/2006

    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.

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    • Research Project

    Human DPPII : biochemical and enzymological characterization. 01/10/2004 - 30/09/2006

    Abstract

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    • Research Project

    Role of cytokine-serotonin, interaction in stress and depression. 01/04/2004 - 31/12/2006

    Abstract

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    • Research Project

    Does the plasma proCPU (TAFI) concentration modulate the clinical expression of hereditary diseases of primary haemostasis ? 01/01/2004 - 31/12/2005

    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)

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    • Research Project

    Novel mechanisms in the physiological and pathophysiological regulation of the fibrinolytic pathway. 01/10/2003 - 30/09/2005

    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)

    Research team(s)

    Project type(s)

    • Research Project

    Molecular characterisation of carboxypeptidase M, an extracellular membrane-bound peptidase involved in the tissue-specific response toward biologically active peptides. 01/10/2003 - 30/09/2004

    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)

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    Development of new fixating entities (NFE's) for tissue processing. 01/04/2003 - 31/03/2005

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    Substrate specificity and structure-function relationships in the prolyl oligopeptidase family of serine proteases. 01/10/2002 - 30/09/2004

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    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.

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    Plasma procarboxypeptidase U: a novel regulator of fibrinolysis and potential risk factor for cardiovascular disease. 01/10/2002 - 30/09/2004

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    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.

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    Dipeptidyl peptidases in human leukocytes : molecular and functional diversity ? 01/10/2002 - 30/09/2004

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    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.

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    Chemokines and chemokine receptors. 01/01/2002 - 31/12/2006

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    Dipeptidyl peptidase IV: identification of natural substrates. 01/01/2001 - 31/12/2004

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    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.

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    • Research Project

    01/01/2001 - 31/12/2004

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    • Research Project