Research team

Medical Biochemistry

Expertise

Molecular basis of host-pathogen interactions through a combination of biochemical, biophysical and structural methodologies. * Recombinant protein production in bacterial and eukaryotic systems * Purification of proteins through a wide range of chromatographic techniques * Biochemical and biophysical techniques: dynamic light scattering (DLS), analytical gel filtration, circular dichroism (CD) spectroscopy, fluorescence spectroscopy * Protein-ligand studies: surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), bio-layer interferometry (BLI) * Structure determination of macromolecules through X-ray crystallography, small-angle X-ray crystallography and the combination thereof.

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 - 31/08/2022

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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High resolution structure determination of therapeutically relevant proteins as a means to validate an affinity grid for cryo-electron microscopy. 01/07/2021 - 30/06/2023

Abstract

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

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Extracellular vesicles of African trypanosomes: novel strategies to study their role in the parasite-host interaction. 01/11/2019 - 31/10/2023

Abstract

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

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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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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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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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