Public defense Nele Geerts 13/12/2024 - Elucidation of the clinical relevance and potential mechanisms of persistence in Streptococcus pneumoniae - Department Pharmaceutical Sciences

Promotors: Prof. Paul Cos en dr. Bram Van den Bergh (KULeuven)

Location: Campus Drie Eiken, gebouw O, Aula O5

Abstract:

Infectious diseases exist already for millennia and so does antimicrobial therapy. The past 100 years, major advances were accomplished into the fight against infectious diseases, especially with the discovery of antimicrobial agents. Unfortunately, these discoveries were accompanied with the fast emergence of antimicrobial resistance. Many efforts were made regarding research into antimicrobial resistance, but bacteria can evade antibiotic treatment also in different ways resulting in therapy failure. One way of how bacteria can evade killing by antibiotics, regardless of resistance, is antibiotic persistence. Persister cells are phenotypic variants that exist as a subpopulation within a clonal culture and can transiently switch to a nongrowing state that enables them to survive treatment with a bactericidal drug concentration. Being tolerant to lethal antibiotics, they underly the chronic nature of a variety of infections and even help in acquiring genetic resistance.

Streptococcus pneumoniae is a common colonizer of the mucosal surfaces of the nasopharynx of children, but can also cause life-threating diseases like pneumonia, sepsis and meningitis. S. pneumoniae remains a major cause of morbidity and mortality worldwide, despite the availability of a vaccine, and is classified as a priority pathogen by the World Health Organization (WHO) in 2024 for the need of novel antibiotics. Many reports were published about antibiotic persistence in a variety of bacterial species, but for Streptococcus pneumoniae, no studies were published.

We were the first to report the presence of persisters in S. pneumoniae cultures for different clinically relevant, bactericidal antibiotics (amoxicillin, cefuroxime, moxifloxacin and vancomycin) using our optimized long-living in vitro model. We detected high persister levels for S. pneumoniae reference strain D39 varying according to the growth phase and the antibiotic. Experimental evolution did not lead to the formation of a highly persistent phenotype which hampered us to investigate the molecular mechanisms behind S. pneumoniae persistence. Furthermore, we employed a large set of S. pneumoniae clinical isolates to prove the clinical relevance of S. pneumoniae persistence as persister cells were widely present in our strain collection. Finally, we made the first steps for the optimization of a long-term in vivo model to enable persistence studies in a clinically relevant setting. Altogether, our work advocates for higher interest for persistence in S. pneumoniae as a contributing factor for therapy failure and resistance development.

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Public defense Lidia Belova 25/11/2024 - Identification of contaminants of emerging concern and their biotransformation products by ion-mobility high-resolution mass spectrometry based screening approaches - Department Pharmaceutical Sciences

Promotors: Prof. Adrian Covaci - Prof. Alexander van Nuijs

Location: Aula Promotiezaal (Q.002), building Q, Campus Drie Eiken, Universiteitsplein 1, 2610 Antwerpen (Wilrijk)

Abstract:

The occurrence of synthetic chemicals in the environment is ubiquitous. Alongside well-studied older contaminants, humans can be exposed to numerous unknown or less characterized contaminants for which the collective term 'contaminants of emerging concern' (CECs) is used indicating that data on the occurrence, toxicity and biotransformation of these chemicals is scarce.

Environmental contaminants are commonly studied by the application of so-called targeted methods which cover only a limited number of compounds leaving most CECs undetected. To fill this gap, suspect and non-targeted screening approaches (SSA/NTS) gained increasing attention in recent years. These methods, commonly based on high-resolution mass spectrometry (HRMS), allow the simultaneous detection and identification (or annotation) of a high number of compounds. The identified compounds can be further expanded by the implementation of ion-mobility MS allowing the calculation of compound specific collision cross section (CCS) values which can be used as an additional identification parameter in CEC annotations improving identification confidence.

The presented thesis aimed at applying SSA and NTS for a holistic assessment of the presence of CECs in the indoor environment, the investigation of their biotransformation and consequently, their detection in human urine. Additionally, methods facilitating the inclusion of IM-MS in SSA and NTS studies were developed and the added value of IM-MS for CEC annotations was investigated.

First, a database containing more than 300 CCS values for over 110 CECs and their metabolites was experimentally compiled. The database allowed the characterization of compound class specific trendlines observed for the relationships between CCS value and mass-to-charge (m/z) ratio. For selected CECs, the reproducibility of CCS calculation was compared between different IM-MS instrumental set-ups and prediction models.

To investigate human indoor exposure to CECs, 46 indoor dust samples collected in Flanders were screened. The focus was laid on quaternary ammonium compounds (QACs) which gained increasing attention during the COVID-19 pandemic due to their high-volume use in disinfecting products. Targeted and suspect screening identified 38 QACs, 24 of which showed detection frequencies > 90%. The same sample set was screened applying a generic workflow focusing on numerous CEC classes. This led to the assignment of 55 CECs. Besides numerous known contaminants, several novel CECs were annotated including emerging phthalates with differing side chains, and two novel organophosphate flame retardants not previously described in indoor dust.

For QACs and emerging phthalates, CCS values were used to increase annotation confidence by matching them with CCS-m/z trendlines established within the CCS database. This process highlighted the added value of IM-MS for the identification of compounds belonging to specific homologue series.

The ubiquitous presence of CECs was further confirmed through the screening of 83 urine samples from Flemish adolescents. 63 compounds were annotated most of which were not included in current biomonitoring programs. This points out the need for the inclusion of screening approaches allowing the detection of metabolized CECs in future biomonitoring campaigns.

Nevertheless, analyses of metabolized CECs are often hampered by limited knowledge of their biotransformation products. Therefore, three QACs widely detected in indoor dust were incubated with human liver microsomes and cytosol (HLM/HLC) simulating Phase I and II metabolism. Thirty-one Phase I metabolites were annotated originating from 19 biotransformation reactions.

All in all, these results will facilitate future human biomonitoring studies ultimately aiming at a comprehensive assessment of human exposure to CECs.

Public defence Olivier Beyens 29/10/2024 - Computational study of DPP8 and DPP9: fundamental insights and inhibitor design - Department Pharamceutical Sciences

Promotors: Prof. Hans De Winter - Prof. Pieter Van der Veken

Location: Aula O1, building O, Campus Drie Eiken

Abstract:

Dipeptidyl peptidyl peptidases (DPP) 8 and 9 are intracellular enzymes with the ability to cleave off a dipeptidyl post-proline from the N-terminal of a substrate. Additionally, DPP9 forms protein-protein complexes with the inflammasome sensors CARD8 and NLRP1. By inhibition of DPP9 the complexes are disrupted and subsequent activation of the pyroptosis pathways occurs. This mechanism leads to cell death in acute myeloid leukemia and HIV-1 cell lines, increasing the interest in DPP9 as a drug target. Research regarding DPP8 is currently in an earlier stage, with less information about the biological function being available.

In this work we focus on a multifaceted computational study on DPP8 and DPP9. In the first objective of this work, we evaluated the structural differences leading to the remarkable selectivity of the DPP9 inhibitor “cpd42” against DPP8. Using an interdisciplinary approach, we validated that the Y280/F253 difference (DPP8/DPP9) is a factor in DPP8 versus DPP9 selectivity of the compound. This result can aid in the further design of selective DPP8/9 inhibitors.

In the second workstream, Hamiltonian Replica Exchange simulations were used to study the R-helix dynamics in DPP9. This R-helix is an α-helix formed by conformational change upon ligand or substrate binding. The R-helix dynamics were compared between a simulation with and without ligand. Removal of the ligand led to a lower occurrence of the R-helix, but even after ligand removal the structured form remained the most prominent conformation of the segment in the simulations. This work can facilitate follow-up research focusing on simulating ligand binding and unbinding pathways to DPP9.

The third objective of this work focusses on the discovery of novel ligands. The chosen methodology to discover new scaffolds was a cosolvent molecular dynamics (MD) informed pharmacophore model. To aid in the setup of cosolvent MD simulations with hydrophobic probes, we developed a new tool called “PART” (Plumed Automatic Restraining Tool), which we released under an open-source license. The resulting cosolvent MD simulations on the targets led to qualitative fragment affinity maps, which were also made freely available. We then used these qualitative fragment affinity maps to perform a pharmacophore screening. Resulting compounds were tested in vitro, but the best compounds were only weak binders.

The final aim focusses on improving the potency of the known DPP8/9 inhibitor (1G244) using relative binding free energy calculations. The optimization of the 1G244 inhibitor led to significant potency improvement, which was validated in vitro.

Public defense Yunita Eka Puspitasari 11/09/2024 - Phytochemical and Antidiabetic Investigations on Marine Natural Products from Indonesia - Department Pharmaceutical Sciences

Promotors:

Prof. dr. N. Hermans (promotor, UAntwerp)

Dr. T. De Bruyne (promotor, UAntwerp)

Prof. dr. A. Aulanni'am (promotor, Universitas Brawijaya)

Prof. dr. ir. A. M. Hariati (promotor, Universitas Brawijaya)

LOCATION: Graduate School Universitas Brawijaya (Indonesia)

Abstract:

Diabetes mellitus is a metabolic disorder characterized by elevated blood glucose levels. Controlling the fluctuation of blood glucose levels by inhibiting α-glucosidase activity is one possible strategy of diabetes management. α-Glucosidase is involved in carbohydrate metabolism by breaking down carbohydrates into their composing monosaccharides such as glucose. Inhibition of α-glucosidase activity slows down the carbohydrate digestion and the subsequent glucose absorption in the intestine.  Marine organisms, such as sea cucumbers and mangrove plants, are important resources of natural products. However, studies on sea cucumbers as a potential source for antidiabetic compounds, are limited. Studies reported that triterpene glycosides have been isolated from the Cuvierian tubules of sea cucumbers. Black sea cucumber (Holothuria atra) however is an exception compared to common sea cucumbers, since it lacks Cuvierian tubules.

In South-East Asia, mangrove (Rhizophora mucronata) bark has been used to treat diabetes, and the most prominent phytochemical compounds thereof, condensed tannins, have been identified. As a result of continuous exploitation of the bark, deforestation of mangrove areas occurs. Bearing this in mind, the leaves, with abundant and faster production, were tested as an alternative diabetes treatment, thus preventing degradation of the marine ecosystem.

This current project was therefore intended to investigate the phytochemical compounds present in the body wall of black sea cucumber (H. atra) and mangrove (R. mucronata) leaves, respectively; and to determine the α-glucosidase inhibitory activity from the body wall of black sea cucumber (H. atra) and mangrove (R. mucronata) leaves by in vitro assays.

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Public defence Natalia Smiejkowska 30/08/2024 - Supporting TB drug discovery through a target-based approach focusing on Mycobacterium tuberculosis mycothione reductase - Department Pharmaceutical Sciences

Promotors: Prof. Yann Sterckx - Prof. Paul Cos

Location:

Aula Q002, gebouw Q

Campus Drie Eiken

Abstract:

Tuberculosis (TB) is an airborne communicable disease that has long been declared a global public health threat. The World Health Organization estimates that a quarter of the world’s population has been infected with Mycobacterium tuberculosis, the etiological agent of TB. Although only 5-10% of infected individuals develop active TB, in 2022, the disease was the second leading cause of death from an infectious agent after COVID-19, with 1.3 million deaths worldwide. Besides its mortality, TB is characterized by a high morbidity, affecting the quality of life and socio-economic development in afflicted regions. A long-term, multidrug treatment and the frequent occurrence of adverse effects complicate patient compliance, leading to an alarming increase in drug resistance, which has become an emerging problem in TB treatment and disease control. There is thus a dire need for the discovery of drugs with novel modes of action and exploration of new drug targets.

M. tuberculosis is an intracellular pathogen that mainly resides in host macrophages. Here, the bacterium encounters dynamic host responses, including the release of reactive oxygen and nitrogen species (ROS and RNS, respectively). Mycothiol (MSH), a low molecular weight thiol, plays a role in maintaining a reducing environment as a key antioxidant. During oxidative stress, MSH acts as a ROS scavenger through its oxidation to mycothiol disulfide (MSSM). The enzyme mycothione reductase (Mtr) recycles MSSM back to MSH, thereby contributing to the maintenance of redox homeostasis.

This thesis explores the early stages of target-based drug discovery, starting from recombinant protein production and purification, through assay development, with the aim to identify and assess the M. tuberculosis Mtr (MtrMtb) inhibitors. The optimisation of MtrMtb production and purification increased protein yield, solubility and stability, thereby ensuring sufficient high-quality material for subsequent stages of the project.

A high-throughput screening (HTS) assay was developed using recombinant MtrMtb, by coupling enzyme activity to a bioluminescent read-out. This assay was applied in a semi-automated setting to screen 137,000 compounds, identifying 19 hits based on their potency, selectivity, and specificity. These hits included two clusters and four fragment molecules, which were further evaluated in whole-cell and intracellular infection assays. Additionally, the role of Mtr in mycobacterial growth, virulence, and stress tolerance was assessed.

The established HTS discovery pipeline offers an opportunity to deliver novel MtrMtb inhibitors and lays the foundation for future efforts in developing robust biochemical assays for the identification and triaging of inhibitors from high-throughput library screens.

Public defence Laurence Van Moll 03/06/2024 - The black soldier fly as a source of new antimicrobials - Department Pharmaceutical Sciences

Promotors:

Prof. dr. Paul Cos (promotor UA)

Prof. dr. Peter Delputte (promotor UA)

Prof. dr. Mik Van Der Borght (promotor KUL)

Prof. dr. Jeroen De Smet (co-promotor KUL)

Location defence:

Aula Q002, gebouw Q

Campus Drie Eiken

Abstract:

With the surge of antimicrobial resistance (AMR), many drugs routinely used in clinical practice are failing to treat critical infectious diseases. Tackling this AMR emergency requires a multifaceted approach, including the discovery and development of new antimicrobial agents that are active against drug-resistant pathogens . Recently, there has been increased interest in natural niches such as insects. Insects are remarkably resilient to microbial infections and possess a wide range of antimicrobial defense tactics. Here, the black soldier fly (BSF, Hermetia Illucens) has been explored as a source of new antimicrobials. A large collection of BSF antimicrobial peptides (AMPs) has been evaluated for its in vitro biological activity using a high-throughput screening platform. Two cecropins (HC1 and HC10) were selected due to their favorable activity profile, being potent action against Gram-negative pathogens and absence of early cytotoxicity indicators. Detailed characterization further elucidated LPS neutralization, membrane permeabilization, fast bactericidal activity, and divalent salt sensitivity. Other characteristics, such as biofilm eradication and synergistic action, were not noted. As these peptides show promising activity against Pseudomonas aeruginosa (with HC10 having confirmed in vivo activity), they can serve as starting points for future antipseudomonal therapeutics. However, some key points of the cecropins, such as their enzymatic stability and biological residency time, remain unaddressed to date. Lastly, this thesis also evaluated the antimicrobial activity of a collection of BSF symbiont strains. The abundant gut isolate Trichosporon asahii showed potent activity against Staphylococcus aureus, but the responsible compounds remain to be investigated.

Public defence Anneleen Robberechts 27/04/2024 - Implementation of type 3 medication review in Flanders: obstacles and opportunities - Department Pharmaceutical Sciences

Promotors: Prof. Hans De Loof - Prof. Guido De Meyer - Prof. Stéphane Steurbaut

Location:

Aula Q002, gebouw Q

Campus Drie Eiken

Inschrijven via: https://www.kava.be/openbareverdediging

Abstract:

Type 3 medication reviews (MR3s) represent a collaborative effort between healthcare providers and patients, aiming to optimize medication therapy and promote better health outcomes. This thesis examines the sustainable integration of MR3s within Flemish community pharmacies, delving into the multifaceted aspects of successful implementation, quality assessment, challenges encountered, and effective strategies for seamless integration into the healthcare system. The thesis begins with a clear introduction, breaking down the multiple complex definitions, categories, and historical development of MR practices.

The thesis subsequently elaborates on the insights obtained from the perspectives of community pharmacists and general practitioners (GPs) who participated in the MR3 pilot projects.  These insights, using a qualitative research methodology, unveil a noticeable eagerness and readiness among healthcare professionals for the incorporation of MR3 in practice. The project faced certain barriers for future implementation, such as a shortage of time and a lack of commensurate compensation.

Moreover, the perspectives of patients who participated in the pilot project were also examined though qualitative research. Although most patients were enthusiastic about this new service, a lack of patients’ understanding of the whole process was also observed. Therefore, better communication to patients by pharmacists and GPs about the goals and components of this type of medication review is needed, with the added benefit of increased efficiency.

Central to the thesis is the imperative of quality assessment, which is instrumental in ensuring the efficacy and sustainability of MR3 interventions. Initially, four target groups were surveyed via an electronic questionnaire to identify the essential quality criteria for a qualitative MR3. There was widespread agreement about the hierarchy of the quality assessment criteria for MR3s. Minor differences were related to the experience of the participants. With these results and a small number of suggested extra criteria, a quality assessment instrument for MR3 was created, the BRANT-MERQS (Brussels Antwerp Medication Review Quality Score) scoring table. This scoring tool was tested in two subprojects, namely in patients with rheumatoid arthritis (subproject 1, RA) and in patients with type 2 diabetes (subproject 2, T2DM). The results demonstrated the feasibility of MR3 quality assessment through a scoring system. However, this study also unveiled the tool's current imperfections and highlighted the ongoing need for refinement, something expected of a new service in an implementation phase.

In conclusion, this research not only demonstrates the feasibility and enthusiasm surrounding MR3 but also offers nuanced insights and pragmatic recommendations reinforcing clinical practice and improving patient care through the judicious integration  of MR3s with quality assurances into the healthcare landscape.

Public defence Pathy Kibungu Kembelo 03/05/2024 - Ethnobotanical study and characterization of medicinal plants used by populations of Kisantu and Mbanza-Ngungu territories, Kongo-Central Province (DR Congo) - Department Pharmaceutical Sciences

Promotors:

Prof. dr. Luc Pieters (promotor UAntwerpen)

Prof. dr. Eduardo de la Pena (promotor UGent)

External promotors:

Prof. dr. Honoré Belesi

Prof. dr. Patrick Van Damme

Location:

Room A0.1 Azalea, Coupure links 653, 9000, Ghent (UGENT)

Online participation: https://www.fbw.ugent.be/doctorate/PathyKibunguKembelo

Abstract:

The DR Congo pharmacopoeia contains monographs on various medicinal plants with pharmacological properties, whose knowledge and use is rapidly eroding. To document the remaining knowledge, we conducted an interdisciplinary study integrating ethnobotany, ecology, and phytochemistry. The study aimed to assess Kongo people's traditional medicinal expertise in Kisantu and Mbanza-Ngungu territories, identify the most important medicinal plants, as well as the key people who hold this knowledge, and to study  the vegetation of Mbanza-Ngungu, with an emphasis on the most important medicinal plant species. In addition, a phytochemical profiling of selected important medicinal plants for which no relevant phytochemical information was available in the literature was performed to provide a scientific basis for their use in the Kongo traditional medicine. Ethnobotanical data were gathered from 188 informants, including herbalists, traditional health practitioners, and curing healers, using snowball sampling and semi-structured interviews. Quantitative ethnobotanical parameters such as UV and IAR allowed identification of 54 important medicinal plant species from the 231 species inventoried. These metrics, combined with ICF, introduced the "Species Therapeutic Potential" (STP), facilitating the selection of 35 Kongo key medicinal plant species, most of which (33) are chemically well documented. Only two native species: Commelina africana L. and Kalaharia uncinata (Schinz) Moldenke showed no relevant phytochemical information and were therefore subjected to further phytochemical investigation. Moreover, statistical analysis revealed that Kongo ethnomedicinal knowledge varies by gender, location, and practitioner categories, with men, curing healer and urban phytotherapists of Mbanza-Ngungu exhibiting advanced expertise compared to women, rural respondents, and traditional health practitioners. The vegetation of the region, which included savannah, anthropized areas, swamps and dry forests, hosted 709 botanical species (113 families, 446 genera). It reflected the degraded Guinean-Congo flora, dominated by Fabaceae, phanerophytes, herbs, sarcochores and species characteristic of the phytosociological group Musango-Terminalietea. The analysis also revealed similarities between adjacent plant formations, especially dryland forests and savannahs. Most plant species showed limited ecological niche and declining availability, especially medicinal plants, that could be considered as rare or endangered. Phytochemical analysis via UPLC-ESI-QTOF-MS identified flavonoids, hydroxycinnamic acids, and lignanamides in Commelina africana, with antioxidant, anti-inflammatory, and antimicrobial effects. Similarly, phenylethanoid glycosides, phenolic acids, and iridoid glycosides were found in K. uncinata extracts, with reported antiviral, anti-inflammatory, and antioxidant properties. Further studies, including NMR spectrometry, can be conducted to elucidate the chemical structure of compounds. Additionally, biological studies can assess additive and synergistic effects to enhance extracts potency and efficacy. Conservation efforts, both in situ and ex situ, are essential to safeguard rare plants and ensure their availability.

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Public defence Rob Geens 19/04/2024 - Investigating the role of intrinsic disorder in the structure-function relationship of the circumsporozoite protein from the malaria parasite - Department Pharmaceutical Sciences

Promotors:

Prof. dr. Yann Sterckx (promotor UA)

Prof. dr. Peter Tompa (promotor VUB)

dr. Oleksandr Volkov (promotor VUB)

Location: Aula Q002, building Q

Abstract:

Malaria is a vector-born disease that poses a global health threat to almost half of the world's population. Annually, about 250 million cases over 600 thousand deaths are reported worldwide. The most afflicted region is Sub-Saharan-Africa, and children under the age of five form the most vulnerable group of the population. This devastating disease is caused by parasites belong to the Plasmodium genus, which are injected in their sporozoite (SPZ) life form into the skin of the human host during blood feeding of an infected female Anopheles mosquito. From there, they travel to the liver and differentiate into tens of thousands of merozoites, which later on re-occur in the circulation to asexually replicate within erythrocytes and cause the notorious malaria pathology.

The circumsporozoite protein (CSP) is the SPZ’s main surface antigen, which uniformly coats its surface and plays a pivotal role in its immunobiology, both in the insect and the human hosts. In addition, it constitutes the immunodominant target for host antibodies raised against the SPZ life stage, and therefore forms the basis of the only two WHO-approved malaria vaccines today. CSP has a conserved modular buildup consisting of: i) an N-terminal domain (CSP_N); ii) a linker region composed of tandem repeats (CSP_rep); iii) a C-terminal domain (CSP_C) that consist of a flexible junctional region that connects the globular part of the domain (αTSR domain) to CSP_rep; and iv) a C-terminal GPI anchor attaching the antigen to the SPZ plasma membrane. Despite significant advances made during the past decades and the clear relevance of CSP in SPZ immunobiology, the structures of full-length CSP (CSP_FL) and CSP_N, and most aspects of its structure-function relationship, remain enigmatic.

The research focus of this doctorate was to investigate the role of intrinsic disorder in the structure-function relationship of CSP, with particular attention devoted to CSP from P. falciparum (PfaCSP) and P. vivax (PviCSP) as the most dominant human-infective species, and P. berghei (PbeCSP) as a commonly used model system. Several constructs corresponding to CSP_FL or truncates thereof were recombinantly produced and purified. Subsequently, an interdisciplinary approach was used to characterize CSP_N and CSP_FL as intrinsically disordered regions and proteins, respectively. In addition, we present the first all-atom conformational ensemble of PfaCSP and validate that the surface antigen is subject to a conformation change during SPZ development in the mosquito. Finally, the molecular details of two CSP-mediated ligand interactions that play important roles in parasite biology were investigated.

Public defence Alan Kerstjens 16/04/2024 - Computational design of synthesizable molecules by imitating reference Chemistry - Department Pharmaceutical Sciences

Promotor: Prof. dr. Hans De Winter

Location: Aula O5, building O, Campus Drie Eiken

Abstract:

De novo molecular design is the practice of generating molecules with desirable properties from scratch. When done computationally the proposed molecules tend to be difficult to synthesize and overall chemically unappealing. In this work we present methods to extract patterns from available data and bias molecular design towards synthetically accessible chemistry. Given a list of known synthesizable compounds, we design molecules with the same chemical features, under the assumption that this resemblance increases the likelihood of them being synthesizable as well.

Molecules were designed using evolutionary algorithms that breed populations of molecules by modifying their molecular graphs. The designed molecules were constrained to be composed of chemical features that are prevalent in reference chemistry. Different ways of defining chemical features were explored, and we determined that mimicking small circular atomic environments allowed us to design reasonably fit and easy to synthesize molecules.

We developed an evolutionary algorithm that constructs molecules with desirable chemical features by assembling molecular fragments in a computationally efficient way, and showed how it outperformed competing algorithms in both the quality of the generated molecules and its ability to navigate chemical space effectively.

We also developed a molecule correction algorithm that can identify flaws in molecules and sanitize them to make the molecules more desirable. Said tool can be used to post-process molecules generated elsehow, or integrated into molecule generators to enforce chemical constraints in a hands-off fashion.

Public defence Anne-Sophie Weyns 11/04/2024 -Exploring the therapeutic potential of French Maritime Pine Bark Extract in Attention-Deficit Hyperactivity Disorder and Arterial Stiffness - Department Pharmaceutical Sciences

Promotors:

Prof. dr. Nina Hermans 

Prof. dr. Luc Pieters

Location: Aula Q002, building Q, Campus Drie Eiken

Abstract:

Non-communicable diseases (NCDs) including neurodevelopmental diseases such as Attention-Deficit Hyperactivity Disorder (ADHD) and cardiovascular diseases, represent a growing global health problem that impacts the quality of life. Polyphenols, found in dietary sources like fruits and vegetables, offer promising health benefits, notably antioxidant and anti-inflammatory properties, potentially alleviating NCD’s burden, associated with oxidative stress and inflammation. This PhD thesis explores a patented polyphenol-rich extract from the French Maritime Pine bark (PBE; Pinus pinaster) as a potential novel treatment option for NCDs due to its antioxidant and anti-inflammatory activities.

Firstly, PBE assesses the role in ADHD, a prevalent neurodevelopmental disorder with implicated dopaminergic dysfunction and immune and oxidant-antioxidant imbalances. Since methylphenidate, the first-choice medication for ADHD, is linked to adverse effects, novel treatment options are highly warranted. A 10-week double-blind, randomised clinical trial compared PBE to placebo and methylphenidate, and revealed that both methylphenidate and PBE significantly improved hyperactivity/impulsivity, and proved that PBE is a good alternative for methylphenidate. Besides, loss of appetite, indicated by the neurochemical parameter neuropeptide Y, and weight loss were observed for methylphenidate, whereas a significant weight gain was established for PBE. Moreover, a potential prebiotic effect of PBE was explored by investigating its impact on the gut microbiota via amplicon sequencing of the full length 16s rRNA ribosomal subunit. Though no clear prebiotic effects were observed, subtle changes emerged.

Secondly, PBE’s therapeutic potential in cardiovascular diseases, particularly arterial stiffness, was studied. Both oxidative stress and inflammation appear to play a role in arterial stiffness and can lead to structural and functional changes in the arterial wall, impair endothelial function, and thus promote the development of cardiovascular diseases. PBE was tested in an array of in vitro assays on oxidative and inflammatory mechanisms contributing to arterial stiffness and results showed PBE’s inhibition of cyclooxygenase enzymes and NF-κB activity and alterations in protein expression levels associated with pathways underlying arterial stiffness.

Lastly, PBE was subjected to in vitro gastrointestinal biotransformation, which mimics the gastric, intestinal and colonic phase, including faecal fermentation in an anaerobic environment. Samples were taken at different timepoints and analysed with UHPLC-ESI-QTOF-MS. Extensive biotransformation of PBE was observed and various biotransformation products could be identified.

To conclude, our study provides valuable insights into the complex interplay between polyphenols, biotransformation, and potential health benefits, contributing to the ongoing efforts to address the global burden of NCDs.

Public defence Jens Van fraeyenhove 15/03/2024 - Activation of ERBB4 Attenuates Atrial Fibrillation in Mice -Department Pharmaceutical Sciences

Promotors: 

Prof. dr. Gilles De Keulenaer 

Prof. dr. Guido De Meyer 

Location: Aula Q002, building Q, Campus Drie Eiken

Abstract:

Atrial fibrillation (AF), the most common arrhythmia, results from electrical and structural remodeling of the atria (atrial myopathy). Although, inflammation and fibrosis are important underlying mechanisms of AF and atrial myopathy, current therapies focus on symptomatic treatment and do not target the structural problem. This implies a high medical need for new AF therapies. The neuregulin-1 (NRG1)/ERBB system plays a compensatory role during heart failure by acting on cardiomyocytes and mitigates fibrosis in different tissues, including the myocardium. In this thesis, we evaluated the effects of ERBB4 stimulation on atrial fibrosis and atrial fibrillation (AF) in a transgenic mouse model and mouse models of clinically important risk factors of AF (hypertension and obesity). Selective ERBB4 stimulation was induced by JK07, a recombinant fusion protein consisting of a receptor-activating fragment of human NRG1 and a humanized antagonistic ERBB3 immunoglobulin G1 antibody. In chapter 4, we confirmed that JK07 more selectively stimulates ERBB4 than ERBB3. As anticipated from its design, JK07 induced ERBB4/4 dimerization with similar potency but significantly higher efficacy than ERBB2/3 dimerization compared to recombinant NRG1. Moreover, we observed that JK07 induced ERBB4 phosphorylation and activation of downstream signaling pathways in cultured atrial cardiomyocytes. Here, we also described that JK07 reduced the upregulation of inflammatory cytokines in cultured macrophages, and the upregulation of collagen mRNA levels (type I and III) in atrial tissue samples, recapitulating the anti-inflammatory and anti-fibrotic effects previously observed with NRG1. In chapter 5, we evaluated the effects of NRG1 and JK07 therapy in a transgenic mouse model of cardiac-specific overexpression of TNF-a. Both NRG1 and JK07 did not prevent inflammatory pathways, fibrosis, and dilation of atria in these transgenic mice. Despite the early onset of severe atrial remodeling in these transgenic mice, we did not observe spontaneous AF (through sustained telemetry monitoring).

To further investigate the effects of ERBB4 activation, AF inducibility -by programmed electrical stimulation- was assessed in two distinct mouse models, as described in chapter 6. In a first model, mice were exposed to a high concentration of angiotensin II (Ang II), and in a second model, mice received a high fat diet (HFD). We demonstrated that JK07 treatment, started upon induction of hypertension or with HFD, prevented AF inducibility and reduced the total AF duration. Despite developing increased AF inducibility, these mice did not show increased atrial fibrosis. This implies that ERBB4 activation, by JK07 treatment, in mice exerts anti-AF effects through mechanisms other than anti-fibrotic effects. Moreover, when treatment with JK07 in the Ang II-induced hypertension model was given for only a brief period (one week, therapeutic regimen), similar effects on AF inducibility were observed endorsing the hypothesis. In chapter 7 we tested if JK07 has antiarrhythmic effects by acting directly on the electrical properties of atrial cardiomyocytes. Following RNA-sequencing experiments, we observed more than 5000 DEGs in atrial cardiomyocytes after ERBB4 activation, of which 1% were distinct ion channel genes. Suggesting that ERBB4 activation may also regulate electrical activity of atrial cardiomyocytes. This was further substantiated in experiments with conditionally immortalized atrial myocyte monolayers, showing that JK07 completely prevented inducibility of reentrant activity (spiral waves). Overall, this study identifies the ERBB4 signaling pathway as a new therapy to treat AF, targeting electrophysiological components of atrial myopathy. These observations may open new avenues to treat AF in human patients.

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Public defence Siham Benramdane 13/02/2024 - Design and synthesis of selective DPP9 inhibitors- Department Pharmaceutical Sciences

Promotors:

Prof. dr. Pieter Van der Veken
Prof. dr. Hans De Winter

Location: Aula R2, gebouw R, Campus Drie Eiken

Abstract:

The aim of this work is to design and synthesize selective inhibitors targeting dipeptidyl peptidase 9 (DPP9), a challenging goal considering the high sequence similarity between DPP9 and other related enzymes, such as DPP4 but especially DPP8. DPP9 has been gaining interest recently as a pivotal role in inflammatory cell death (pyroptosis) could be attributed to DPP9, which lead to the identification of DPP9 as a therapeutic target in acute myeloid leukemia (AML).

The thesis primarily concentrates on crafting dipeptide-derived DPP9 inhibitors, inspired by the N-terminal dipeptides of known substrates. By using the marketed DPP4 inhibitor Vildagliptin as a reference, the design strategy involves a bimodular structure incorporating a proline-mimetic as the P1 unit, with or without a nitrile electrophilic warhead, while allowing certain flexibility in the P2 fragment. Vildagliptin's N-(3-hydroxyadamantyl)glycine moiety was thoroughily investigated, by replacing the alcohol group with a variety of functional groups. As a result, several promising compounds have been obtained, exhibiting promising potency and selectivity for DPP9. Notably, compounds 42 and 47 emerged as top candidates, exhibiting low nanomolar DPP9 affinity (3 – 4 nM) alongside significant DPP9-to-DPP8 selectivity indices, in the magnitude of two orders.

This comprehensive study not only presents a strategic evolution in designing DPP9-selective inhibitors, but also sheds light on the structural nuances influencing selectivity, providing valuable insights for future drug development and understanding enzyme differentiation in this class.

Public defence Karuna Adhikari 30/01/2024 - Design, synthesis, and evaluation of trans-cyclooctene (TCO) and cis- dioxolane fused TCO (d-TCO) probes for bioorthogonal pretargeted PET imaging - Department Pharmaceutical Sciences

Promotors:

Prof. dr. Koen Augustyns
Prof. dr. Sigrid Stroobants
Prof. dr. Filipe Elvas

Location: Aula O.05, gebouw O, Campus Drie Eiken

Abstract:

Immuno-positron emission tomography (PET) imaging using radiolabeled monoclonal antibodies (mAbs) is a powerful tool with high specificity and sensitivity for cancer diagnosis and therapy response monitoring. However, slow mAb pharmacokinetics require the use of long-lived radioisotopes, resulting in suboptimal target-to-background ratios and high radiation exposure in patients. To address this, a pretargeting strategy based on the bioorthogonal inverse electron demand Diels-Alder (IEDDA) reaction between trans-cyclooctene (TCO) and tetrazine was proposed. In this approach, an mAb-conjugated bioorthogonal tag is initially administered, allowing clearance from circulation. Subsequently, a radiolabeled reaction partner is injected, which covalently binds to the tag on the mAb. This allows the use of short-lived radioisotopes such as Fluorine-18, consequently minimizing radiation exposure while enhancing in vivo imaging contrast. The TCO-tetrazine ligation is a perfect candidate and has been extensively studied for this application. In this project, we focused on the development and characterization of fluorinated TCO and cis-dioxolane-fused TCO (d-TCO) derivatives as radiolabeled reaction partners with CC49 (anti-TAG-72) mAb-conjugated tetrazine tags for in vivo pretargeted imaging in mice bearing LS174T colorectal tumor xenografts. Based on previous studies, a novel [¹⁸F]FTCO derivative with a chelator linker was developed and evaluated in a mouse model of colorectal cancer xenografts. The ¹⁸F-labeled TCO derivative showed improved in vivo stability and demonstrated clear visualization of tumor tissue in the pretargeted group when compared to the control. Furthermore, a small library of d-TCO derivatives with enhanced reaction kinetics and diverse linkers was developed. Two of these compounds demonstrated excellent in vivo ligation in a pre-targeted blocking assay. Further radiochemical development and analysis revealed higher in vivo stability and favorable clearance of the ¹⁸F-labeled d-TCO derivative containing a squaramide linker. This radiotracer also showed a significant difference in tumor uptake compared with the control group in pretargeted imaging. Additionally, d-TCO derivatives were tested in a turn-on fluorescence-based cell permeability assay to visualize internalizing or intracellular targets. A d-TCO derivative containing a cyclobutyl fluoride linker exhibited excellent intracellular ligation in this assay. Although further fine-tuning of the linkers is required to obtain improved image contrast, this project demonstrates the potential of TCO- and d-TCO-based radiotracers in pretargeted PET imaging and provides insights for future probe development.