Public defence Bahaa Shaqour 15/02/2022 - Can 3D printing technologies advance the production of drug delivery systems? - Department Pharmaceutical Sciences

Promotors: Prof. dr. Paul Cos - dr. Koen Beyers

ONLINE DEFENCE: https://eu.bbcollab.com/guest/5a5ed7b9cffe4031a7cad5ab8ab1d89d

Abstract:

Can 3D printing technologies advance the production of drug delivery systems?

Since the invention of 3D printing technologies, huge interest has been allocated to exploring their potential in many fields. Fabricating objects in a customized and a cost-effective manner was the biggest advantage that drew the attention of the industrial and the scientific communities. The medical field benefited from 3D printing in many aspects such as educational training and personalized medicine. In this study, 3D printing technologies were used as a tool for producing drug delivery systems and for producing tools by which medical devices such as scaffolds or catheters can be fabricated.

First, fused filament fabrication (FFF) was used as a tool for producing drug delivery systems. The replacement of the conventional filament-based extrusion system by a screw driven extrusion system was investigated. Results from this part of the project illustrated the performance of different feedstock preparation techniques in term of the drug loading efficiency, homogeneity among others. The use of the mini single screw extruder showed how we can broaden the formulations by eliminating the reliance on the filament mechanical properties during printing.

Afterward, selective laser melting was used for producing a novel nozzle design by which hollow fibers may be extruded. This nozzle, when fitted into an FFF 3D printer, allows the fabrication of scaffolds with hollow fibers that may be used in tissue regeneration and drug delivery applications. Moreover, this nozzle was used to fabricate catheters loaded with Niclosamide, a repurposed drug with antibacterial and antibiofilm properties. Niclosamide shows good thermal stability during processing and the produced catheters showed good antibacterial and antibiofilm activity in vitro.

In conclusion, future research shall focus on adapting 3D printing technologies to meet the strict requirements set by legislators. Additionally, exploring more polymers that are better suited for FFF 3D printing along with screening the relevant properties of incorporated drugs.

Public defence Maarten Degreef 09/02/2022 - Routine forensic analysis of psychoactive substances: In-depth assessment of instrumentation, confirmation and quantification - Department Pharmaceutical Sciences

Promotors: prof. dr. Alexander van Nuijs - dr. Kristof Maudens

ONLINE DEFENCE 4pm: https://eu.bbcollab.com/guest/09d6eb1dbf374944b252e0e57352b315

Abstract:

Routine forensic analysis of psychoactiv e substances:
In-depth assessment of instrumentation, confirmation and quantification.

Toxicological analysis requires broad screening for both therapeutically prescribed and illicit compounds, including their metabolites. This research sought to develop reliable, fast and easy-to-use analytical methods, focussing on three types of psychoactive substances: antidepressants, antipsychotics and benzodiazepines and Z-drugs. They are increasingly prescribed for long-term use, frequently encountered in routine forensic samples and often require close monitoring due to their potential for serious adverse events.
The first objective was to develop targeted confirmation methods using triple quadrupole mass spectrometry (QQQ). A liquid-liquid extraction on 200 μL sample yielded sufficient sensitivity for the intended subtherapeutic detection limits. The methods were fully validated according to international guidelines and tested on internal and external quality control samples. These methods allow for unequivocal identification and accurate concentration determination of the substances under investigation in blood.
The second objective was to develop screening methods: one using QQQ operated in triggered multiple reaction monitoring mode (tMRM) and another using untargeted, quadrupole time-of-flight mass spectrometry (QTOF). The performance of both methods regarding correct identifications was comparable and within acceptable error margins. The limited number of false negative results predominantly consisted of compounds present at low to sub-ng/mL concentrations. More false positive results were obtained for the QTOF versus the tMRM method, the majority being misidentifications of phenelzine and prothipendyl. Semi-quantitation was feasible for tMRM analysis only, where it resulted in sufficiently accurate concentration determinations at a reduced cost compared to routine methods.
The third objective involved the critical assessment of method implementation in routine analyses. The semi-quantitative screening methods performed well and – following continued validation on case samples – could be implemented in routine toxicological analysis, without the need for highly trained, specialised personnel. QTOF screening is recommended over targeted tMRM screening, as it allows for easy expansion of the number of analytes included and for retrospective data analysis, should new information become available. Positive QTOF findings could be verified and semi-quantified by tMRM, without the need for additional sample preparation or further use of, often limited, sample volume. The high accuracy of the semi-quantitative tMRM results may often suffice for interpretation of case findings, particularly given the limitations associated with post-mortem samples. Confirmation of findings and accurate concentration determination may be required depending on the case, for which purpose fully validated, targeted QQQ methods have been developed.

Pulic Defence Michelle De bruyn 28/01/2022 - Profiling of proteases as potential therapeutic targets – Focus on inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) - Department Pharmaceutical Sciences

Promotors: Prof. Ingrid De Meester - Prof. Benedicte De Winter

ONLINE DEFENCE: https://eu.bbcollab.com/guest/227ec13e6fd14195ab7a60ef3fbf60fe

Abstract:

Profiling of proteases as potential therapeutic targets – Focus on inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS)
De bruyn Michelle
Proteases are potential therapeutic targets in several diseases such as gastrointestinal disorders and lung infections. However, the role of proteases in the pathophysiology of these diseases is often not fully elucidated yet. Therefore, this doctoral research explored the role of proteases in biological tissues and investigated these enzymes as therapeutic targets in inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS).
Insight into the involvement of proteases in IBD and IBS was first gained by assessing the proteolytic activity using fluorogenic substrates in colonic tissue and fecal samples from a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model in the acute (IBD) and post-inflammatory (IBS) phase. Obtained results showed an elevated trypsin-like activity in colonic tissue from rats in the post-inflammatory phase of colitis. Furthermore, intracolonic treatment of these rats with the broad-spectrum serine protease inhibitor UAMC-00050 decreased this trypsin-like activity. Therefore, these results highlighted trypsin-like proteases as important enzymes and potential therapeutic targets in IBS.
Next to the fluorogenic assays, a peptide-based mass spectrometry assay was used to further identify potential therapeutic targets in the same animal models for IBD and IBS. A set of peptides relevant in pain and inflammation was incubated with a protease source for fragmentation and the cleavage products were identified with mass spectrometry to obtain specific cleavage patterns. First, differential cleavage patterns were observed for a set of trypsin- and elastase-like proteases present in the human colon. These patterns could be used to understand which trypsin-like or elastase-like enzymes are active in a given biological sample. Second, the processing of the same peptides by colon samples from the TNBS-induced colitis model was investigated and pointed to a set of proteases as potential targets for therapy. An increased cleavage of several peptides was observed in the colon from acute colitis rats and suggested thrombin, chymotrypsin-like proteases and a set of proteases with different specificities as potential therapeutic targets in IBD. Finally, the value of the peptide-based mass spectrometry assay was proven in a different sample type by applying it to lung tissue of mice infected with Pseudomonas aeruginosa.
In conclusion, this doctoral research contributed to the unraveling of the role of proteases in the pathophysiology of IBD and IBS and identified a set of proteases for which further investigation as potential therapeutic targets is worthwhile. However, further research is required to elucidate the exact mechanisms underlying IBD and IBS and the function of proteases therein.

Public defence Juliana Aizawa Porto de Abreu 12/01/2022 - 4pm- Biofilm-related pneumonia: Evaluation of anti-infective devices and in vivo bacterial persistence - Department Pharmaceutical Sciences

Promotor: Prof. Paul Cos

ONLINE DEFENCE: https://eu.bbcollab.com/guest/8596b0b6bc074b6895c41cebedf220b3​

Abstract:

Biofilm-related infections are a major threat to healthcare as they underlie treatment failure. The main question we pose is: Can we improve strategies to prevent biofilm-related lung infections? To prevent such infections, we must first understand how biofilms contribute to their chronic nature. The in vitro biofilm differs from the in vivo biofilm and we aimed at evaluating both. For in vitro evaluation, an interlaboratory effort was made to compare three methods of quantification of Staphylococcus aureus biofilm formation in 96-well microtiter plates: crystal violet, resazurin, and plate counts. Viable plate counts had the best reproducibility and responsiveness. For in vivo evaluation, the development and optimization of mouse models of pneumonia was conducted. We addressed questions related to biofilm lung infections that occur in diseases such as ventilator-associated pneumonia and cystic fibrosis. A S. aureus ventilator-associated pneumonia mouse model was validated with ciprofloxacin loaded thermoplastic polyurethane tubes, since ciprofloxacin is a reference drug with known antibacterial and antibiofilm activity. This model was then used to evaluate commercial tubes with polylactic acid coating loaded with fingolimod, a repurposed drug with antibacterial and antibiofilm potential. The model was useful to evaluate the anti-infective properties of personalized endotracheal tubes. Furthermore, an established Pseudomonas aeruginosa alginate bead lung infection mouse model was applied for the first time to study antibacterial persistence, which contributes to chronic infections such as cystic fibrosis, and a positive correlation between the in vitro and in vivo survival levels was found. Therefore, the pre-clinical work developed in this thesis was an important step towards improving strategies to prevent biofilm-related lung infections.

Public defence Ella Dendooven 21/12/2021 - Contact allergy caused by natural and synthetic components in medical devices, adhesives in particular - Department Pharmaceutical Sciences

Promotors: Prof. dr. Kenne Foubert - prof. dr. Olivier Aerts

ONLINE DEFENCE: https://eu.bbcollab.com/guest/60a4394c133e4c6c891bfb37bfa98e5a

Abstract: 

The number and use of “medical devices” is increasing rapidly in several para(medical) disciplines. These devices are used to aid in the diagnosis, treatment, monitoring and prevention of several human diseases. Although they are of great importance in health care, adverse cutaneous reactions, including allergic contact dermatitis (ACD), may occur when using  them. Such allergic skin reactions can be caused by particular chemicals present in these devices, adhesives in particular, i.e., synthetic substances (e.g., “acrylates” and “isocyanates”), natural components (e.g., “colophonium”, “terpenes such as D-limonene” and “sesquiterpene lactones”) or other substances (e.g., sulphites). A well-known and recent example are contact-allergic skin reactions caused by acrylates, such as isobornyl acrylate (IBOA), used as adhesives (tackifiers) in glucose sensors (e.g., Freestyle^® Libre) and insulin infusion sets (pumps), used by many diabetes patients worldwide.

This research project concerns a cooperation between a chemical analytical laboratory (NatuRA, University of Antwerp) and a clinical department (Dermatology, University Hospital Antwerp/UZA).

Apart from giving a general overview of contact allergies from medical devices (epidemiology), the main objective of this project is to reveal the (qualitative/quantitative) presence of some important contact allergens in particular types of medical devices, notably by means of chemical analyses, including, among others, gas chromatography-mass spectrometry (GC/MS). Furthermore, we aim to give insights into optimization of patch test preparations with (some of) the identified substances (e.g., how to patch test them, how to interpret results). Such patch tests are important diagnostic skin tests that can be used in the clinic to diagnose ACD caused by medical devices.

Public defence Besa Emini Veseli 14/12/2021 - Search for new therapeutic options to prevent and treat plaque neovascularization and atherogenesis - Department Pharmaceutical Sciences

Promotors: Prof. dr. Guido De Meyer - Prof. dr. Mirela Delibegovic

ONLINE DEFENCE: https://eu.bbcollab.com/guest/20f57b1466de4f28b2cbc0cd14bc97dd

ABSTRACT:

Atherosclerosis, the most common cause of cardiovascular diseases (CVD), is characterized by progressive inflammation and accumulation of lipids in the arterial vessel wall starting early in life. When the disease reaches an advanced stage, one of the detrimental factors triggering acute end-point events such as myocardial infarction and stroke, is the formation of new blood vessels within the plaque. These vessels are formed through a complex orchestrated growth known as angiogenesis. In this thesis we aimed at, on one hand, stabilizing the atherosclerotic plaque by targeting intraplaque angiogenesis, and on the other, reducing atherosclerosis by the use of natural compounds.

Key players in the process of angiogenesis are endothelial cells, and recently, endothelial cell metabolism has gained attention in being the target in treating pathological angiogenesis. Among, it is PFKFB3 protein which is a key regulatory enzyme of the glycolytic pathway. A variety of inhibitors of this important target have been studied, and a plethora of biological effects related to the process of angiogenesis have been reported. The most well studied one is small molecule 3PO. However, since recent studies have disputed its mechanism of action, we sought at defining the thermodynamic profile of the 3PO-PFKFB3 interaction. To this end, we revealed that the biological effects leading to reduced angiogenesis upon 3PO use were PFKFB3-independent as there is no biomolecular interaction between these two components. In order to reiterate the role of PFKFB3 in angiogenesis, we made use of a bona fide PFKFB3 inhibitor, compound AZ67. This allowed us to demonstrate that pharmacological inhibition of PFKFB3 enzyme leads to reduced angiogenesis in vitro and in vivo, making this a worthwhile target to investigate, not only from a therapeutic perspective in atherosclerosis, but also in oncology and other angiogenesis-related disorders.

On the other hand, the new approaches in targeting atherosclerosis development by use of natural compounds like chalcones (belonging to calorie-restriction group) showed beneficial effects. The use of 4,4’-dimethoxychalcone (possessing a wide range of health benefits) resulted in reduced atherosclerotic plaque formation in an in vivo model of atherosclerosis. Other in vivo, and in vitro parameters were also altered in favor of atheroprotection. These observations reveal an additional beneficial property of DMC, which, as a natural compound can be regarded for further therapeutic possibilities in treating atherosclerotic cardiovascular disease.

Public Defence Olga Balabon 17/11/2021: Profiling and structural investigation of dihydrotriazine-based DHFR inhibitors and hydantoin-derived DprE1 inhibitors in search of novel antimycobacterials - Department Pharmaceutical Sciences

Promotors: 

Prof. dr. Koen Augustyns 

Prof. dr. Pieter Van der Veken 

dr. Robert Bates

ONLINE DEFENCE:  https://eu.bbcollab.com/guest/e05271d533074dfbb1059e1bdfc629dc

Abstract:

The presented research addresses the urgent need to discover new anti-TB agents with novel mechanism of actions. It was performed within the OpenMedChem EID-ITN project (FP7) between the University of Antwerp and GlaxoSmithKline and had a focus on early hit-to-lead anti-tubercular drug design and development.

The project was concentrated on hit exploration and hit-to-lead optimization of two novel chemical series of Mtb DHFR and DprE1 inhibitors to provide potent and selective compounds against M. tuberculosis. The initial hits identified during two different high throughput screening (HTS) campaigns performed by GSK formed the starting point for the research.

The first compound family, identified in the whole-cell HTS campaign against M. bovis with hit confirmation in M. tuberculosis, was proven to inhibit the MtbDHFR (dihydrofolate reductase) enzyme. The initial hits demonstrated very good whole-cell and enzymatic potency, coupled however with significant human-cell toxicity. A scaffold hopping approach was then applied in order to overcome toxicity and selectivity issues while preserving the crucial pharmacophore. Unfortunately, the obtained analogues showed no cellular or DHFR inhibition activity. Therefore, this line of research was discontinued.

A target-based HTS campaign against the mycobacteria-specific enzyme flavo-enzyme deca-prenylphosphoryl-beta-D-ribose 2-epimerase (DprE1) revealed a novel hydantoin-based family of potent DprE1 inhibitors. The follow-up hit-to-lead optimization program concentrated on removal of potential liabilities and safety evaluation of the series was performed in two rounds producing an extensive SAR around the initial hit. A number of highly active DprE1 inhibitors with sub-micromolar cellular potencies and balanced physicochemical profiles were delivered. Moreover, the discussed series showed no cytotoxicity and was found selective against mycobacteria. The proof-of-concept of in vivo activity was demonstrated.

ONLINE DEFENCE: https://eu.bbcollab.com/guest/5d1877069c6e4d65967b9d5e5fa7f8f7

Since 2011, the World Health Organization has encouraged a global phase-down of the use of dental amalgam and actively supported the use of alternative, resin-based dental materials. Resin-based dental materials usually consist of a mixture of inorganic filler particles attached to a resin matrix. The organic resin-based matrix consists most commonly of (meth)acrylate monomers and functional additives (photo- and co-initiators, stabilizers and others). Upon restoration, the resin-based materials need to be cured by polymerization of the resin matrix, but this process is not complete, resulting in the release of chemicals after restorative treatments.

This thesis aimed to answer three important questions regarding resin-based dental materials:

1.       What are the ingredients of dental composites and sealants?

2.       To which chemicals are patients possibly exposed during and after dental restoration surgery?

3.       To what extent are dental composites degraded after restoration, and which degradation products do they release?

The composition of resin-based dental materials was investigated using liquid chromatography coupled to high-resolution mass spectrometry. Major components, such as the monomers BisEMA, BisGMA, TEGDMA and UDMA, were identified although they were not always stated in the MSDSs. Minor components included photoinitiators, such as ethyl 4-dimethyl aminobenzoate (EDMAB) and (meth)acrylate impurities originating from production of main ingredients.

Unreacted monomers and not covalently bound additives can leach out of the polymer matrix after restorative treatment, a process which can be aggravated by degradation of the polymer due to exposure to various stressors in the mouth, such as pH and enzymes in saliva. To test this hypothesis, dental monomers and polymerized dental resin-based materials were immersed for 24 h in chemical media and human pooled saliva in order to identify leached monomers and degradation products.

During in vitro chemical degradation, uncured monomers were rapidly hydrolyzed to mono- and demethacrylated degradation products. Degradation in human pooled saliva for 24 h to mimic the in vivo situation, resulted in the identification of both monomers and their degradation products.

The liver metabolism of leached monomers was simulated using an in-house assay using human liver microsomes and human liver cytosols. During Phase I incubation the (meth)acrylic acid in the monomers was rapidly removed followed by oxidative and hydroxylation pathways. For BisPMA, an O-dealkylation pathway resulted in the formation of BPA. Carbamate groups present in TCD-DI-HEA and UDMA were resistant to biotransformation reactions. Phase II biotransformation products were only observed for BisPMA and included conjugation reactions with sulphate and glucuronic acid.

In the final part of the thesis, a study was carried out for the first time aiming to quantitatively and qualitatively monitor the release of parent compounds and their degradation products in saliva from patients undergoing multiple restorations. Saliva samples were collected at various time points after restorative treatment. Monomers are only present in saliva shortly after restoration, but several degradation products can be detected weeks after the restoration confirming a long-term release.

Results from this thesis show that patients may be exposed to monomers and their degradation products after restorative surgery, for which little to no toxicological data exists. If possible, it can be considered to limit the number of teeth treated per session and to plan multiple restorations with several weeks in between in order to limit exposure to chemicals from resin-based dental materials.

Sinds 2011 moedigt de Wereldgezondheidsorganisatie een wereldwijde geleidelijke stopzetting van het gebruik van amalgaam aan en steunt zij actief het gebruik van alternatieve, op hars gebaseerde tandheelkundige materialen. Deze materialen bestaan meestal uit een mengsel van anorganische vulstofdeeltjes die aan een harsmatrix zijn bevestigd. De organische harsmatrix bestaat meestal uit (meth)acrylaatmonomeren en functionele additieven (foto- en co-initiatoren, stabilisatoren en andere). Bij restauratie moeten de materialen worden uitgehard door polymerisatie van de matrix, maar dit proces is niet volledig waardoor chemicaliën vrijkomen na restauratieve behandelingen. Dit proefschrift had tot doel drie belangrijke vragen te beantwoorden:

1.       Wat zijn de ingrediënten van tandheelkundige composieten en verzegelingsmaterialen?

2.       Aan welke chemicaliën worden patiënten mogelijk blootgesteld tijdens en na de tandheelkundige restauratie?

3.        In welke mate worden tandheelkundige composieten afgebroken na restauratie, en welke afbraakproducten komen daarbij vrij?

De samenstelling van tandheelkundige materialen op harsbasis werd onderzocht met behulp van vloeistofchromatografie gekoppeld aan hoge-resolutie massaspectrometrie. Belangrijke componen-ten, zoals de monomeren BisEMA, BisGMA, TEGDMA en UDMA, werden geïdentificeerd, hoewel ze niet altijd in de MSDS'en werden vermeld. Voorts werden fotoinitiatoren, zoals ethyl-4-dimethyl aminobenzoaat (EDMAB) en (meth)acrylaatonzuiverheden afkomstig van de productie van de hoofdingrediënten geïdentificeerd.

Niet-gereageerde monomeren en niet covalent gebonden additieven kunnen na de restauratie uit de polymeermatrix lekken, een proces dat kan worden verergerd door degradatie van het polymeer als gevolg van blootstelling aan diverse stressfactoren zoals de pH en enzymen in het speeksel. Om deze hypothese te testen, werden monomeren en gepolymeriseerde materialen gedurende 24 u blootgesteld aan chemische media en menselijk speeksel om uitgeloogde monomeren en afbraakproducten te identificeren. Tijdens de chemische degradatie werden niet-uitgeharde monomeren snel gehydrolyseerd tot mono- en demethacrylaatdegradatieproducten. Blootstelling aan speeksel gedurende resulteerde in de identificatie van zowel monomeren als hun afbraakproducten.

Het levermetabolisme van uitgeloogde monomeren werd gesimuleerd met behulp van humane levermicrosomen en levercytosolen. Tijdens fase I incubatie werd het (meth)acrylzuur in de monomeren snel verwijderd, gevolgd door oxidatie- en hydroxylatie. Voor BisPMA resulteerde een O-dealkylering in de vorming van BPA. Carbamaatgroepen aanwezig in TCD-DI-HEA en UDMA waren resistent tegen biotransformatiereacties. Fase II-biotransformatieproducten werden alleen waargenomen voor BisPMA en omvatte conjugatiereacties met sulfaat en glucuronzuur.

In het laatste deel van dit proefschrift werd voor het eerst een studie uitgevoerd om het vrijkomen van monomeren en hun afbraakproducten in speeksel van patiënten die restauraties ondergingen, kwantitatief en kwalitatief te controleren. Speekselstalen werden verzameld op verschillende tijdstippen na de restauratie. Monomeren zijn slechts kort na de restauratie in het speeksel aanwezig, maar verscheidene afbraakproducten kunnen weken na de restauratie worden gedetecteerd, hetgeen bevestigt dat zij op lange termijn vrijkomen.

Uit de resultaten van dit proefschrift blijkt dat patiënten na restauratieve chirurgie kunnen worden blootgesteld aan monomeren en hun afbraakproducten, waar vaak weinig tot geen toxicologische data beschikbaar van zijn. Indien mogelijk kan worden overwogen het aantal behandelde tanden per sessie te beperken en meerdere restauraties te plannen met enkele weken ertussen om de blootstelling aan chemicaliën van tandheelkundige materialen op harsbasis te beperken.

ONLINE DEFENCE: https://eu.bbcollab.com/guest/d821b56c7cb84ed581cea538d2c7348f

Prolyl carboxypeptidase and its substrates: a potential role in metabolic disorders and the cardiovascular system 

English abstract: 

Prolyl carboxypeptidase (PRCP) is a serine protease with a function in metabolic disorders and the cardiovascular system because of its role as angiotensin II- and III-, des-Arg9-bradykinin-, α-MSH 1-13- and (pyr)-apelin-13-cleaving enzyme.During this doctoral research, the physiological role of PRCP was further explored. To guarantee a solid supply, we produced recombinant human PRCP by use of the baculovirus insect cell expression system. Using this in-house produced recombinant enzyme, it was confirmed that compound 8o is a potent, reversible and selective inhibitor of PRCP, although the earlier reported potency (IC50 = 1 nM) could not be confirmed.Compound 8o was used to further explore the role of PRCP in peptide turnover in human umbilical vein (HUVEC) and aortic (HAoEC) endothelial cells. The selective inhibitors compound 8o, DX600 and KYP-2047 were used to study the contribution of PRCP, angiotensin converting enzyme 2 (ACE2) and prolyl oligopeptidase (PREP). For the first time, the role of PRCP was investigated by use of a selective inhibitor. In HUVEC, the C-terminal cleavage of (pyr)-apelin-13 was mediated only by PRCP, while in HAoEC, also ACE2 contributed to this cleavage. PREP was not involved in the cleavage of (pyr)-apelin-13 and the studied enzymes did not contribute to the C-terminal cleavage of α-MSH 1-13 in endothelial cells. The C-terminal cleavage of both angiotensin II and III was PRCP-dependent in HUVEC and HAoEC, while the C-terminal cleavage of des-Arg9-bradykinin was PRCP-dependent in HUVEC and PRCP- and ACE2-dependent in HAoEC. To unravel where exactly PRCP exerts its function in this peptide turnover, we studied the subcellular location of PRCP in endothelial cells. We did confirm lysosomal location of PRCP, while we did not observe its location at the cell membrane, contrary to an earlier report. Additionally, pro-inflammatory stimulation of endothelial cells leads to the secretion of PRCP in the extracellular environment.In the context of its role in metabolic disorders, PRCP activity was measured in human subcutaneous and visceral adipose tissue and corresponding serum samples and muscle tissue of obese, type 2 diabetic and non-diabetic, and lean men. For the first time, PRCP activity was detected in adipose tissue. Moreover, the PRCP activity was significantly higher in adipose tissue in comparison with the activity in muscle tissue, in both lean and obese individuals. The serum PRCP activity was significantly higher in obese type 2 diabetic individuals in comparison with lean and obese non-diabetic individuals.Additionally, the serum PRCP activity was positively correlated with biochemical parameters important in the diagnosis of diabetes. 

Prolyl carboxypeptidase en zijn substraten: een potentiële rol in metabole aandoeningen en het cardiovasculair systeem 

Dutch abstract: 

Prolyl carboxypeptidase (PRCP) is een serine protease met een functie in metabole aandoeningen en het cardiovasculair systeem door het knippen van angiotensin II en III, des-Arg9-bradykinin, α-MSH 1-13 en (pyr)-apelin-13.Tijdens dit doctoraal onderzoek werd de fysiologische rol van PRCP verder onderzocht. Om over voldoende kwaliteitsvol PRCP te beschikken gedurende heel het onderzoek, werd recombinant human PRCP geproduceerd met behulp van het baculovirus insect cel expressie systeem. Door gebruik van dit in-huis geproduceerde recombinant enzym, werd bevestigd dat compound 8o een potente, reversibele en selectieve PRCP-inhibitor is. Nochtans kon de eerder gerapporteerde sterkte (IC50 = 1 nM) niet bevestigd worden.Compound 8o werd vervolgens gebruikt om de rol van PRCP in peptide turnover verder te onderzoeken in humane endotheelcellen van de navelstreng en de aorta (HUVEC en HAoEC). De selectieve inhibitoren compound 8o, DX600 en KYP-2047 werden gebruikt om de bijdrage van respectievelijk PRCP, angiotensin converting enzym 2 (ACE2) en prolyl oligopeptidase (PREP) na te gaan. De rol van PRCP werd voor het eerst onderzocht met behulp van een selectieve inhibitor. In HUVEC kon de C-terminale knipping van (pyr)-apelin-13 enkel toegeschreven worden aan PRCP, terwijl in HAoEC ook ACE2 betrokken was bij deze knipping. PREP was niet betrokken bij de knipping van (pyr)-apelin-13 en de bestudeerde enzymen waren niet betrokken bij de C-terminale knipping van α-MSH 1-13 in endotheelcellen. De C-terminale knipping van zowel angiotensin II als III was PRCP-afhankelijk in HUVEC en HAoEC, terwijl de C-terminale knipping van des-Arg9-bradykinin PRCP-afhankelijk was in HUVEC en PRCP- en ACE2-afhankelijk in HAoEC. Om na te gaan waar in de cellulaire context PRCP zijn functie in peptide turnover uitvoert, werd de subcellulaire locatie van PRCP in endotheelcellen onderzocht. We bevestigden de lysosomale locatie van PRCP terwijl we geen PRCP op de celmembraan detecteerden, in tegenstelling tot een eerder rapport. Verder leidde de pro-inflammatoire stimulatie van endotheelcellen tot de secretie van PRCP in het extracellulair milieu.In het kader van zijn rol in metabole aandoeningen, werd de PRCP activiteit gemeten in humaan subcutaan en visceraal vetweefsel en overeenkomstige serum en spierweefselstalen van gezondecontroles en obese mannen, al dan niet gediagnosticeerd met diabetes type 2. Voor de eerste keer werd PRCP activiteit gemeten in vetweefsel. Bovendien was de PRCP activiteit significant hoger in vetweefsel in vergelijking met de activiteit in spierweefsel, in zowel gezonde controles als obese mannen. De serum PRCP activiteit was significant hoger in obese mannen met type 2 diabetes in vergelijking met de gezonde controles en obese, niet-diabete mannen. Aanvullend was de PRCP activiteit positief gecorreleerd met biochemische parameters belangrijk in de diagnose van diabetes.

ONLINE DEFENCE: https://eu.bbcollab.com/guest/5f83128a360049ad90754df047c34186

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Computational investigation of the catalytic mechanism and dynamics of Staphylococcus aureus glycosyltransferase towards development of novel antibiotics 

Kenneth Goossens 

Bacterial glycosyltransferases of the GT51 family are key enzymes in the bacterial cell wall synthesis. Inhibiting cell wall synthesis is a very effective approach for development of antibiotics, as this can lead to either bacteriostatic or bactericidal effects. However, drug development focused on bacterial transglycosylase has been hampered due to little being know about its structure and reaction mechanism. We have addressed this issue by computationally investigating the S. aureus monoglycosyltransferase enzyme, which is one of the main catalysts for peptidoglycan synthesis in S. aureus. Using molecular dynamics simulations, it was possible to gain new insights on the dynamics of the protein and the binding mode of the natural substrate, lipid II. The function of the different protein domains was explored this way, as well as the function of metal ions in facilitating catalysis. It was also found that the binding mode of the natural substrate appears to be different from the commonly proposed binding mode, and that lipid II subunits bind a shared pocket between the flexible flap domain and motif 3. Following these findings, a detailed study of the catalytic mechanism using hybrid quantum mechanics/molecular mechanics provided further insight on the functionality of the protein. One particular mechanism was discovered in which lipid II linkage is catalyzed by a metal-bound hydroxyl ion. With newfound structural information, the binding mode of existing drug-like inhibitors was reevaluated. Through molecular dynamics simulations, a new binding mode was proposed and provided the basis for an extensive virtual screening campaign in which molecular docking was combined with umbrella sampling calculations. The campaign did not lead to the discovery of novel inhibitors, but have paved the way for future virtual screening campaigns. With advancements in X-ray crystallography and cryogenic electron microscopy, high resolution structures of glycosyltransferases in complex with inhibitors should become available. With these new structures, virtual screening campaigns augmented by fragment screening methodologies could very well lead to novel, high potency inhibitors of Staphylococcus aureus glycosyltransferase which could be further developed into novel antibiotics. 

Computationele studie van het katalytisch mechanisme en de dynamica van Staphylococcus aureus glycosyltransferase voor het ontwikkelen van nieuwe antibiotica 

Kenneth Goossens 

De bacteriële glycosyltransferasen van de GT51 familie zijn cruciale enzymen voor de synthese van de bacteriële celwand. Inhibitie van celwandsynthese is een zeer effectieve aanpak voor het ontwikkelen van antibiotica, omdat dit kan leiden tot bacteriostatische of bactericide werking. De evolutie in geneesmiddelenontwikkeling met bacteriële glycosyltransferasen als doelwit is echter gelimiteerd door het gebrek aan kennis over de structuur en het reactiemechanisme van deze eiwitten. Dit probleem wordt nu aangekaart door het eiwit S. aureus monoglycosyltransferase computationeel te onderzoeken. Dit eiwit is een van de belangrijkste katalysatoren van de synthese van peptidoglycaan in S. aureus. Nieuwe inzichten zijn gevonden over de dynamica van het eiwit en de bindingswijze van het natuurlijke substraat, lipid II, door het uitvoeren van moleculaire dynamicasimulaties. Op deze manier was het mogelijk om de functie van de individuele regio’s van het eiwit te bestuderen, alsook de functie van metaalionen in katalyse. De voornaamste bevinding was dat de bindingswijze van het natuurlijke substraat lijkt te verschillen van de algemeen voorgestelde bindingswijze; namelijk in een gemeenschappelijke pocket tussen het flexibele flapdomein en domein 3. Deze bevindingen werden gevolgd door een gedetailleerde studie van het katalytische mechanisme van het eiwit met behulp van hybride kwantummechanische-/moleculaire mechanicaberekeningen. Hiermee werd een dieper inzicht vergaard over de functionaliteit van het eiwit. Eén mechanisme werd ontdekt waarbij de verbinding van lipid II eenheden werd gekatalyseerd door een metaalgebonden hydroxide-ion. Met de nieuwe structurele informatie werd de bindingswijze van bestaande inhibitoren opnieuw onderzocht. Via moleculaire dynamicasimulaties werd er een nieuwe bindingswijze voorgesteld, welke de basis vormde voor een uitgebreide virtuele screeningcampagne waarin moleculaire docking werd gecombineerd met umbrella sampling. Dit onderzoek leidde niet tot het ontdekken van nieuwe inhibitoren, maar heeft wel de weg opengelegd naar toekomstige screeningcampagnes. Met toekomstige vooruitgang in X-stralenkristallografie en cryogene elektronmicroscopie zullen nieuwe glycosyltransferasestructuren van hoge resolutie en gebonden door inhibitoren beschikbaar worden. Met deze nieuwe structuren kunnen virtuele screeningcampagnes in combinatie met fragment screening leiden tot de eventuele ontdekking van nieuwe, potente inhibitoren van Staphylococcus aureus glycosyltransferase, wat op zich kan leiden tot het ontwikkelen van nieuwe antibiotica.

Promotors: Prof. Nina Hermans - Prof. Alexander LN van Nuijs

Abstract:

During the past decade, researchers have shown an increased interest in the fate of orally ingested exogenous compounds, such as food supplements and pharmaceuticals in the gastrointestinal system. The gastrointestinal tract contains diverse compartments with varying physiological properties which could influence the bioavailability, activity and/or toxicity of these compounds. Until recently, it was believed that the biotransformation of xenobiotics occurs mainly by the cytochrome P450 enzyme system (CYP450) in the liver. However, recent data clearly show that the gut microbiome play a significant role in the biotransformation of oral administered xenobiotics, leading to a potential influence of this microbiotic biotransformation on activation, inactivation and possible toxicity of these compounds. A thorough knowledge on how the gut microbiome influences the bioavailability of xenobiotics and if the microbiotic biotransformation varies between individuals and different disease-states is essential further research on formulation and dosing strategies. Application of relevant in vitro gastrointestinal models can aid in the development of this research field.

In the first part of this work a ready-to-use in vitro gastrointestinal platform, including (i) the gastrointestinal dialysis model with colon phase (GIDM-Colon) and (ii) bioanalytical strategies to elucidate the gastrointestinal biotransformation of xenobiotics, was optimized using chlorogenic acid as a model compound. The influence different incubation media and different incubation times on bacterial composition and concentration of the pooled fecal slurry suspension were evaluated. The use of a sterile phosphate buffer and a one-hour incubation time ensured a closer resemblance to the in vivo composition. Furthermore, different sample preparation procedures and data-analysis workflows were evaluated and optimized.

Secondly, the in vitro gastrointestinal platform was successfully applied to investigate inter-population differences in microbiotic biotransformation of xenobiotics between a lean and obese population. The obese gut microbiome presented a lower metabolic activity in comparison to the lean population. The presented results in this thesis confirmed that changes in gut microbiota related to obesity are associated with differences in microbiotic biotransformation of xenobiotics.

Finally, the in vitro gastrointestinal platform was expanded with an in vitro intestinal permeability assay using a Caco-2 cell line and an in vitro intestinal first-pass biotransformation assay using human intestinal microsomes and cytosol respectively. The involvement of an active efflux mechanism was observed for the intestinal absorption of both chlorogenic acid and quinic acid. Regarding the intestinal first-pass effect, no phase I biotransformation products of chlorogenic acid, caffeic acid and quinic acid were observed. For both chlorogenic acid and caffeic acid two sulfate-conjugate isomers and low-abundance features corresponding to the glucuronide-conjugates were identified. No phase II biotransformation products of quinic acid were detected.

Abstract (NL) – PhD Mortelé Olivier

Het afgelopen decenium hebben onderzoekers meer belangstelling getoond voor het gastro-intestinale lot van oraal ingenomen exogene verbindingen, zoals voedingssupplementen en geneesmiddelen. Het gastro-intestinale stelsel bevat verschillende compartimenten met variërende fysiologische eigenschappen die de biologische beschikbaarheid, de activiteit en/of de toxiciteit van deze verbindingen kunnen beïnvloeden. Tot voor kort werd aangenomen dat de biotransformatie van xenobiotica voornamelijk door het cytochroom P450 enzymsysteem (CYP450) in de lever gereguleerd werd. Recente gegevens toonden echter aan dat het darmmicrobioom een belangrijke rol speelt bij de biotransformatie van oraal toegediende xenobiotica, wat leidt tot een potentiële invloed van deze microbiotische biotransformatie op de activering, inactivatie en mogelijke toxiciteit van deze verbindingen. Een grondige kennis over hoe het darmmicrobioom de biologische beschikbaarheid van xenobiotica beïnvloedt en of de microbiotische biotransformatie varieert tussen individuen en verschillende ziektebeelden is essentieel voor verder onderzoek naar formulerings- en doseringsstrategieën. Toepassing van relevante in vitro gastro-intestinale modellen kan helpen bij de ontwikkeling van dit onderzoeksgebied.

In het eerste deel van dit werk werd een in vitro gastro-intestinaal platform, inclusief (i) het gastro-intestinale dialyse model met colonfase (GIDM-Colon) en (ii) bio-analytische strategieën om de gastro-intestinale biotransformatieproducten van xenobiotica op te helderen, geoptimaliseerd met behulp van chlorogeenzuur als modelverbinding. De invloed van verschillende incubatiemedia en verschillende incubatietijden op de bacteriële samenstelling en de concentratie van de gepoolde fecale suspensie werd geëvalueerd. Het gebruik van een steriele fosfaatbuffer en een incubatietijd van één uur zorgden voor een nauwere gelijkenis met de in vivo samenstelling. Verder werden verschillende staalvoorbereidingsprocedures en data-analyse workflows geëvalueerd en geoptimaliseerd.

Ten tweede werd het in vitro gastro-intestinale platform met succes toegepast om verschillen in microbiotische biotransformatie van xenobiotica tussen een slanke en obese populatie te onderzoeken. Het obese darmmicrobioom had een lagere metabolische activiteit in vergelijking met de slanke populatie. De gepresenteerde resultaten in dit proefschrift bevestigen dat veranderingen in de darmmicrobiota in verband met obesitas geassocieerd zijn met verschillen in microbiotische biotransformatie van xenobiotica.

Ten slotte werd het in vitro gastro-intestinale platform uitgebreid met een in vitro intestinale permeabiliteits assay met behulp van een Caco-2 cellijn en een in vitro intestinale first-pass biotransformatie assay met behulp van humane  intestinale microsomen en cytosol respectievelijk. De betrokkenheid van een actief effluxmechanisme werd waargenomen voor de intestinale absorptie van zowel chlorogeenzuur als kininezuur. Met betrekking tot het intestinale first-pass effect werden geen fase I biotransformatieproducten van chlorogeenzuur, koffiezuur en kininezuur waargenomen. Voor zowel chlorogeenzuur als koffiezuur werden twee sulfaat-isomeren en twee lage-abundantie features, die overeenkomen met de glucuronide-conjugaten, geïdentificeerd. Er werden geen fase II-biotransformatieproducten van kininezuur gedetecteerd.

Promotors: Prof. Paul Cos - Prof. Peter Delputte​

Abstract:

The pneumococcus Streptococcus pneumoniae is considered one of the most important human pathogens. Infection can lead to diseases such as meningitis, pneumonia and otitis media. Especially children, the elderly and immunocompromised patients are at risk for developing severe infections. While several vaccines exist, they do not cover the entire pneumococcal spectrum. Moreover, a switch towards non-vaccine serotypes has been observed. Also, pneumococci are developing resistance towards commonly used antibiotics. With both prophylactic and therapeutic treatments failing, there is a dire need for novel antimicrobials to combat these infections. Apart from the development of novel antimicrobials, improving the reaction of the host’s immune system by lowering bacterial virulence has been proposed.

This dissertation focusses on the validation of a novel anti-virulence drug target against pneumococci. In order to adequately evaluate such a novel target, animal models are indispensable. However, in early research stages the costs of optimizing and implementing a novel model do not always outweigh the benefits. Therefore, a novel alternative in vivo model using Galleria mellonella larvae was chosen. These larvae were infected with several pneumococcal strains, after which parameters such as larval survival, bacterial burden, effect of antibiotic treatment and others were assessed. Also, the model’s use in studying the pneumococcal polysaccharide capsule was demonstrated.

The polysaccharide capsule is considered the most important pneumococcal virulence factor, as it is involved in adherence to epithelial cells and biofilm formation in the nasopharynx and in evasion of the innate immune system. However, due to its high heterogeneity, using this capsule or its associated enzymes as drug target has been challenging. Prior to this dissertation, an enzyme present in all pneumococci regardless of capsular serotype, uridine diphosphate glucose pyrophosphorylase (UDPG:PP), was discovered to be involved in capsule production.

First, the effect of a mutation in the corresponding UDPG:PP gene was assessed. The abolishment of a thick polysaccharide capsule in mutants was confirmed microscopically and mutants were more prone to phagocytosis by macrophages in vitro. Moreover, their virulence in the alternative G. mellonella infection model was significantly lowered compared to non-mutated pneumococci. Next, in silico predicted UDPG:PP inhibitors were assessed. In vivo virulence was significantly lowered and a decrease of capsule was observed. While the molecules were probably not capable of fully inhibiting the UDPG:PP enzyme, they did alter pneumococcal virulence using UDPG:PP as a target. With this knowledge, UDPG:PP inhibitors should be further studied as potential novel anti-virulence therapy.