Design and synthesis of metacaspase inhibitors as potential lead compounds for anti-parasitic chemotherapeutics

PhD in Pharmaceutical sciences

This research focuses on a novel cysteine protease of Trypanosoma brucei: metacaspase (MCA). To date, the function of this enzyme remains largely unknown and no MCA inhibitors have been published yet. MCA has been validated by RNA interference and gene knockout experiments and showed to be essential for bloodstream form T. brucei. [ 1 ] This project aims the identification of a first “hit compound” via  a rational design on the basis of known S1 subsite specificity (Arg/Lys) and the catalytic mechanism of the enzyme. Further optimisation efforts are then envisioned to draw a structure-activity-relationship between different series of MCA inhibitors. Our goal is to obtain one or more chemically stable inhibitors with high potency, selectivity towards human caspases and absence of cytotoxicity.  

References

1.  M.J. Helms, A. Ambit, P. Appleton, L. Tetley, G.H. Coombs, J.C. Mottram; J. Cell Sci., 119, 1105-1117 (2006).

Promotor(s)

Prof. Dr. K. Augustyns

Illustration

Figure 1: Schematic representation of TbMCA inhibitors based on L-Arginine or L-Lysine.

Induction of macrophage death as a strategy to stabilize atherosclerotic plaques

PhD in Pharmaceutical sciences

Since macrophages play a key role in atherosclerotic plaque destabilization and rupture, it is generally assumed that macrophage removal stabilizes the plaque. Yet, the mechanisms whereby macrophages can be eliminated from plaques without influencing other cell types, including SMCs, are largely unknown. Therefore, the objective was to evaluate several promising compounds for the ability to selectively induce macrophage death without altering SMC viability.

Because arterial macrophage-derived foam cells consume three times more oxygen than SMCs, it is conceivable that macrophages are metabolically highly active and thus more sensitive to protein synthesis inhibitors as compared to SMCs. Moreover, inhibition of mammalian target of rapamycin (mTOR) by everolimus decreased the macrophage content of plaques, associated with decreased protein synthesis. Therefore, we investigated whether selective induction of macrophage cell death by everolimus was due to inhibition of protein synthesis. The effect of protein synthesis inhibitors cycloheximide, puromycin and anisomycin, on macrophages and smooth muscle cells in vitro as well as on rabbit plaques was evaluated. Also the consequences of inducing macrophage apoptosis in atherosclerotic plaques were assessed.

Secondly, the effects of statins and phytosterols on the viability of macrophages and smooth muscle cells both in vitro and in rabbit atherosclerotic plaques were evaluated. These agents are used to decrease blood cholesterol levels. Since lipid lowering is associated with reduced plaque macrophage numbers and both statins and phytosterols have also been shown to induce cell death of macrophages in vitro, they are very attractive candidates in the quest for drugs that selectively induce macrophage death.

Promotor(s)

Promotor: Prof. Dr. Guido R.Y. De Meyer, Co-promotor: Prof. Dr. Wim Martinet

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Selective induction of macrophage death as innovative strategy to stabilize atherosclerotic plaques

PhD in Pharmaceutical sciences

Rupture of atherosclerotic plaques leads to atherothrombotic complications such as myocardial infarction. Therefore, stabilisation of atherosclerotic plaques is an important pharmacological target. Macrophages play an important role in plaque destabilization and rupture, whereas smooth muscle cells contribute to plaque stability. Pharmacological agents that can reverse plaque composition by depletion of macrophages are believed to stabilize rupture-prone plaques. Induction of macrophage death with unwanted complications or further plaque development mediated by the cell death process itself should be avoided. The preferred type of cell death (apoptosis, autophagy or necrosis) to remove macrophages remains to be investigated. Given that necrosis and apoptosis is characterized by a release of inflammatory molecules, we believe that autophagy is the preferred type of cell death to deplete macrophages in atherosclerotic plaques.

Drugs that can induce autophagy are tested in vitro (cell culture, explants of atherosclerotic plaques) and in vivo (local administration of drugs to atherosclerotic plaques). To explain the possible mechanism of selective induction of cell death in macrophages versus smooth muscle cells and endothelial cells viability tests, quantitative PCR and western blotting are performed. The type of cell death is analyzed using methods that are based on molecular markers that are characteristic for one type of cell death: western blotting, immunohistochemistry, flowcytometry, terminal deoxynucletoidyl transferase mediated dUTP nick end (TUNEL) labelling and electron microscopy. Vascular reactivity studies are performed to examine the drug effect on functionality of smooth muscle cells and endothelial cells.

Promotor(s)

Promoter:Prof. dr. G.R.Y. De Meyer

Co-promoter: Prof. dr. W. Martinet

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Carboxypeptidase M, substrates and ligands

PhD in Pharmaceutical sciences

The purpose of this doctoral thesis is the detection of interaction partners of human carboxypeptidase M (CPM). This metallo-protease exhibits a strict specificity towards C-terminal arginines and lysines of peptides/proteins. Site-specific mutagenesis of amino acids in the catalytic centre of CPM is performed to understand the interaction of this enzyme with its substrates. Potential CPM substrates found in the literature and available databases are incubated in vitro with CPM; if processed, an analysis of the effect on several biological properties of the molecule is performed using techniques such as flow cytometry, ELISA, and molecule-specific assays (e.g. cell cancer model). Furthermore, we search for a possible role of this membrane-anchored protease in cell-cell interaction, extracellular matrix interactions and cell migration.

CPM can be found on different cell types such as macrophages, endothelial cells and alveolar epithelial cells. Expression and/or activity of the CPM protein have been detected in the respiratory, reproductive, hematopoietic, renal system and several other systems. Currently, the presence and function(s) of CPM in the kidney and adipocyte tissue are being investigated by means of immunohistochemistry, activity-based assays, real-time PCR, confocal fluorescence microscopy, knockdown and over expression studies, and functional assays with specific cell lines.

Promotor(s)

Prof. A-M Lambeir

Prof. D Hendriks (co-promotor)

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Optimization of methods for in vitro quantification of oxidative stress in Leishmania infected macrophages

PhD in Pharmaceutical sciences

Oxidative stress is a pivotal factor in inflammatory processes, but research is much hampered by the difficulties to detect and quantify free radicals, i.e. reactive oxygen species (ROS). Objectives of this thesis include: (1) optimization of methods for in vitro quantification of ROS using electron paramagnetic resonance spectroscopy (EPR) and (2) application of these novel platforms to study ROS in inflammatory processes with focus on macrophage function in intracellular infections.

In the first part of the thesis, EPR methodology for the detection of the free radicals NO and superoxide is optimized. Both radicals are quantified in different cell cultures, including macrophage cell lines (RAW 264.7 and J774 A1), a monocyte cell line (U937) and primary mouse macrophages.

In the second part, this novel EPR technique is used to investigate the actual role of ROS in Leishmania spp. infected macrophages. NO and superoxide levels in uninfected and infected macrophages and drug-treated macrophages are compared to determine the role of these radicals in the defense system against the parasite.

Promoter(s)

Natural dipeptidyl peptidase 9: purification, biochemical characterization and immunohistochemical detection

PhD in Pharmaceutical sciences

The proline-selective dipeptidyl peptidases (DPPs), such as DPPIV, fibroblast activation protein α (FAP), DPPII, DPP8 and DPP9, cleave N-terminal dipeptides from peptides with proline at the penultimate position. Until recently, DPP8 and DPP9 were only available as recombinant proteins. Some contradiction exists concerning their biochemical and enzymatic characteristics. Therefore, we purified and characterized DPP9 from bovine testes [1]. We report its identification and N-terminal sequence analysis by MALDI TOF/TOF MS. Using DITC glass beads, a peptide that corresponds to the N-terminal peptide of the short form of bovine DPP9 was isolated. Other properties of DPP9 are reported, including molecular mass, functional stability, substrate specificity, inhibitor and pH profile. Our results indicate that the short form of DPP9 can be isolated from bovine testes and that it behaves as a stable enzyme suitable for further functional and biochemical characterization as well as for inhibitor screening and characterization.

The mRNA expression pattern of DPP8 and DPP9 was already studied in detail, in contrast to their protein expression and enzyme activity. The latter was investigated in the male reproductive system of different mammals [2]. The proline-selective DPP activity in the bovine and rat testis could predominantly be attributed to DPP8/9 and not to DPPIV. Using immunohistochemical experiments, a specific staining for DPP9 was found associated with spermatozoids embedded in the epithelium, just before their release into the lumen, and in spermatids (Figure). DPP8 was localized in spermatozoids in an earlier stage of maturation. These results lay the foundation of further study to the physiological role of DPPIV-like enzymes in the male reproductive system.

References

1. V. Dubois, A. M. Lambeir, P. Van der Veken, K. Augustyns, J. Creemers, X. Chen, S. Scharpé, I. De Meester; Front. Biosci. 13, 3558-3568 (2008).

2. V. Dubois, C. Van Ginneken, H. De Cock, A. M. Lambeir, P. Van der Veken, K. Augustyns, X. Chen, S. Scharpé, I. De Meester; J. Histochem. Cytochem. 57, 531-541 (2009).

Promotor(s)

Prof. Dr. I. De Meester

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Human exposure to the endocrine disruptor Bisphenol-A

PhD in Pharmaceutical sciences

Bisphenol-A (BPA) is a monomer mainly used in the production of the polymers polycarbonate and epoxy resins and is ubiquitous in the environment. These polymers are used in drinking bottles, electrical and electronic equipment, construction materials (polycarbonate) and adhesives, coatings in food and drinking cans (epoxy resins). BPA is an endocrine disruptor with mainly estrogenic activity. Recently, exposure to BPA was linked with endocrine diseases such as obesity and diabetes.

This study aims to model the human exposure to BPA. Different analytical methods will be developed and optimized based on gas or liquid chromatography coupled to mass spectrometry. The human exposure can be assessed by two different approaches. Firstly, BPA will be determined in the different exposure routes (food, dust, air). Special attention will be paid to food exposure and in particular the exposure through the consumption of canned foods. Mechanisms and factors affecting the release of BPA into the food will be investigated. These data will gain insight into the major human exposure sources and their relative contribution. A second approach is the assessing of the overall exposure by biomonitoring. For this purpose, urine is the most appropriate matrix since BPA is rapidly and almost completely urinary excreted. However recent findings suggest a longer half-life than previously expected. Therefore the tissue distribution and the metabolism after chronic exposure will be further investigated.

Promotor(s)

Prof Dr H Neels, Prof Dr A Covaci

Illustration

Carboxypeptidase U: a metallocarboxypeptidase with a specific role in haemostasis and a possible risk factor for thrombotic events.

PhD in Pharmaceutical Sciences

Carboxypeptidase U (CPU), also referred to as active Thrombin-Activatable Fibrinolysis Inhibitor (TAFIa), is a recently discovered attenuator of the fibrinolytic rate and is considered to be the molecular link between coagulation and fibrinolysis [1-3]. By removing the C-terminal lysine residues on partially degraded fibrin, CPU prevents the up-regulation of plasminogen binding and activation and promotes the inhibition of plasmin by α₂-antiplasmin [4,5]. CPU circulates in plasma as an inactive precursor, namely procarboxypeptidase U (proCPU or TAFI). The activation of the pro-enzyme occurs by the action of thrombin or plasmin, but most efficiently by thrombin in the presence of its cofactor thrombomodulin [6]. Inactivation of the enzyme occurs as a result of a conformational change consequent to its intrinsic instability [7], which is highly dependent on a polymorphism at position 325 of the protein[8,9]. Several clinical studies have indicated that a relation between the proCPU plasma concentration and a thrombotic tendency might exist [10].

In the first part of the project, our goal was to elucidate the role of the proCPU/CPU system in ischemic stroke. We were able to demonstrate that active CPU could be detected in the circulation of ischemic stroke patients receiving thrombolytic therapy [11]. Moreover, our results have shown that CPU can decrease the efficacy of thrombolytic therapy in ischemic stroke patients [12]. A large follow-up study was performed, where we a proCPU consumption was seen in the first 72h after stroke, followed to a return to baseline at day 7. Moreover, this proCPU consumption is related to stroke severity, evolution and outcome [13].

The measurement of active CPU in plasma is associated with mayor pitfalls, the most important one being the interference of CPN. Recent screening of Bz-Xaa-Arg peptides with an aromatic amino acid at the P1 position and further modifications in this position, revealed a selective CPU substrate, Bz-o-cyano-Phe-Arg [14], which will allow straight forward determination of CPU in plasma without the interference of CPN. In the second part of this project, we were able to develop an assay for the fast and sensitive determination of the active enzyme CPU in plasma.

In a third part of this project, the assay is being used to identify patient groups (MI, PE, DVT, ischemic stroke, sepsis, …) were the CPU system is being activated and thus could affect the outcome or severity of the pathology.

References

1.    Hendriks D, Scharpé S, van Sande M, Lommaert MP; J Clin Chem Clin Biochem, 27, 277-85 (1989).

2.    Bajzar L, Manuel R, Nesheim ME; J Biol Chem, 270 , 14477-84 (1995).

3.    Hendriks DF, Willemse JL; J Thromb Haemost,3, 2139-46 (2005).

4.    Wang W, Boffa MB, Bajzar L, Walker JB, Nesheim ME; J Biol Chem, 273, 27176-81 (1998).

5.    Schneider M, Nesheim ME. J Biol Chem, 279, 13333-9 (2004).

6.    Bazjar L, Morser J, Nesheim M; J Biol Chem, 271, 16603-8 (1996).

7.    Marx PF, hacking TM, Dawson PE, Griffin JH, Meijers JC, Bouma BN; J Biol Chem, 275, 12410-5 (2000).

8.    Schneider M, Boffa M, Stewart R, Rahman M, Koschinsky M, Nesheim M; J Biol Chem, 277, 1021-30 (2002).

9.    Knecht W, Willemse J, Stenhamre H, Andersson M, Berntsson P, Furebring C, Harrysson A, Malmborg Hager AC, Wissing BM, Hendriks D, Cronet P; Febs J, 273, 778-92 (2006).

10. Willemse JL, Hendriks DF; Front Biosci, 12, 1973-87 (2007).

11. Willemse JL, Brouns R, Heylen E, De Deyn PP, Hendriks DF; J Thromb Haemost, 6, 200-2 (2008).

12. Brouns R, Heylen E, Sheorajpanday R, Willemse JL, Kunnen J, De Surgeloose D, Hendriks DF, De Deyn PP. Clin Neurol Neurosurg, 111, 165-70 (2009).

13. Brouns R, Heylen E, Willemse J, Sheorajpanday R, De Surgeloose D, Verkerk R, DeDeyn PP, Hendriks D;  J Thromb Haemost, in press (2009).

14. Willemse JL, Polla M, Olsson T, Hendriks DF. Clin Chim Acta, 387, 158-60 (2008).

Promotor(s)

Prof. Dr. D. Hendriks

Illustration

Generation of CPU in ischemic stroke patients receiving IV rt-PA. Values represent the average (SD) of a triplicate measurement.

under construction

The role of dipeptidyl peptidases in ischemia/reperfusion injury

PhD in Pharmaceutical sciences

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

References

1. Zhai W, Cardell M, De Meester I, et al. Intragraft DPP IV inhibition attenuates post-transplant pulmonary ischemia/reperfusion injury after extended ischemia. J Heart Lung Transplant, 26,174-180 (2007) .

2. Belyaev A, Zhang X, Augustyns K, et al. Structure-activity relationship of diaryl phosphonate esters as potent irreversible dipeptidyl peptidase IV inhibitors. J Med Chem, 42,1041-1052 (1999).

Promotor(s)

Prof. dr. I. De Meester

Prof. dr. A.M. Lambeir (co-promotor)

Human exposure to new persistent contaminants, especially brominated flame retardants.

PhD in Pharmaceutical sciences

Brominated flame retardants (BFRs) comprise a diverse group of chemical classes such as polybrominated diphenyl ethers (PBDEs) and Hexabromocyclododecane (HBCD). These compounds share some common features such as their use in commercial and industrial applications for the purpose of fire prevention. They can be detected in a wide variety of appliances, including electric/electronic equipment, soft furnishing, textiles and insulation materials. Recently, these indoor contaminants have been receiving more attention as their widespread usage combined with the increasing time spend indoors might be a reason for human health concern. Many studies have reported on the ubiquitous presence of BFRs in the environment [1], their accumulation in human tissues [2], and their potential toxicity, including neurotoxicity and endocrine disruptive properties [3]. Human exposure to PBDEs and HBCD varies widely throughout the world as it depends on country-related usage, production and legislation of these chemicals. US and UK exposure assessments show very diverse levels and patterns which in turn, are likely to differ from those in background exposed countries such as Belgium, where environmental levels tend to be about an order of magnitude lower. This PhD aims to give a comprehensive overview of human exposure to PBDEs and HBCD in Belgium through a variety of exposure pathways. The total exposure is assessed for all age groups and the contribution of possible exposure pathways such as food, dust, air and soil is elucidated.

References

1. A. Covaci, L. Roosens, A.C. Dirtu, N. Waegeneers, I. Van Overmeire, H. Neels, L. Goeyens. Science of the total environment, 407, 4387-4396 (2009).

2. J. Doucet, B. Tague, D.L. Arnold, G.M. Cooke, S. Hayward, C.G. Goodyer. Environmental Health Perspectives, 117, 605-610 (2009).

3. L.T.M. Van der Ven, T. Van de Kuil, P.E.G. Leonards, W. Slob, H. Lilienthal, S. Litens, M. Herlin, H. Håkansson, R.C. Cantón, M. Van den Berg, T.J. Visser, H. Van Loveren, J.G. Vos, A.H. Piersma. Toxicology Letters, 185, 51–62 (2009).

Promotor(s)

Prof. dr. Hugo Neels

Prof. dr. Adrian Covaci

Illustration: structure PBDEs (left) and HBCD (right)

Development of colloidal carriers for the antileishmanial triterpene saponin PX-6518

PhD in Pharmaceutical sciences

Visceral leishmaniasis (VL) is a systemic disease caused by the obligate intra-macrophage protozoa Leishmania donovani and Leishmania infantum. VL is endemic in large areas of the tropics, subtropics and the Mediterranian basin and is transmitted by several species of sandflies. There are an estimated 500 000 new cases of VL and more than 50 000 deaths from the disease each year, a death toll that is surpassed among the parasitic diseases only by malaria [1]. Currently existing drugs such as the pentavalent antimonials, liposomal amphotericin B and miltefosin have several disadvantages in terms of tolerability, therapeutic regimen, duration of treatment, specificity and patient compliance. Moreover, resistance to these drugs has emerged in the past decade. Since the rate of new drug discovery in the segment of parasitic diseases is very low due to a lack of finances, current research focuses on altering the existing drugs’ biodistribution by entrapping them in colloidal carriers such as liposomes, polymeric nanoparticles (NPs) and solid lipid nanoparticles (SLNs) [2]. Due to their nanometer size range, these carriers are readily taken up by the infected reticuloendothelial cells of the human host. This phenomenon of passive targeting enhances the therapeutic efficacy and selectivity of leishmanicidal drugs, limiting their side effects [3]. This PhD work consists of the development of such colloidal carriers for the hepatotoxic triterpene saponin PX-6518 [4,5]. Poly(D,L-lactide-co-glycolide) (PLGA) NPs, SLNs and drug NPs will be investigated with respect to their physicochemical properties size and zeta potential, cytotoxicity and in vitro and in vivo antileishmanial activity.   

References

1. F. Chappuis, S. Sundar, A. Hailu, H. Ghalib, S. Rijal, R.W. Peeling, J. Alvar, M. Boelaert; Nat. Rev. Microbiol., 5, S7-S16 (2007).

2. A.A. Date, M.D. Joshi, V.B. Patravale; Adv. Drug. Deliv. Rev., 59, 505-521 (2007).

3. E. Briones, C.I. Colino, J.M. Lanao; J. Control. Release, 125, 210-227 (2008).

4. N. Germonprez, L. Van Puyvelde, L. Maes, M. Van Tri, N. De Kimpe; Tetrahedron, 60, 223-232 (2004).

5. L. Maes, D. Vanden Berghe, N. Germonprez, L. Quirijnen, P. Cos, N. De Kimpe, L. Van Puyvelde; Antimicrob. Agents & Chemotherap., 48 , 130-136 (2004).

Promotor(s)

Prof. Dr. A. Ludwig

Illustration

Synthesis and biochemical evaluation of selective inhibitors of dipeptidyl peptidase 8 and 9

PhD in Pharmaceutical sciences

Over the last years, dipeptidyl peptidase (DPP) family members DPP8 and DPP9 have garnered much attention following research results from Merck suggesting that the use of the DPP8/9 inhibitor Allo-Ile-isoindoline (fig.1) is associated with severe toxicity in animal models.[1] Contrasting with these findings, a recent study suggests that DPP8/9 inhibition alone cannot account for these toxic symptoms.[2] In addition, another study claims the observation that DPP8 and DPP9 are upregulated in experimentally induced asthma and that these peptidases specifically respond to the inflammatory stimulus.[3] In order to verify whether the reported toxicity observations are related to DPP8/9 inhibition and to further study the clinical relevance of potential enzyme involvement in pathologies, there is an urgent need for new, structurally distinct inhibitors of these enzymes. My thesis will report on the synthesis and biochemical evaluation of P1- and P2- modified analogs of allo-Ile-isoindoline. The influence of these modifications on the inhibitors’ affinity towards other DPPs and more specifically, their potential to discriminate between DPP8 and DPP9 will be discussed.

References

1. G.R. Lankas; Diabetes, 54, 2988-2994 (2005).
2. J.S. Rosenblum; Diabetes, 56, A138 (2007).
3. J. Schade; J. Histochem. Cytochem., 56, 147-155 (2008).

Promotor(s)

Prof. Augustyns

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Evaluation of drug abuse in Belgium by analysis of wastewater

PhD in Pharmaceutical sciences

This projects consists of two parts. In the first part, methods of analysis for the determination of several drugs (cocaine, amphetamines, cannabis,...) and their metabolites in waste and surface water are being developped and validated. In the second part, the concentrations of drugs and metabolites found in watersamples will be used to estimate the abuse of drugs in Belgium.

Promotor(s)

Hugo Neels

Adrian Covaci