Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medical Sciences University of Vienna, Faculty of Life Sciences, Department of Pharmacognosy Candidate: Lenka Kovářová Supervisor: Pharmdr. Miroslav Kovařík, Ph.D. Consultant: Dr. Angela Ladurner Title of the diploma thesis: Characterization of a synthetic leoligin derivative, with agonistic FXR and enhancing macrophage cholesterol efflux activity Atherosclerosis is a pathologic multifactorial process triggering the development of cardiovascular diseases, which are the leading causes of death in the western world. The initial phase of atherosclerosis is characterized by the accumulation of lipid particles, mainly low-density lipoproteins (LDL) and macrophage-derived foam cells in large arteries, leading to the gradual thickening of the vessel wall. These progressive alterations elicit plaque formation, followed by rupture, thrombosis and finally can lead to a cardiovascular event. Reverse cholesterol transport is an important preventive mechanism, which ensures removal of excessive atherogenic lipoproteins from macrophages. This efflux is facilitated by ATP binding cassette transporters, mainly ABCA1 and ABCG1 and in part by scavenger receptor B1 (SR-B1). Several nuclear receptors, including PPARγ, LXRα and LXRβ...

The heterodimeric ATP-binding cassette (ABC) transporter, ABCG5/G8, is responsible for direct secretion of cholesterol and dietary sterols into the gut lumen and the bile. Inactivating mutations of ABCG5/G8 cause sitosterolemia, a rare autosomal recessive disease characterized by the accumulation of plant sterols in plasma, hypercholesterolemia and development of premature coronary heart disease. Functional and structural characterization of ABCG5/G8 is necessary to understand its mechanism and how the genetic defects impact its function. In this thesis, I expressed seventeen constructs of various disease-causing or catalytically deficient missense mutations in Pichia pastoris yeast. This establishes reagents for in vitro functional and structural studies. Secondly, I focused on two disease mutants (ABCG5-E146Q and ABCG8-R543S) and a sterol binding mutation (ABCG5-A540F) and established large-scale purification of these mutants. Using a cholesterol hemisuccinate (CHS)-dependent ATPase assay, I determined ATP hydrolysis by these three mutants and analyze their kinetic parameters. All missense mutants showed a significantly impaired ATPase activity, but the ability of ATP binding appeared unchanged between the WT and the mutants. This work demonstrates an intimate structure-function relationship in ABCG5/G8 and sheds some light on the mechanistic details of this important cholesterol-regulating ABC transporter.

Background The Breast Cancer Resistance Protein (BCRP) drug transporter is important for drug disposition and plays a critical role in regulating drug entry into the brain. Its substrate spectrum overlaps with substrates of Multi Drug Resistance Protein 1 (MDR1, P-gp), which influences and complicates the interpretation of data on drug distribution into tissues (e.g. brain). Distinguishing BCRP mediated transport from the transport by the MDR1 is often problematic. However, with new in vitro tools, this is now possible. In this project, two drug compounds, i.e. Dantrolene and Ritonavir, were investigated using these new in vitro models. The results from the experimental in vitro assay were matched with molecular dynamics (MD) simulations. Using coarse-grained (CG) simulations, a model of the BCRP transporter in a lipid bilayer was built, this model is based on the human BCRP structure revealed by Taylor et al (2017). Simulations were run for Dantrolene (a known substrate of BCRP) independently three times, and another with Ritonavir (a non-substrate) three times. Aim To determine substrate specificity for the BCRP transporter for two compounds, and to construct a CG model of BCRP transporter to see whether in silico methods can be used as an alternative for assessing substrate specificity.  Methods Madin-Darby canine kidney (MDCK) II cell line with no endogenous canine MDR1 (cMDR1) expression (MDCKcMDR1-KO), overexpressing human MDR1 (hMDR1) (MDCK-hMDR1cMDR1-KO) and stable expression of human BCRP (hBCRP) (MDCK-hBCRPcMDR1-KO) cells were cultured and used in Transwell experiments. Samples were analyzed using LC-MS/MS to determine the substrate concentrations. Apparent permeability and efflux ratio was calculated and evaluated.  MD simulations used the Martini 3 CG force field, and were run with Gromacs (version 2020.4). Tools including MODELLER, INSANE and others were used to construct the initial model (Webster, 2000; Wassenaar et al., 2015), for parameterization of substrate and non-substrate molecules. And visual inspection was done with the visual molecular dynamics (VMD) program and PyMOL. Results In vitro transport experiment confirmed that Dantrolene is a BCRP specific substrate, and Ritonavir is MDR1 specific substrate. Following simulations of these two compounds, Dantrolene is observed to stay in the transmembrane domains (TMD) for a certain period (on average several hundreds of nanoseconds), while Ritonavir is not found to bind in the TMD, which provides a proof of concept for future studies.

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McMaster University MASTER OF SCIENCE (2019) Hamilton, Ontario (Medical Sciences) TITLE: Modulation of cyclic adenosine monophosphate for potentiation of long-acting β2-agonist and glucocorticoids in human airway epithelial cells AUTHOR: Yechan Kim, B.HSc. (McMaster University) SUPERVISOR: Dr. Jeremy Alexander Hirota NUMBER OF PAGES: xiv, 81 / In Canada, asthma is the third most common chronic disease resulting in 250 premature deaths annually and related healthcare expenses exceeding $2.1 billion/year. It is estimated that around 50-80% of asthma exacerbations are due to viral infections. Despite an advanced understanding on how to treat and manage the symptoms of asthma, current therapy is sub-optimal in 35-50% of moderate-severe asthmatics around the world resulting in lung inflammation, persistent impairment of lung function, and increased risk of mortality. Combination of long-acting β2 agonists (LABA) for bronchodilation and glucocorticoids (GCS) to control lung inflammation represent the dominant strategy for the management of asthma. Increasing intracellular cyclic adenosine monophosphate (cAMP) beyond existing combination LABA/GCS are likely to be beneficial for the management of difficult to control asthmatics that are hypo-responsive to mainstay therapy. In human airway epithelial cells (HAEC), cAMP is either exported by transporters or broken down by enzymes, such as phosphodiesterase 4 (PDE4). We have demonstrated that HAEC express ATP Binding Cassette Transporter C4 (ABCC4), an extracellular cAMP transporter. We also show that ABCC4 and PDE4 inhibition can potentiate LABA/GCS anti-inflammatory responses in a human epithelial cell line in a cAMP-dependent mechanism validating the pursuit of novel ABCC4 inhibitors as a cAMP elevating agent for asthma. / Thesis / Master of Science in Medical Sciences (MSMS) / Asthma is a common chronic lung disease characterized by narrow and inflamed airways that cause breathing difficulties. Current management includes the combination of bronchodilators, to relax the airway, and steroids, to decrease inflammation. Unfortunately, this combination therapy is suboptimal in 35-50% of users, increasing the risk of asthma attacks, hospitalization rate, and health care costs. Recently, there have been studies theorizing that we can improve the therapy’s ability to decrease inflammation by increasing cAMP, an important molecule for biological activities. We tested this claim by blocking the breakdown and export of cAMP to increase its levels and measured inflammatory cytokines, molecules that direct the action of immune cells. Our results show that in a model of viral infection, administering the combination therapy while increasing cAMP levels can further decrease inflammatory cytokines prompting further investigation for its potential implication in the clinic.

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Cytokinins are a large group of phytohormones. Since their discovery in the 1950s, they have shown to play a pivotal role in plant physiology. Most studies so far focused on cytokinin action mechanisms and their metabolic regulation. Identification of AtABCG14 and AtPUP14 as cytokinin-specific membrane carriers brought researchers' attention to cytokinin membrane transport, too. In this thesis, we performed experiments with radio-labelled cytokinin tracers. We show that trans-zeatin and isopentenyladenine, two major biologically active cytokinins, are readily transported across the plasma membrane in tobacco BY-2 cell suspension. Making use of mathematical modelling, we show that BY-2 cells possess a membrane transport system with an affinity toward cytokinins. Next, we show that atabcg14 and atpup14 mutations affect cytokinin metabolism in Arabidopsis thaliana plants. Keywords: cytokinin, Arabidopsis thaliana, tobacco BY-2 cell lines, membrane transport, purine permease, ATP-binding cassette, radio-labelling

<p>The aims of this thesis were to study the in vivo relevance of membrane transporters for intestinal absorption and the hepatic disposition of drugs in humans and preclinical models. Fexofenadine is a substrate for ABCB1 (P-glycoprotein) and members of the organic anion transporting polypeptide (OATP/SLCO) family. It is marginally metabolised in humans. </p><p>The influence of known inhibitors of ABCB1 and OATPs on the membrane transport and pharmacokinetics of fexofenadine was investigated in Caco-2 and porcine models and in humans. The permeability of fexofenadine remained low, even when significantly altered by the addition of an inhibitor. Using the Loc-I-Gut^® technique in vivo in humans, it was possible to see that the jejunal effective permeability of fexofenadine was unchanged when given with verapamil. However, the systemic exposure and apparent absorption rate of fexofenadine increased. This suggests that the first-pass liver extraction of fexofenadine was reduced by verapamil, probably through the inhibition of sinusoidal OATP-mediated and/or canalicular ABCB1-mediated secretion. The unchanged permeability can be explained by simultaneous inhibition of jejunal apical OATP-uptake and ABCB1-efflux, which would leave fexofenadine to be transported by passive trancellular diffusion. A Loc-I-Gut^® perfusion in the porcine model enabling blood sampling in the portal and hepatic veins and bile collection revealed increased jejunal permeability, but no subsequent verapamil-induced elevation in the systemic exposure of fexofenadine. This indicates a species-related difference in the localisation of and/or the substrate specificity of fexofenadine for the transporters involved. The absence of an effect on the first-pass liver extraction in the porcine model might be caused by the observed lower liver exposure of verapamil.</p><p>Finally, a novel intubation technique enabling dosing of fexofenadine in the jejunum, ileum and the colon showed that fexofenadine was absorbed less along the length the intestine in agreement with the properties of a low permeability drug.</p>