In this thesis a combined approach is described to investigate the constitutive activity of G protein protein-coupled receptors (GPCRs) using human adenosine A2B receptors and to evaluate disease... Show moreIn this thesis a combined approach is described to investigate the constitutive activity of G protein protein-coupled receptors (GPCRs) using human adenosine A2B receptors and to evaluate disease-related constitutive GPCR activity as a target for treatment. To this end a yeast expression system together with pharmacological and theoretical receptor models have been applied. In Chapter 2 the advantages of yeasts as tools to study GPCRs are reviewed. Adapted yeast cells able to communicate with mammalian GPCRs have become available and provide a very convenient system to express mutated receptors. A major advantage of yeast cells over mammalian cells extends from easy culturing conditions to the characteristic of yeast cells to allow entry of only a single plasmid. This latter property in combination with the robust screening assay based on yeast growth makes them an ideal test system to study randomly as well as site-directed mutated receptors. In chapters 3 to 5 this yeast growth assay is the main experimental tool to evaluate the functional properties of random and site-directed mutant receptors. In chapters 3 and 4 the yeast system is exploited to study inverse agonism of the human adenosine A2B receptor. At first, constitutively active mutant (CAM) human adenosine A2B receptors have been used to discriminate inverse agonists of the adenosine A2B receptor from A2B receptor antagonists. As a result, three inverse agonists ZM241385, DPCPX and MRS1706 were identified and their rank order of efficacy determined. Moreover, an interesting system-dependent phenomenon was noticed, that is the intrinsic activities of the inverse agonists were affected by the level of constitutive activity. It was demonstrated that inverse agonists show the greatest intrinsic activity on receptors displaying a medium level of constitutive activity. To further investigate the relationship between the effectiveness of an inverse agonist and the level of constitutive activity of the receptor, the two-state receptor model was introduced in both Chapter 3 and Chapter 4. According to this two-state model, both the receptor isomerization constant (L) and the intrinsic efficacy (_) of the inverse agonist determines the sensitivity to detect the intrinsic activity of an inverse agonist which is reflected by the observed experimental window. The biggest experimental window can be achieved on receptors with an L value equaling the reciprocal square root of _. Our experiments show that mutant A2B receptors with an intermediate level of constitutive activity possess the greatest experimental window, whereas mutants with a low level of constitutive activity showed small experimental windows and highly constitutively active mutants did not respond to our tested inverse agonists. Based on these findings we conclude that receptors with intermediate levels of constitutive activity should be the most sensitive screening tools for detecting inverse agonists. In Chapter 5 the activation of the human adenosine A2B receptor was investigated. To investigate the role of the NxxxNPxxY motif and the potential salt bridge between TM1 (E14) and TM7 (H280) in receptor activation, site-directed mutagenesis was applied to yield 15 mutant A2B receptors. The mutations were selected based on an adenosine A2B receptor model using the structure of bovine rhodopsin as a template. The expression levels of these mutants were determined by western blot analysis and the activation of the receptors was measured in the presence or absence of the following agonists NECA, CPA, CGS21680, IBMECA and LUF5833. None of the mutant receptors displayed constitutive activity. On the contrary, most mutants had a reduced potency and/or efficacy, e.g. mutants N282Q, N282R, N286A, N286Q, N286R, and Y290F showed impaired activation and mutants Y290N, E14H, H280E, E14H/H280E, I61A, I61D and I61K could not be activated by any of the agonists tested. Among all the mutants constructed, only N282R and N286R receptors behaved similarly to the wild-type receptor. Moreover, mutant N286A reduced receptor expression and H280E and E14H/H280E abolished receptor expression. These results suggest an important role for the NxxxNPxxY motif and the potential salt bridge in receptor expression and activation. The recent publication of the _2-adrenergic receptor structure enabled us to construct a second model of the adenosine A2B receptor. Comparison of the two A2B receptor models based on these two different templates is also described in Chapter 5. The various effects caused by mutations in the NxxxNPxxY motif and the potential salt bridge of different receptors in both our experiments and from literature do suggest that receptor activation is a receptor-specific phenomenon. In Chapter 6 we provide a theoretical investigation of the treatment of disease-related constitutively active receptor mutations. Comparison of the characteristics of allosteric ligands with traditional orthosteric ligands using a two-state allosteric model predicts that allosteric ligands display a more complicated interaction with a receptor/endogenous ligand pair and are able to cooperatively modify receptor binding and function. As a result allosteric modulators may affect the level of constitutive activity without changing the potency of the endogenous ligand. Thus allosteric modulators may provide advantages over orthosteric ligands in the treatment of diseases caused by constitutively active GPCR mutations. Finally in Chapter 7, general conclusions about the research described in this thesis are drawn. This is also supplemented by an outlook on some potential aspects of research to be pursued, based upon the application of receptor models, pharmacology models and functional receptor assays. Show less
Each protein is characterized by its unique sequential order of amino acids, the so-called protein sequence. Biology__s paradigm is that this order of amino acids determines the protein__s... Show moreEach protein is characterized by its unique sequential order of amino acids, the so-called protein sequence. Biology__s paradigm is that this order of amino acids determines the protein__s architecture and function. In this thesis, we introduce novel algorithms to analyze protein sequences. Chapter 1 begins with the introduction of amino acids, proteins and protein families. Then fundamental techniques from computer science related to the thesis are briefly described. Making a multiple sequence alignment (MSA) and constructing a phylogenetic tree are traditional means of sequence analysis. Information entropy, feature selection and sequential pattern mining provide alternative ways to analyze protein sequences and they are all from computer science. In Chapter 2, information entropy was used to measure the conservation on a given position of the alignment. From an alignment which is grouped into subfamilies, two types of information entropy values are calculated for each position in the MSA. One is the average entropy for a given position among the subfamilies, the other is the entropy for the same position in the entire multiple sequence alignment. This so-called two-entropies analysis or TEA in short, yields a scatter-plot in which all positions are represented with their two entropy values as x- and y-coordinates. The different locations of the positions (or dots) in the scatter-plot are indicative of various conservation patterns and may suggest different biological functions. The globally conserved positions show up at the lower left corner of the graph, which suggests that these positions may be essential for the folding or for the main functions of the protein superfamily. In contrast the positions neither conserved between subfamilies nor conserved in each individual subfamily appear at the upper right corner. The positions conserved within each subfamily but divergent among subfamilies are in the upper left corner. They may participate in biological functions that divide subfamilies, such as recognition of an endogenous ligand in G protein-coupled receptors. The TEA method requires a definition of protein subfamilies as an input. However such definition is a challenging problem by itself, particularly because this definition is crucial for the following prediction of specificity positions. In Chapter 3, we automated the TEA method described in Chapter 2 by tracing the evolutionary pressure from the root to the branches of the phylogenetic tree. At each level of the tree, a TEA plot is produced to capture the signal of the evolutionary pressure. A consensus TEA-O plot is composed from the whole series of plots to provide a condensed representation. Positions related to functions that evolved early (conserved) or later (specificity) are close to the lower left or upper left corner of the TEA-O plot, respectively. This novel approach allows an unbiased, user-independent, analysis of residue relevance in a protein family. We tested the TEA-O method on a synthetic dataset as well as on __real__ data, i.e., LacI and GPCR datasets. The ROC plots for the real data showed that TEA-O works perfectly well on all datasets and much better than other considered methods such as evolutionary trace, SDPpred and TreeDet. While positions were treated independently from each other in Chapter 2 and 3 in predicting specificity positions, in Chapter 4 multi-RELIEF considers both sequence similarity and distance in 3D structure in the specificity scoring function. The multi-RELIEF method was developed based on RELIEF, a state-of-the-art Machine-Learning technique for feature weighting. It estimates the expected __local__ functional specificity of residues from an alignment divided in multiple classes. Optionally, 3D structure information is exploited by increasing the weight of residues that have high-weight neighbors. Using ROC curves over a large body of experimental reference data, we showed that multi-RELIEF identifies specificity residues for the seven test sets used. In addition, incorporating structural information improved the prediction for specificity of interaction with small molecules. Comparison of multi-RELIEF with four other state-of-the-art algorithms indicates its robustness and best overall performance. In Chapter 2, 3 and 4, we heavily relied on multiple sequence alignment to identify conserved and specificity positions. As mentioned before, the construction of such alignment is not self-evident. Following the principle of sequential pattern mining, in Chapter 5, we proposed a new algorithm that directly identifies frequent biologically meaningful patterns from unaligned sequences. Six algorithms were designed and implemented to mine three different pattern types from either one or two datasets using a pattern growth approach. We compared our approach to PRATT2 and TEIRESIAS in efficiency, completeness and the diversity of pattern types. Compared to PRATT2, our approach is faster, capable of processing large datasets and able to identify the so-called type III patterns. Our approach is comparable to TEIRESIAS in the discovery of the so-called type I patterns but has additional functionality such as mining the so-called type II and type III patterns and finding discriminating patterns between two datasets. From Chapter 2 to 5, we aimed to identify functional residues from either aligned or unaligned protein sequences. In Chapter 6, we introduce an alignment-independent procedure to cluster protein sequences, which may be used to predict protein function. Traditionally phylogeny reconstruction is usually based on multiple sequence alignment. The procedure can be computationally intensive and often requires manual adjustment, which may be particularly difficult for a set of deviating sequences. In cheminformatics, constructing a similarity tree of ligands is usually alignment free. Feature spaces are routine means to convert compounds into binary fingerprints. Then distances among compounds can be obtained and similarity trees are constructed via clustering techniques. We explored building feature spaces for phylogeny reconstruction either using the so-called k-mer method or via sequential pattern mining with additional filtering and combining operations. Satisfying trees were built from both approaches compared with alignment-based methods. We found that when k equals 3, the phylogenetic tree built from the k-mer fingerprints is as good as one of the alignment-based methods, in which PAM and Neighborhood joining are used for computing distance and constructing a tree, respectively (NJ-PAM). As for the sequential pattern mining approach, the quality of the phylogenetic tree is better than one of the alignment-based method (NJ-PAM), if we set the support value to 10% and used maximum patterns only as descriptors. Finally in Chapter 7, general conclusions about the research described in this thesis are drawn. They are supplemented with an outlook on further research lines. We are convinced that the described algorithms can be useful in, e.g., genomic analyses, and provide further ideas for novel algorithms in this respect. Show less
Protein binding can have major impact on a drug__s pharmacokinetics (PK) and pharmacodynamics (PD). At present the theoretical basis of the influence of (alterations in) plasma protein binding on... Show moreProtein binding can have major impact on a drug__s pharmacokinetics (PK) and pharmacodynamics (PD). At present the theoretical basis of the influence of (alterations in) plasma protein binding on pharmacokinetics is well-established. The role of plasma-protein binding on pharmacodynamics however has sofar not been established in a systematic manner. As such there is no scientific basis for the __free drug hypothesis__ which states that the pharmacological activity is correlated with unbound drug concentrations in plasma. The objective of the research described in this thesis is to determine, in a strictly quantitative manner, the influence of plasma protein binding on in vivo pharmacodynamics. To this end both in silico and in vivo investigations were performed using the beta-blockers as model compounds. In contrast to the PK, the free drug concentration is the main determinant of the PD under equilibrium conditions for both target and plasma protein. Moreover for the beta-blockers, the free plasma concentration appears to be the best predictor of in vivo PD. In case of a dynamic interaction, the probability of non-restrictive protein binding is (theoretically) higher with regard to the PD under certain conditions, but more extensive research is needed to confirm this statement. Show less
Currently, the raising awareness of the role of glucocorticoids in the onset of numerous (neuro)-pathologies constitutes the increasing necessity of understanding the mechanisms of action of... Show moreCurrently, the raising awareness of the role of glucocorticoids in the onset of numerous (neuro)-pathologies constitutes the increasing necessity of understanding the mechanisms of action of glucocorticoids in bodily processes and brain functioning. Glucocorticoids mediate their effects by binding to intracellular receptors which act as transcription factors. A remarkable and yet unexplained phenomenon described more than two decades ago, is the cell-specific effects glucocorticoids bring about on gene expression in brain. For example, while glucocorticoids suppress corticotrophin-releasing hormone (CRH) synthesis in the hypothalamus, production of CRH in the central nucleus of the amygdala (CeA) is stimulated by increased hormone levels. Inasmuch as the neuroanatomical distribution of the corticosteroid receptors does not satisfactorily explain these effects, it is of interest to decipher the role of recently discovered coregu lator proteins that modulate the direction and the magnitude of steroid receptor-driven transcription. Therefore, in the current thesis the expression and function of central coregulators was studied: the coactivators SRC1a and SRC1e along with the corepressors N-CoR and SMRT were found to be expressed in brain and involved in regulation of CRH gene expression. Finally, a method that allows detection of coregulator recruitment by steroid receptors in brain tissue was developed. Show less
Despite the availability of a wide range of antiepileptic drugs, in about one third of people who suffer from epilepsy, seizures cannot be adequately controlled by pharmacotherapy. The mechanisms... Show moreDespite the availability of a wide range of antiepileptic drugs, in about one third of people who suffer from epilepsy, seizures cannot be adequately controlled by pharmacotherapy. The mechanisms underlying this pharmacoresistance are still unclear. This thesis investigates the relationship between the time course of epileptogenesis, the associated changes in GABA mediated inhibition and the development of pharmacoresistance. Impairment of GABA, the brain's major inhibiting neurotransmitter, may lead to excessive activation of excitatory neuronal circuits, as is the case in epilepsy. In vivo studies using an animal model for epilepsy show that the GABA(A) receptor changes early in the epileptogenic process. Alterations include decreased expression and altered subunit composition, resulting in a receptor that is less sensitive to some ligands and more sensitive to others. Show less
Hart- en vaatziekten zijn, ondanks de toepassing van cholesterol verlagende medicijnen, nog steeds de meest voorkomende doodsoorzaak in de westerse wereld, en manifesteren zich onder andere door... Show moreHart- en vaatziekten zijn, ondanks de toepassing van cholesterol verlagende medicijnen, nog steeds de meest voorkomende doodsoorzaak in de westerse wereld, en manifesteren zich onder andere door hartinfarcten en hersenbloedingen. Cholesterol, en ook fosfolipiden zijn essentieel voor de opbouw van celmembranen, maar ook voor de synthese van bijvoorbeeld hormonen. Een evenwichtige cholesterol huishouding is dus niet alleen belangrijk voor het hele lichaam maar ook voor individuele cellen. Dit evenwicht wordt, in een gezonde situatie, in stand gehouden door een strak gereguleerde balans tussen de opname van cholesterol uit het voedsel en de novo cholesterol synthese. Het zogenaamde goede cholesterol, het HDL, dankt zijn beschermende werking aan het zogenaamde reverse cholesterol transport, het transport van cholesterol uit de periferie naar de lever, waar het kan worden verwerkt en afgevoerd via de gal naar de feaces. Dit proefschrift richt zich op de verschillende stappen van het reverse cholesterol transport. Concluderend: therapeutische modulatie van het __reverse cholesterol transport__ moet zich niet richten op __n specifiek gen/eiwit, maar er dient gestreefd te worden naar een simultane activatie van cholesterol transporteiwitten wat zou kunnen leiden tot een optimale bescherming tegen vaatvernauwing en dientengevolge hart- en vaatziekten. Show less
To fail or not to fail __ Clinical trials in depression investigates the causes of the high failure rate of clinical trials in depression research. Apart from the difficulties in the search for new... Show moreTo fail or not to fail __ Clinical trials in depression investigates the causes of the high failure rate of clinical trials in depression research. Apart from the difficulties in the search for new antidepressants during drug discovery, faulty clinical trial designs hinder their evaluation during drug development. This thesis focuses on three important aspects of clinical trials in depression: clinical endpoints, data analysis and trial design-related factors. Show less
This thesis describes new, non-ribose ligands for the human Adenosine A1 Receptor (hA1R). An introduction to the four adenosine receptors subtypes, their history and cloning, occurrence,... Show moreThis thesis describes new, non-ribose ligands for the human Adenosine A1 Receptor (hA1R). An introduction to the four adenosine receptors subtypes, their history and cloning, occurrence, functioning, trafficking and therapeutic potential is given in Chapter 1. The process of desensitization and internalization of adenosine receptors in cell systems, tissues and in vivo studies is described in Chapter 2. An overview of the current literature concerning desensitization and internalization of the adenosine receptors is given and the regulation of the different subtypes upon agonist binding is discussed. In Chapter 3, human adenosine A1 receptors fused to 351Cys mutated Gi_-subunits are used as tools to study inverse agonism. In addition, the enhancing effect of the allosteric modulator PD81,723 on agonist affinity is shown. The influence of allosteric modulators on the internalization of the A1R is detailed in Chapter 4. The synthesis, affinity and activity of twelve non-ribose ligands, all ___2-amino-4-(3 and/or 4-disubstituted phenyl)-6-(substituted)sulfanyl-pyridine-3,5-dicarbonitriles, are described in Chapter 5. Chapter 6 evaluates the characteristics of LUF6037 a representative of these 3,4-methylenedioxyphenyl substituted compounds. One of the other non-ribose agonists with high affinity for the A1R, LUF5834, was chosen to be radioactively labelled. Chapter 7 describes the evaluation of [3H]LUF5834 as a new agonist radioligand. Show less
