@phdthesis{Volpato2021,
  author    = {Volpato, Daniela},
  title     = {Bitopic Ligands and their molecular fragments for the study of the M1 Muscarinic Receptor},
  doi       = {10.25972/OPUS-24881},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-248815},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The past decades have witnessed the development of new pharmaceutical compounds that modulate receptor function by targeting allosteric sites. Allosteric sites are, by definition, domains topographically distinct from the orthosteric binding pocket where the natural ligand binds. Exploring the possibilities of linking orthosteric and allosteric pharmacophores in one compound to yield 'bitopic' compounds is a strategy derived from the "message-address" concept by Schwyzer , first applied to GPCRs by Portoghese et al. This concept explicitly underlines the orthosteric/allosteric combination, in opposite to the more general umbrella term bivalent. The broad possibilities of bitopic ligands in the pharmaceutical field are under continuous study. Bitopic compounds are promising pharmaceutical tools for taking advantage of the allosteric binding to achieve subtype selectivity while preserving high affinity at the receptor. The development of bitopic ligands, based on the idea of combining high affinity (via orthosteric sites) with high selectivity (via allosteric sites), have led to the development of highly selective bivalent ligands for GPCRs , such as for the opioid receptors , muscarinic acetylcholine receptors (mAChRs), serotonin receptors, cannabinoid receptors, and gonadotropin-releasing hormone receptors. This concept has even been extended to other receptors, for examples nicotinic receptors and other proteins, such as acetylcholinesterases and the tyrosine kinase receptors TrkA and TrkC. The reasons to pursue a bitopic ligand approach are various. An improved affinity for the target GPCR and/or an improved selectivity either at the level of receptor subtype, or at the level of signaling pathway. Another advantage of bitopic ligands over purely allosteric ligands is that the former rely on the appropriate presence of endogenous agonist tone to mediate their effects, whereas a bitopic ligand would engage the orthosteric site irrespective of the presence or absence of endogenous tone. By way of introduction to the hybrid approach, a review of the concept of hybrids compounds targeting the cholinergic system is presented in section A of this thesis. Recent updates in hybrid molecule design as a strategy for selectively addressing multiple target proteins involved in Alzheimer's disease (AD) is here reported . This represents the potential and the growing interest in hybrid compound as pharmacological tools to achieve receptor subtype selectivity and/or, to study the overall functional activity of the receptor. Until now, muscarinic acetylcholine receptors (mAChRs) have proved to be a particularly fruitful receptor model for the development and characterization of bitopic ligands. In this thesis, several examples of new muscarinic bitopic approach are reported in the results section. A study of bipharmacophoric ligands composed of the muscarinic positive allosteric modulators (BQCAderived compounds) linked with chain of various lengths to different orthosteric building blocks is reported in the result part 1. Synthesis and examination of the potential pharmacological characteristic of Oxotremorine-BQCAd compounds and Xanomeline-BQCAd hybrid derivatives are described in results parts 2 and 4, respectively. Moreover, the bitopic concept has even been extended to other proteins, such as acetylcholinesterase. In the result part 5 an overview of the new Tacrine-Xanomeline hybrids aiming to improve the inhibitory potency of the acetylcholinesterase and simultaneously to increase the cholinergic tone, via the xanomelinic portion acting on the M1 receptor is given. A new trivalent approach is presented for the first time to deepen the study of the M1 muscarinic receptor in the result part 6. Moreover, the synthesis of a new series of iperoxo-derived alkane, bis(ammonio)alkane-type and rigidified chain ligands is given in the result part 7 together with some prospects for further research.},
  subject      = {Ligand <Biochemie>},
  language  = {en}
}
@phdthesis{Riad2019,
  author    = {Riad, Noura},
  title     = {The Development of Dualsteric Ligands for the Elucidation of Mode of Activation of Muscarinic Receptors and their Selective Signaling},
  doi       = {10.25972/OPUS-17928},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179282},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {GPCRs, particularly muscarinic receptors (mAChRs), are significant therapeutic targets in many physiological conditions. The significance of dualsteric hybrids selectively targeting mAChR subtypes is their great advantage in avoiding undesired side effects. This is attained by exploitation of the high affinity of ligand-binding to the orthosteric site and the structural diversity of the allosteric site to target an individual mAChR subtype, as well as offering signal bias to avoiding undesired transduction pathways. Furthermore, dualsteric targeting of mAChR subtypes helps in the elucidation of the physiological role of each individual mAChR subtype. The first project was the attempt of synthesis of the M2-preferring ligand AFDX-384. AFDX-384 is known to preferentially bind to the M2 receptor subtype as an orthosteric antagonist, with partial interaction with residues in the allosteric site. This project aimed to re-trace the synthesis route of AFDX-384, to open the door to its upscaling and the future synthesis of AFDX-type dualsteric ligands. The multi-step synthesis of AFDX-384 is achieved through the synthesis of its 2 precursors, the chloro acyl derivative VIII and the piperidinyl derivative IV. Upscaled synthesis of the piperidinyl derivative IV was attained. Synthesis of the chloro acyl compound VIII was attempted. Several trials to synthesize the benzopyridodiazepine nucleus as well as its chloro-acylation resulted in the production of the novel crystal structures V and VI. X-ray crystallography was also done for crystallized molecules of the closed-ring benzopyridodiazepine VII that was previously synthesized. Chloro-acylation reactions of compound VII using phosgene seem to be attainable when done using reflux overnight. However, the use of methanol to aid in elution during silica gel column chromatography converted the expected product to the carbamate analogue IX. Hence, further attempts in purification should refrain from the use of methanol. The use of triphosgene instead of phosgene demonstrates a cleaner route for further upscaled synthesis. The second project was the synthesis of dualsteric ligands involving variable orthosteric and allosteric moieties. Four different types of hybrids have been created over multiple steps. Dualsteric ligands have been synthesized using either a phthalimido- or 1,8-naphthalimidopropylamino moiety as the allosteric-binding group, coupled to either N-desmethyl pirenzepine or N-desmethyl clozapine using variable chain lengths. Furthermore, the synthesis of the dualsteric ligands involving N-desmethyl clozapine linked to either the super-agonist iperoxo or acetylcholine, and being connected using variable alkane chain lengths. Several reaction conditions have been investigated throughout the analysis of the optimal condition to conduct the critical final step of synthesis of these dualsteric hybrids, which involves the linking of the two segments of the hybrid together. The optimal method, which produced the least side products and highest yield, was to connect the two intermediates of the compound in absence of base, catalyst or microwaves while stirring at 35 °C for several days using acetonitrile as solvent (silica gel TLC monitoring, 0.2 M aqueous KNO3/MeOH 2:3). The ideal purification methods for the final compounds were found to be either crystallization from the reaction medium or using C18 reverse phase silica gel flash chromatography (using H2O/MeOH solvent system). All the hybrids will be subjected to pharmacological testing using the appropriate FRET assays.},
  subject      = {Muscarinrezeptor},
  language  = {en}
}
@phdthesis{Dolles2018,
  author    = {Dolles, Dominik},
  title     = {Development of Hybrid GPCR Ligands: Photochromic and Butyrylcholinesterase Inhibiting Human Cannabinoid Receptor 2 Agonists},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163445},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {While life expectancy increases worldwide, treatment of neurodegenerative diseases such as AD becomes a major task for industrial and academic research. Currently, a treatment of AD is only symptomatical and limited to an early stage of the disease by inhibiting AChE. A cure for AD might even seem far away. A rethinking of other possible targets is therefore necessary. Addressing targets that can influence AD even at later stages might be the key. Even if it is not possible to find a cure for AD, it is of great value for AD patients by providing an effective medication. The suffering of patients and their families might be relieved and remaining years may be spent with less symptoms and restrictions. It was shown that a combination of hCB2R agonist and BChE inhibitor might exactly be a promising approach to combat AD. In the previous chapters, a first investigation of dual-acting compounds that address both hCB2R and BChE was illustrated (figure 6.1). A set of over 30 compounds was obtained by applying SARs from BChE inhibitors to a hCB2R selective agonist developed by AstraZeneca. In a first in vitro evaluation compounds showed selectivity over hCB1R and AChE. Further investigations could also prove agonism and showed that unwanted off-target affinity to hMOP receptor could be designed out. The development of a homology model for hCB2R (based on a novel hCB1R crystal) could further elucidate the mode of action of the ligand binding. Lastly, first in vivo studies showed a beneficial effect of selected dual-acting compounds regarding memory and cognition. Since these first in vivo studies mainly aim for an inhibition of the BChE, it should be the aim of upcoming projects to proof the relevance of hCB2R agonism in vivo as well. In addition, pharmacokinetic as well as solubility studies may help to complete the overall picture. Currently, hybrid-based dual-acting hCB2R agonists and selective BChE inhibitors are under investigation in our lab. First in vitro evaluations showed improved BChE inhibition and selectivity over AChE compared to tacrine.78 Future in vitro and in vivo studies will clarify their usage as drug molecules with regard to hepatotoxicity and blood-brain barrier penetration. Since the role of hCB2R is not yet completely elucidated, the use of photochromic toolcompounds becomes an area of interest. These tool-compounds (and their biological effect) can be triggered upon irradiation with light and thus help to investigate time scales and ligand binding. A set of 5-azobenzene benzimidazoles was developed and synthesized. In radioligand binding studies, affinity towards hCB2R could be increased upon irradiation with UV-light (figure 6.2). This makes the investigated compounds the first GPCR ligands that can be activated upon irradiation (not vice versa). The aim of upcoming research will be the triggering of a certain intrinsic activity by an "efficacy-switch". For this purpose, several attempts are currently under investigation: an introduction of an azobenzene moiety at the 2-position of the benzimidazole core already led to a slight difference in efficacy upon irradiation with UV light. Another approach going on in our lab is the development of hCB1R switches based on the selective hCB1R inverse agonist rimonabant. First in vitro results are not yet available (figure 6.3).},
  subject      = {Ligand <Biochemie>},
  language  = {en}
}
@phdthesis{Mohsen2017,
  author    = {Mohsen, Amal Mahmoud Yassin},
  title     = {Structure Activity Relationships of Monomeric and Dimeric Strychnine Analogs as Ligands Targeting Glycine Receptors},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142228},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The inhibitory glycine receptors are one of the major mediators of rapid synaptic inhibition in the mammalian brainstem, spinal cord and higher brain centres. They are ligand-gated ion channels that are mainly involved in the regulation of motor functions. Dysfunction of the receptor is associated with motor disorders such as hypereklepxia or some forms of spasticity. GlyR is composed of two glycosylated integral membrane proteins α and β and a peripheral membrane protein of gephyrin. Moreover, there are four known isoforms of the α-subunit (α1-4) of GlyR while there is a single β-subunit. Glycine receptors can be homomeric including α subunits only or heteromeric containing both α and β subunits. To date, strychnine is the ligand that has the highest affinity as glycine receptor ligand. It acts as a competitive antagonist of glycine that results in the inhibition of Cl- ions permeation and consequently reducing GlyR-mediated inhibition. For a long time, the details of the molecular mechanism of GlyRs inactivation by strychnine were insufficient due to the lack of high-resolution structures of the receptor. Only homology models based on structures of other cys-loop receptors have been available. Recently, 3.0 {\AA} X-ray structure of the human glycine receptor- α3 homopentamer in complex with strychnine, as well as electro cryo-microscopy structures of the zebra fish α1 GlyR in complex with strychnine and glycine were published. Such information provided detailed insight into the molecular recognition of agonists and antagonists and mechanisms of GlyR activation and inactivation. Very recently, a series of dimeric strychnine analogs obtained by diamide formation of two molecules of 2-aminostrychnine with diacids of different chain length was pharmacologically evaluated at human α1 and α1β glycine receptors. None of the dimeric analogs was superior to strychnine. The present work focused on the extension of the structure-activity relationships of strychnine derivatives at glycine receptors All the synthesized compounds were pharmacologically evaluated at human α1 and α1β glycine receptors in a functional FLIPRTM assay and the most potent analogs were pharmacologically evaluated in a whole cell patch-clamp assay and in [3H]strychnine binding studies. It was reported that 11-(E)-isonitrosostrychnine displayed a 2-times increased binding to both α1 and α1β glycine receptors which prompted us to choose the hydroxyl group as a suitable attachment point to connect two 11-(E)-isonitrosostrychnine molecules using a spacer. In order to explore the GlyR pocket tolerance for oxime extension, a series of oxime ethers with different spacer lengths and sterical/lipophilic properties were synthesized biologically evaluated. Among all the oxime ethers, methyl, allyl and propagyl oxime ethers were the most potent antagonists displaying IC50 values similar to that of strychnine. These findings indicated that strychnine binding site at GlyRs comprises an additional small lipophilic pocket located in close proximity to C11 of strychnine and the groups best accommodated in this pocket are (E)-allyl and (E)-propagyl oxime ethers. Moreover, 11-aminostrychnine, and the corresponding propionamide were prepared and pharmacologically evaluated to examine the amide function at C11 as potential linker. A series of dimeric strychnine analogs designed by linking two strychnine molecules through amino groups in position 11 with diacids were synthesized and tested in binding studies and functional assays at human α1 and α1β glycine receptors. The synthesized bivalent ligands were designed to bind simultaneously to two α-subunits of the pentameric glycine receptors causing a possibly stronger inhibition than the monomeric strychnine. However, all the bivalent derivatives showed no significant difference in potency compared to strychnine. When comparing the reference monomeric propionamide containing ethylene spacer to the dimeric ligand containing butylene spacer, a 3-fold increase in potency was observed. Since the dimer containing (CH2)10 spacer length was found to be equipotent to strychnine, it is assumed that one molecule of strychnine binds to the receptor and the 'additional' strychnine molecule in the dimer probably protrudes from the orthosteric binding sites of the receptor.},
  subject      = {Strychnin},
  language  = {en}
}
@phdthesis{Messerer2017,
  author    = {Messerer, Regina},
  title     = {Synthesis of Dualsteric Ligands for Muscarinic Acetylcholine Receptors and Cholinesterase Inhibitors},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-149007},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The study is dealing with the synthesis and pharmacological investigation of newly designed dualsteric ligands of muscarinic acetylcholine receptors belonging to the superfamily of G protein-coupled receptors. Such bipharmacophoric ligands combine the advantages of the orthosteric binding site (high-affinity) and of the topographically distinct allosteric binding site (subtype-selectivity) resulting in compounds with reduced side effects. This opens the way to a new therapeutic approach in the treatment of e.g. chronic pain, drug withdrawal, Parkinson`s and Alzheimer`s disease. Furthermore, the newly synthesized dualsteric compounds were pharmacologically investigated in order to get a better understanding of the activation and signaling processes in muscarinic acetylcholine receptors, especially with regard to partial agonism. The development of the "dynamic ligand binding" concept offers new perspectives for ligand binding and signaling at G protein-coupled receptors. GPCRs are no longer considered as simple on/off switches. Dualsteric ligands can bind in a dualsteric pose, reflecting an active receptor state as well as in a purely allosteric binding pose, characterized by an inactive receptor state resulting in partial agonism. The degree of partial agonism depends on the ratio of active versus inactive receptor populations. On this basis, orthosteric/orthosteric hybrid ligands consisting of the antagonist atropine and scopolamine, respectively, as well as of the agonist iperoxo and isoxazole, respectively, linked via different alkyl chain length were synthesized in order to investigate partial agonism (Figure 1). Figure 1: Structures of the synthesized iperoxo/isoxazole-atropine/scopolamine-hybrids. Furthermore, different sets of quaternary and tertiary homodimers consisting either of two iperoxo or two acetylcholine units were synthesized in order to study their extent on partial agonism (Figure 2). The two agonists were connected by varying alkyl chain length. Binding studies on CHO-hM2 cells of the quaternary compounds revealed that dimerization of the agonist results in a loss of potency. The iperoxo-dimers reached higher maximum effects on the Gi- as well as on the Gs pathway in comparison to the acetylcholine-dimers. Besides the choice of the orthosteric building block (potency of the agonist), the alkyl chain length is also crucial for the degree of partial agonism. Figure 2: Structures of the synthesized quat./tert. iperoxo/acetylcholine-homodimers. Quinolone-based hybrids connected to the superagonist iperoxo and to the endogenous ligand acetylcholine, respectively, linked through an alkyl chain of different length were synthesized in order to develop further partial agonists (Figure 3). FRET studies confirmed M1 subtype-selectivity as well as linker dependent receptor response. The greatest positive FRET signal was observed with quinolone-C6-iper resulting from a positive cooperativity between the two separated moieties, alloster and orthoster. However, the corresponding hybrids with a longer linker led to an inverse FRET signal indicating a different binding mode, e.g. purely allosteric, in contrast to the shorter linked hybrids. Furthermore, the flexible alkyl spacer was replaced by a rigidified linker resulting in the hybrid quinolone-rigid-iperoxo (Figure 3). FRET studies on the M1 receptor showed reduced FRET kinetics, resulting from interactions between the bulky linker and the aromatic lid, located between the orthosteric and allosteric binding site. A bitopic binding mode of the rigidified hybrid is presumed. For further clarity, mutational studies are necessary. Figure 3: M1-selective hybrid compounds. Another aim of this work was the design and synthesis of new hybrid compounds, acting as agonists at the M1 and M2 receptor and as inhibitors for AChE and BChE in the context of M. Alzheimer. Several sets of hybrid compounds consisting of different pharmacophoric units (catalytic active site: phthalimide, naphthalimide, tacrine; peripheric anionic site: iperoxo, isoxazole) linked through a polymethylene chain of varying length were synthesized. Tac-C10-iper (Figure 4), consisting of tacrine and the superagonist iperoxo linked by a C10 polymethylene spacer, was found to have excellent anticholinesterase activity for both AChE (pIC50 = 9.81) and BChE (pIC50 = 8.75). Docking experiments provided a structural model to rationalize the inhibitory power towards AChE. Additionally, the tacrine related hybrids showed affinity to the M1 and M2 receptor. Such compounds, addressing more than one molecular target are favorable for multifactorial diseases such as Alzheimer. Figure 4: Structure of the most active compound regarding anticholinesterase activity. In summary, the choice of the pharmacophoric units, their connecting point as well as the nature, length, and flexibility of the linker play an important role for the activity of designed bivalent ligands. A shorter linker length cannot bridge both binding sites simultaneously in contrast to longer linker chains. On the other hand, too long linker chains can result in unwanted steric interactions. Further investigations with respect to structural variations of hybrid compounds, with or without quaternary ammonium groups, are necessary in the light of drug development.},
  subject      = {Cholinesteraseinhibitor},
  language  = {en}
}
@phdthesis{Zilian2014,
  author    = {Zilian, David},
  title     = {Neuartige, empirische Scoring-Modelle f{\"u}r Protein-Ligand-Komplexe und computergest{\"u}tzte Entwicklung von Hsp70-Inhibitoren},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-105055},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Techniken des computergest{\"u}tzten Wirkstoffdesigns spielen eine wichtige Rolle bei der Entwicklung neuer Wirkstoffe. Die vorliegende Arbeit befasst sich sowohl mit der Entwicklung als auch mit der praktischen Anwendung von Methoden des strukturbasierten Wirkstoffdesigns. Die Arbeit glieder sich daher in zwei Teile. Der erste Teil besch{\"a}ftigt sich mit der Entwicklung von empirischen Scoring-Funktionen, die eine Schl{\"u}sselrolle im strukturbasierten computergest{\"u}tzen Wirkstoffdesign einnehmen. Grundlage dieser Arbeiten sind die empirischen Deskriptoren und Scoring-Funktionen aus dem SFCscore-Programmpaket. Dabei wurde zun{\"a}chst untersucht, wie sich die Zusammensetzung der Trainingsdaten auf die Vorhersagen von empirischen Scoring-Funktionen auswirkt. Durch die gezielte Zusammenstellung eines neuen Trainingsdatensatzes wurde versucht, die Spannweite der Vorhersagen zu vergr{\"o}ßern, um so vor allem eine bessere Erkennung von hoch- und niedrig-affinen Komplexen zu erreichen. Die resultierende Funktion erzielte vor allem im niedrig-affinen Bereich verbesserte Vorhersagen. Der zweite Themenkomplex besch{\"a}ftigt sich ebenfalls mit der verbesserten Separierung von aktiven und inaktiven Verbindungen. Durch den Einsatz der Machine Learning-Methode RandomForest wurden dazu Klassifizierungsmodelle abgeleitet, die im Unterschied zu den klassischen Scoring-Funktionen keinen genauen Score liefern, sondern die Verbindungen nach ihrer potentiellen Aktivit{\"a}t klassifizieren. Am Beispiel des mykobakteriellen Enzyms InhA konnte gezeigt werden, dass derartige Modelle den klassischen Scoring-Funktionen im Bezug auf die Erkennung von aktiven Verbindungen deutlich {\"u}berlegen sind. Der RandomForest-Algorithmus wurde im n{\"a}chsten Schritt auch verwendet, um eine neue Scoring-Funktion zur Vorhersage von Bindungsaffinit{\"a}ten abzuleiten. Diese Funktion wurde unter dem Namen SFCscoreRF in das SFCscore-Programmpaket implementiert. Die Funktion unterschiedet sich in einigen wesentlichen Punkten von den urspr{\"u}nglichen SFCscore-Funktionen. Zum einen handelt es sich beim RF-Algorithmus um eine nicht-lineare Methode, die im Unterschied zu den klassischen Methoden, die zur Ableitung von Scoring-Funktionen eingesetzt werden, nicht von der Additivit{\"a}t der einzelnen Deskriptoren ausgeht. Der Algorithmus erlaubt außerdem die Verwendung aller verf{\"u}gbaren SFCscore-Deskriptoren, was eine deutlich umfassendere Repr{\"a}sentation von Protein-Ligand-Komplexen als Grundlage des Scorings erm{\"o}glicht. F{\"u}r die Ableitung von SFCscoreRF wurden insgesamt 1005 Komplexe im Trainingsdatensatz verwendet. Dieser Datensatz ist somit einer der gr{\"o}ßten, die bisher f{\"u}r die Ableitung einer empirischen Scoring-Funktion verwendet wurden. Die Evaluierung gegen zwei Benchmark-Datens{\"a}tze ergab deutlich bessere Vorhersagen von SFCscoreRF im Vergleich zu den urspr{\"u}nglichen SFCscore-Funktionen. Auch im internationalen Vergleich mit anderen Scoring-Funktion konnten f{\"u}r beide Datens{\"a}tze Spitzenwerte erreicht werden. Weitere ausgiebige Testungen im Rahmen einer Leave-Cluster-Out-Validierung und die Teilnahme am CSAR 2012 Benchmark Exercise ergaben, dass auch SFCscoreRF Performanceschwankungen bei der Anwendung an proteinspezifischen Datens{\"a}tzen zeigt - ein Ph{\"a}nomen, dass bei Scoring-Funktionen immer beobachtet wird. Die Analyse der CSAR 2012-Datens{\"a}tze ergab dar{\"u}ber hinaus wichtige Erkenntnisse im Bezug auf Vorhersage von gedockten Posen sowie {\"u}ber die statistische Signifikanz bei der Evaluierung von Scoring-Funktionen. Die Tatsache, dass empirische Scoring-Funktionen innerhalb eines bestimmten chemischen Raums trainiert wurden, ist ein wichtiger Faktor f{\"u}r die protein-abh{\"a}ngigen Leistungsschwankungen, die in dieser Arbeit beobachtet wurden. Verl{\"a}ssliche Vorhersagen sind nur innerhalb des kalibrierten chemischen Raums m{\"o}glich. In dieser Arbeit wurden verschiedene Ans{\"a}tze untersucht, mit denen sich diese ``Applicability Domain'' f{\"u}r die SFCscore-Funktionen definieren l{\"a}sst. Mit Hilfe von PCA-Analysen ist es gelungen die ``Applicability Domain'' einzelner Funktionen zu visualisieren. Zus{\"a}tzlich wurden eine Reihe numerischer Deskriptoren getestet, mit den die Vorhersageverl{\"a}sslichkeit basierend auf der ``Applicability Domain'' abgesch{\"a}tzt werden k{\"o}nnte. Die RF-Proximity hat sich hier als vielversprechender Ausgangspunkt f{\"u}r weitere Entwicklungen erwiesen. Der zweite Teil der Arbeit besch{\"a}ftigt sich mit der Entwicklung neuer Inhibitoren f{\"u}r das Chaperon Hsp70, welches eine vielversprechende Zielstruktur f{\"u}r die Therapie des multiplen Myeloms darstellt. Grundlage dieser Arbeiten war eine Leitstruktur, die in einer vorhergehenden Arbeit entdeckt wurde und die vermutlich an einer neuartigen Bindestelle in der Interface-Region zwischen den beiden großen Dom{\"a}nen von Hsp70 angreift. Die Weiterentwicklung und Optimierung dieser Leitstruktur, eines Tetrahydroisochinolinon-Derivats, stand zun{\"a}chst im Vordergrund. Anhand detaillierter Docking-Analysen wurde der potentielle Bindemodus der Leitstruktur in der Interfaceregion von Hsp70 untersucht. Basierend auf diesen Ergebnissen wurde eine Substanzbibliothek erstellt, die von Kooperationspartnern innerhalb der KFO 216 synthetisiert und biologisch getestet wurde. Die Struktur-Wirkungsbeziehungen, die sich aus diesen experimentellen Daten ableiten lassen, konnten teilweise gut mit den erstellten Docking-Modellen korreliert werden. Andere Effekte konnten anhand der Docking-Posen jedoch nicht erkl{\"a}rt werden. F{\"u}r die Entwicklung neuer Derivate ist deswegen eine umfassendere experimentelle Charakterisierung und darauf aufbauend eine Verfeinerung der Bindungsmodelle notwendig. Strukturell handelt es sich bei Hsp70 um ein Zwei-Dom{\"a}nen-System, dass verschiedene allostere Zust{\"a}nde einnehmen kann. Um die Auswirkungen der daraus folgenden Flexibilit{\"a}t auf die Stabilit{\"a}t der Struktur und die Bindung von Inhibitoren zu untersuchen, wurden molekulardynamische Simulationen f{\"u}r das Protein durchgef{\"u}hrt. Diese zeigen, dass das Protein tats{\"a}chlich eine {\"u}berdurchschnittlich hohe Flexibilit{\"a}t aufweist, die vor allem durch die relative Bewegung der beiden großen Dom{\"a}nen zueinander dominiert wird. Die Proteinkonformation die in der Kristallstruktur hscaz beobachtet wird, bleibt jedoch in ihrer Grundstruktur in allen vier durchgef{\"u}hrten Simulationen erhalten. Es konnten hingegen keine Hinweise daf{\"u}r gefunden werden, dass die Mutationen, welche die f{\"u}r die strukturbasierten Arbeiten verwendete Kristallstruktur im Vergleich zum Wildtyp aufweist, einen kritischen Einfluss auf die Gesamtstabilit{\"a}t des Systems haben. Obwohl die Interface-Region zwischen NBD und SBD also in allen Simulationen erhalten bleibt, wird die Konformation in diesem Bereich doch wesentlich durch die Dom{\"a}nenbewegung beeinflusst und variiert. Da dieser Proteinbereich den wahrscheinlichsten Angriffspunkt der Tetrahydroisochinolinone darstellt, wurde der Konformationsraum detailliert untersucht. Wie erwartet weist die Region eine nicht unerhebliche Flexibilit{\"a}t auf, welche zudem, im Sinne eines ``Induced-Fit''-Mechanismus, durch die Gegenwart eines Liganden (Apoptozol) stark beeinflusst wird. Es ist daher als sehr wahrscheinlich anzusehen, dass die Dynamik der Interface-Region auch einen wesentlichen Einfluss auf die Bindung der Tetrahydroisochinolinone hat. Molekuardynamische Berechnungen werden deswegen auch in zuk{\"u}nftige Arbeiten auf diesem Gebiet eine wichtige Rolle spielen. Die Analysen zeigen zudem, dass die Konformation der Interface-Region eng mit der Konformation des gesamten Proteins - vor allem im Bezug auf die relative Stellung von SBD und NBD zueinander - verkn{\"u}pft ist. Das untermauert die Hypothese, dass die Interface-Bindetasche einen Angriffspunkt f{\"u}r die Inhibtion des Proteins darstellt.},
  subject      = {Arzneimittelforschung},
  language  = {de}
}
@phdthesis{Wyzgol2012,
  author    = {Wyzgol, Agnes},
  title     = {Generierung und Charakterisierung rekombinanter TNF-Liganden},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-76449},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Liganden und Rezeptoren der TNF-Familie regulieren eine Vielzahl zellul{\"a}rer Prozesse, darunter Apoptose und Immunprozesse. TNF-Liganden kommen in Form l{\"o}slicher und membranst{\"a}ndiger trimerer Molek{\"u}le vor, wobei die trimere Organisation durch die konservierte THD vermittelt wird. Im Gegensatz zu den membranst{\"a}ndigen Molek{\"u}len k{\"o}nnen l{\"o}sliche TNF-Liganden nicht immer an ihren TNF-Rezeptor binden oder ihn effektiv aktivieren. F{\"u}r zwei solcher inaktiven TNF-Liganden, n{\"a}mlich TRAIL und CD95L, konnte gezeigt werden, dass durch sekund{\"a}re Oligomerisierung oder durch artifizielle Herstellung einer Membranst{\"a}ndigkeit mittels Antik{\"o}rperdom{\"a}nen gegen zelloberfl{\"a}chenexprimierte Proteine hochaktive Ligandenvarianten generiert werden k{\"o}nnen. Inwieweit sich diese Verfahren auf die T-Zell-kostimulatorischen TNF-Liganden OX40L, 41BBL und CD27L {\"u}bertragen lassen, wurde in dieser Arbeit untersucht. L{\"o}sliche Flag- und Flag-TNC-Varianten von OX40L und 41BBL zeigten eine gute Bindung an die Rezeptoren OX40 und 41BB. Die l{\"o}sliche Variante Flag-CD27L konnte nicht an ihren Rezeptor CD27 binden. Dies war aber nach Einf{\"u}hrung der trimerstabilisierenden TNC-Dom{\"a}ne m{\"o}glich. Eine effektive Aktivierung ihres Rezeptors, nachgewiesen durch Analyse der IL8-Induktion, bewirkten die l{\"o}slichen TNF-Ligandenvarianten nur nach sekund{\"a}rer Oligomerisierung mittels des Flag-spezifischen Antik{\"o}rpers M2. Eine {\"a}hnlich gute TNFR-Aktivierung ließ sich durch Einf{\"u}hrung der hexamerisierenden Fc-Dom{\"a}ne erzielen. Fc-Flag-OX40L und Fc-Flag-41BBL induzierten bereits ohne sekund{\"a}re Quervernetzung effektiv IL8. Die Hexamerisierung alleine reichte f{\"u}r die l{\"o}sliche CD27L-Variante nicht aus, hier war zus{\"a}tzlich zur Fc- wiederum auch die TNC-Dom{\"a}ne erforderlich, um die Bindung an CD27 und eine schwache IL8-Induktion zu erzielen. F{\"u}r die FAP-bindenden Fusionsproteine antiFAP-Flag- OX40L, antiFAP-Flag-41BBL und antiFAP-Flag-TNC-CD27L war die Bindung an OX40, 41BB und CD27 sowie an FAP nachweisbar. Erst durch die artifizielle Membranst{\"a}ndigkeit nach Bindung an FAP konnten diese Fusionsproteine {\"u}ber ihren Rezeptor effektiv IL8 induzieren. Zusammenfassend ließ sich somit zeigen, dass sich schwach oder nicht aktive l{\"o}sliche Ligandenvarianten von OX40L und 41BBL durch sekund{\"a}re Oligomerisierung, durch die Fc-Hexamerisierungsdom{\"a}ne und durch artifizielle Membranst{\"a}ndigkeit in hochaktive Liganden verwandeln lassen. L{\"o}sliche CD27L-Varianten ben{\"o}tigen zus{\"a}tzlich die trimerstabilisierende TNC-Dom{\"a}ne, um CD27 binden und aktivieren zu k{\"o}nnen. F{\"u}r das bessere Verst{\"a}ndnis der Ligand-Rezeptor-Interaktionen wurden zus{\"a}tzlich OX40L-, 41BBL- und CD27L-Fusionsproteine mit der hochaktiven Gaussia princeps Luziferase (GpL) generiert, um Gleichgewichtsbindungs-, Dissoziationsstudien und homologe Kompetitionsassays durchf{\"u}hren zu k{\"o}nnen. F{\"u}r die Fusionsproteine GpL-Flag-TNC-OX40L, GpL-Flag-TNC-41BBL und GpL-Flag-TNC-CD27L konnte gezeigt werden, dass die IL8-Induktion nicht von der Rezeptorbelegung abh{\"a}ngt, sondern von der sekund{\"a}ren Oligomerisierung, da bei gleicher Rezeptorbelegung durch sekund{\"a}r quervernetzte TNF-Liganden mehr IL8 induziert wird, die Rezeptoraktivierung also qualitativ besser sein muss.},
  subject      = {Tumor-Nekrose-Faktor},
  language  = {de}
}
@phdthesis{Cappel2011,
  author    = {Cappel, Daniel},
  title     = {Computersimulationen zur Untersuchung von Wassermolek{\"u}len in Protein-Ligand Komplexen am Beispiel einer Modellbindetasche},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-55003},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2011},
  abstract  = {Wassermolek{\"u}le spielen oft eine entscheidende Rolle bei der Bindung von Liganden an Proteine. Zum einen ist dies in ihrer Eigenschaft als Wasserstoffbr{\"u}ckendonor und -akzeptor begr{\"u}ndet, die es erm{\"o}glicht Wechselwirkung zwischen Ligand und Rezeptor zu vermitteln. Zum anderen stellen die Desolvatisierungsenthalpie und -entropie einer Bindetasche w{\"a}hrend der Ligandbindung einen entscheidenden Anteil der Bindungsaffinit{\"a}t dar. Obwohl man sich dieser Einfl{\"u}sse seit langem bewusst ist, sind aktuelle Methoden des computerbasierten Wirkstoffdesigns nur in sehr begrenztem Umfang in der Lage, die entsprechenden Effekte zu erfassen und vorherzusagen. Da experimentelle Daten {\"u}ber die Effekte von Wassermolek{\"u}len in Protein-Ligand Komplexen von Natur aus schwierig zu erhalten sind, untersucht die vorliegende Arbeit eine Modellbindetasche einer Cytochrom c Peroxidase Mutante (CCP W191G) mit Hilfe von Molecular Modeling Techniken. Diese polare und solvatisierte Kavit{\"a}t ist strukturell sehr gut charakterisiert und bindet kleine, kationische Heterozyklen zusammen mit unterschiedlichen Mengen an Wassermolek{\"u}len. F{\"u}r die Untersuchungen wurden strukturell {\"a}hnliche Liganden mit einem unterschiedlichen Wechselwirkungsmuster ausgew{\"a}hlt. Davon ausgehend wurde die M{\"o}glichkeit zweier Docking-Programme, den Grad der Wasserverdr{\"a}ngung durch den Liganden zusammen mit dem Bindungsmodus vorherzusagen, untersucht. Die dynamischen Eigenschaften der Bindetaschenwassermolek{\"u}le wurden mittels Molekulardynamiksimulationen studiert. Schließlich wurden diese rein strukturellen Betrachtungen durch eine energetische/thermodynamische Analyse komplettiert. Die Anwendung dieser unterschiedlichen Verfahren liefert einige neue Erkenntnisse {\"u}ber die untersuchte Modellbindetasche. Trotz der relativen Einfachheit der kleinen Kavit{\"a}t der CCP W191G Mutante war die vollst{\"a}ndige Charakterisierung und eine korrekte (retrospektive) Vorhersage des Wasser-Wechselwirkungsmuster der Ligand-Komplexe nicht trivial. Zusammenfassend kann man festhalten, dass insgesamt eine gute {\"U}bereinstimmung zwischen den durch Computersimulationen erhaltenen Ergebnissen und den kristallographischen Daten erzielt wurde. Unerwartete Befunde, die auf den ersten Blick mit den kristallographischen Beobachtungen nicht {\"u}bereinstimmen, k{\"o}nnen ebenso durch Limitationen in den Kristallstrukturen bedingt sein. Dar{\"u}ber hinaus gaben die Ergebnisse auch eine Hilfestellung, welches Verfahren zur Beantwortung einer Fragestellung im Rahmen von Wassermolek{\"u}len im Wirkstoffdesign geeignet sind. Schließlich wurden ebenso die Begrenzungen der jeweiligen Methoden aufgezeigt.},
  subject      = {Cytochromperoxidase},
  language  = {de}
}
@phdthesis{Duechs2011,
  author    = {D{\"u}chs, Matthias},
  title     = {Effects of Toll-like receptor agonists on the pathogenesis of atopic asthma in mice},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-66369},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2011},
  abstract  = {In the last decades, both the incidence and the severity of asthma have steadily increased. Furthermore, available therapies only treat the symptoms but do not cure the disease. Immune modulation induced by TLR agonists may be a promising novel approach to effectively treat asthma as it targets the underlying immunopathology directly rather than one mediator alone. The aim of this thesis was to investigate if the immunostimulatory properties of Toll-like receptor (TLR) agonists can be utilized to develop novel therapeutic intervention strategies for the treatment of asthma using murine models of allergic inflammation. For this purpose five different TLR agonists were tested in preclinical mouse models of acute and chronic asthma, both in preventive and therapeutic settings. Firstly, TLR-2, 3, 4, 7/8 and 9 agonists were delivered intratracheally at different doses before pulmonary allergen exposure in the asthma model of acute inflammation. TLR9 agonist CpG-containing oligodeoxynucleotides (CpG) > TLR7 agonist Resiquimod (R848) > TLR3 agonists poly(I:C) strongly reduced allergen induced airway eosinophilia and IL-4 levels in a dose-dependent manner. All TLR agonists increased neutrophil numbers, TLR4 agonist lipopolysaccharide (LPS) > TLR2 agonist lipoteichonic acid (LTA) > poly(I:C) > CpG > R848 and, with the exception of R848, the amount of pro-inflammatory cytokines in the airways. Suppressive effects were not dependent upon IFN-γ and IL-10 or associated with increased numbers of regulatory T cells in the airways. All TLR agonists, except LTA, similarly reduced airway eosinophilia and IL-4 levels when applied therapeutically after allergen challenge. These results show that the TLR agonists have different suppressive effects on TH2 responses in the airways which further depend on the dose and the experimental setup in which they were tested. Interestingly, all agonists induced airway neutrophilia, albeit to different degrees, raising the question if TLR ligands are safe for human use when applied directly into the lung. Different TLR agonists are also being developed for human use as adjuvants combined with allergen in specific immunotherapy. Recent clinical data suggest that this may be achieved by induction of allergen-specific TH1 responses. For this reason, the ability of different TLR agonists to induce allergen-specific TH1 and suppress allergen-specific TH2 responses in a preclinical setting was investigated in this thesis. Different doses of the TLR agonists were applied together with allergen, then mice were exposed to allergen aerosol. CpG > LPS >LTA dose-dependently strongly suppressed the development of airway eosinophilia with poly(I:C) and R848 having no effect. The decrease in eosinophilic numbers was associated withincreased neutrophils present in the airways. IL-4 and IL-5 levels in the bronchoalveolar lavage fluid were also decreased when poly(I:C), LPS, and CpG were used. All TLR agonists increased allergen-specific IgG2a, and with the exception of poly(I:C), reduced allergen-specific IgE levels in the serum. Cutaneous anaphylaxis to allergen was completely prevented when LPS or CpG were given as adjuvant. The strongest TH1 responses were induced by CpG and poly(I:C), characterized by the presence of IFN-γ in the bronchoalveolar lavage and the highest allergen-specific IgG2a levels in the serum. This data supports approaches to use TLR9 or TLR4 agonists for human therapy as adjuvant in combination with allergen in novel specific immunotherapy formulations. In the last part of the thesis, it was investigated if TLR activation can also affect the pathology of severe chronic asthma. Therapeutic administration of R848 or CpG reduced features of inflammation and remodeling. Both agonists showed superior effects to dexamethasone, with CpG being more efficient than R848. This result again supports a TLR9-based therapy as a viable option for the treatment of severe chronic asthma which may present a potential alternative for anti-inflammatory therapy with steroids. Taken together, the results of this thesis support the use of TLR agonists to treat asthma. The most favorable efficacy/safety ratio is to be expected from TLR-based therapies combining TLR4 or TLR9 agonists with allergen in specific immunotherapy. In regard to TLR agonist monotherapy, R848 and CpG showed the most promising profiles, CpG particularly in a model of severe chronic asthma. However, since all TLR agonists used in this study also showed pro-inflammatory potential, the safety aspect of such an approach needs to be taken into account.},
  subject      = {Toll-like Rezeptor},
  language  = {en}
}
@phdthesis{Harth2010,
  author    = {Harth, Stefan},
  title     = {Molecular Recognition in BMP Ligand-Receptor Interactions},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-52797},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {Bone Morphogenetic Proteins (BMPs) are secreted multifunctional signaling proteins that play an important role during development, maintenance and regeneration of tissues and organs in almost all vertebrates and invertebrates. BMPs transmit their signals by binding to two types of serine-/threonine-kinase receptors. BMPs bind first to their high affinity receptor, thereby recruiting their low affinity receptor into the complex. This receptor assembly starts a Smad (Small mothers against decapentaplegic) protein signaling cascade which regulates the transcription of responsive genes. Up to date, only seven type I and five type II receptors are known for more than 30 ligands. Therefore, many BMP ligands can recruit more than one receptor subtype. Vice versa, receptors can bind to several ligands, indicating a highly promiscuous ligand-receptor interaction. This raises the following questions: (i) How are BMPs able to induce ligand-specific signals, despite forming complexes with identical receptor composition and (ii) how are they able to recognize and bind various binding partners in a highly specific manner. From the ligand's point of view, heterodimeric BMPs are valuable tools for studying the interplay between different sets of receptors, thereby providing new insights into how the various BMP signals can be generated. This study describes the expression and purification of the heterodimers BMP-2/6 and -2/7 from E.coli cells. BIAcore interaction studies and various in vitro cell activity assays revealed that the generated heterodimers are biologically active. Furthermore, BMP-2/6 and -2/7 exhibit a higher biological activity in most of the cell assays compared to their homodimeric counterparts. In addition, the BMP type I receptor BMPR-IA is involved in heterodimeric BMP signaling. However, the usage of other type I receptor subtypes (e.g. ActR-I) building a heteromeric ligand-receptor type I complex as indicated in previous works could not be determined conclusively. Furthermore, BMP heterodimers seem to require only one type I receptor for signaling. From the receptors' point of view, the BMP type I receptor BMPR-IA is a prime example for its promiscuous binding to different BMP ligands. The extracellular binding interface of BMPR-IA is mainly unfolded in its unbound form, requiring a large induced fit to adopt the conformation when bound to its ligand BMP-2. In order to unravel whether the binding promiscuity of BMPR-IA is linked to structural plasticity of its binding interface, the interaction of BMPR-IA bound to an antibody Fab fragment was investigated. The Fab fragment was selected because of its ability to recognize the BMP-2 binding epitope on BMPR-IA, thus neutralizing the BMP-2 mediated receptor activation. This study describes the crystal structure of the complex of the extracellular domain of BMPR-IA bound to the antibody Fab fragment AbyD1556. The crystal structure revealed that the contact surface of BMPR-IA overlaps extensively with the contact surface of BMPR-IA for BMP-2 interaction. Although the contact epitopes of BMPR-IA to both binding partners coincide, the three-dimensional structures of BMPR-IA in both complexes differ significantly. In contrast to the structural differences, alanine-scanning mutagenesis of BMPR-IA showed that the functional determinants for binding to both the antibody and BMP-2 are almost identical. Comparing the structures of BMPR-IA bound to BMP-2 or to the Fab AbyD1556 with the structure of unbound BMPR-IA revealed that binding of BMPR-IA to its interaction partners follows a selection fit mechanism, possibly indicating that the ligand promiscuity of BMPR-IA is inherently encoded by structural adaptability.},
  subject      = {Knochen-Morphogenese-Proteine},
  language  = {en}
}