@article{RauertStuehmerBargouetal.2011, author = {Rauert, H. and St{\"u}hmer, T. and Bargou, R. and Wajant, H. and Siegmund, D.}, title = {TNFR1 and TNFR2 regulate the extrinsic apoptotic pathway in myeloma cells by multiple mechanisms}, series = {Cell Death and Disease}, volume = {2}, journal = {Cell Death and Disease}, doi = {10.1038/cddis.2011.78}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-133486}, pages = {e194}, year = {2011}, abstract = {The huge majority of myeloma cell lines express TNFR2 while a substantial subset of them failed to show TNFR1 expression. Stimulation of TNFR1 in the TNFR1-expressing subset of MM cell lines had no or only a very mild effect on cellular viability. Surprisingly, however, TNF stimulation enhanced cell death induction by CD95L and attenuated the apoptotic effect of TRAIL. The contrasting regulation of TRAIL- and CD95L-induced cell death by TNF could be traced back to the concomitant NFjBmediated upregulation of CD95 and the antiapoptotic FLIP protein. It appeared that CD95 induction, due to its strength, overcompensated a rather moderate upregulation of FLIP so that the net effect of TNF-induced NFjB activation in the context of CD95 signaling is pro-apoptotic. TRAIL-induced cell death, however, was antagonized in response to TNF because in this context only the induction of FLIP is relevant. Stimulation of TNFR2 in myeloma cells leads to TRAF2 depletion. In line with this, we observed cell death induction in TNFR1-TNFR2-costimulated JJN3 cells. Our studies revealed that the TNF-TNF receptor system adjusts the responsiveness of the extrinsic apoptotic pathway in myeloma cells by multiple mechanisms that generate a highly context-dependent net effect on myeloma cell survival}, language = {en} } @phdthesis{Schmidt2015, author = {Schmidt, Thomas Christian}, title = {Theoretical Investigations on the Interactions of Small Compounds with their Molecular Environments}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-127860}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {Im ersten Teil dieser Arbeit wird eine Kombination theoretischer Methoden f{\"u}r die strukturbasierte Entwicklung neuer Wirkstoffe pr{\"a}sentiert. Ausgehend von der Kristallstruktur eines kovalenten Komplexes einer Modellverbindung mit dem Zielprotein wurde mit Hilfe von quantenmechanischen und QM/MM Rechnungen die genaue Geometrie des vorausgehenden nicht-kovalenten Komplexes betimmt. Letztere ist der bestimmende Faktor f{\"u}r die Reaktivit{\"a}t des Inhibitors gegen{\"u}ber der katalytisch aktiven Aminos{\"a}ure und damit f{\"u}r die Ausbildung einer kovalenten Bindung. Aus diesem Grund wurde diese Geometrie auch f{\"u}r die Optimierung der Substitutionsmusters des Ihnibitors verwendet, um dessen Affinit{\"a}t zum Zielenzyme zu verbessern ohne dass dieser seine F{\"a}higkeit kovalent an das aktive Zentrum zu binden verliert. Die Optimierung des Substitutionsmuster wurde doch Methode des Molekularen Dockings unterst{\"u}tzt, das diese optimal dazu geeignet sind, Bindungsaffinit{\"a}ten vorherzusagen, die durch eine Modifikation der chemischen Struktur entstehen. Eine Auswahl der besten Strukturen wurde anschließend verwendet, um zu {\"u}berpr{\"u}fen, ob die ver{\"a}nderten Molek{\"u}le noch gen{\"u}gen Reaktivit{\"a}t gegen{\"u}ber dem Zielprotein aufweisen. Molek{\"u}ldynamik Simulationen der neuen Verbindungen haben jedoch gezeigt, dass die ver{\"a}nderten Verbindungen nur so and das Protein binden, dass die Bilung eine kovalenten Bindung zum Enzym nicht mehr m{\"o}glich ist. Daher wurden in einem weiteren Schritt die Modellverbindungen weiter modifiziert. Neben {\"A}nderungen im Substitutionsmuster wurde auch die chemische Struktur im Kern ver{\"a}ndert. Die Bindungsaffinit{\"a}ten wurde wieder mittels Docking {\"u}berpr{\"u}ft. F{\"u}r die besten Bindungsposen wurden wieder Simulationen zur Molek{\"u}ldynamik durchgef{\"u}hrt, wobei diesmal die Ausbildung einer kovalenten Bindung zum Enzyme m{\"o}glich erscheint. In einer abschließenden Serie von QM/MM Rechnungen unter Ber{\"u}cksichtigung verschiedener Protonierungszust{\"a}nde des Inhibitors und des Proteins konnten Reaktionspfade und zugeh{\"o}rige Reaktionsenergien bestimmt werden. Die Ergebnisse lassen darauf schließen, dass eines der neu entwickelten Molek{\"u}le sowohl eine stark verbesserte Bindungsaffinit{\"a}t wie auch die M{\"o}glichkeit der kovalenten Bindung an Enzyme aufweist. Der zweite Teil der Arbeit konzentriert sich auf die Umgebungseinfl{\"u}sse auf die Elektronenverteilung eines Inhibitormodells. Als Grundlage dient ein vinylsulfon-basiertes Moek{\"u}l, f{\"u}r das eine experimentell bestimmte Kristallstruktur sowie ein theoretisch berechneter Protein Komplex verf{\"u}gbar sind. Ein Referendatensatz f{\"u}r diese Systeme wurde erstellt, indem der Konformationsraum des Inhibitors nach m{\"o}glichen Minimumsstrukturen abgesucht wurde, welche sp{\"a}ter mit den Geometrien des Molek{\"u}ls im Kristall und im Protein verglichen werden konnten. The Geometrie in der Kristallumgebung konnte direkt aus den experimentellen Daten {\"u}bernommen werden. Rechnungen zum nicht-kovalenten Protein Komplex hingegen haben gezeigt, dass f{\"u}r das Modellsystem mehrere Geometrien des Inhibiors sowie zwei Protonierungszust{\"a}nde f{\"u}r die katalytisch aktiven Aminos{\"a}uren m{\"o}glich sind. F{\"u}r die Analyse wurden daher alle m{\"o}glichen Proteinkomplexe mit der Kristallstruktur verglichen. Ebenso wurden Vergleiche mit der Geometrie des isolierten Molek{\"u}ls im Vakuum sowie der Geometrie in w{\"a}ssriger L{\"o}sung angestellt. F{\"u}r die Geometrie des Molek{\"u}ls an sich ergab sich eine gute {\"U}bereinstimmung f{\"u}r alle Modellsysteme, f{\"u}r die Wechselwirkungen mit der Umgebung jedoch nicht. Die Ausbildung von Dimeren in der Kristallumgebung hat einen stark stablisierenden Effekt und ist einer der Gr{\"u}nde, warum dieser Kristall so gut wie keine Fehlordungen aufweist. In den Proteinkomplexen hingegen ergibt sich eine Abstoßung zwischen dem Inhibitor und einer der katalytisch aktiven Aminos{\"a}uren. Als Ursache f{\"u}r diese Abstoßung konnte die Einf{\"u}hrung der Methylaminfunktion ausgemacht werden. Vermutlicherweise f{\"u}hrt diese strukturelle {\"A}nderung auch dazu, dass der Modellinhibitor nicht in der Lage ist, so wie die Leitstruktur K11777 an das aktive Zentrum des Enzyms zu binden.}, subject = {Theoretische Chemie}, language = {en} } @phdthesis{Gelmedin2008, author = {Gelmedin, Verena Magdalena}, title = {Targeting flatworm signaling cascades for the development of novel anthelminthic drugs}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-33334}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2008}, abstract = {Echinococcus multilocularis verursacht die Alveol{\"a}re Echinokokkose (AE), eine lebendsbedrohliche Krankheit mit limitierten chemotherapeutischen M{\"o}glichkeiten. Die jetzige Anti-AE Chemotherapie basiert auf einer einzigen Wirkstoffklasse, den Benzimidazolen. Obwohl Benzimidazole in vitro parasitozid wirken, wirken sie in vivo bei AE-Behandlung lediglich parasitostatisch und rufen schwere Nebenwirkungen hervor. In F{\"a}llen operabler L{\"a}sionen erfordert die Resektion des Parasitengewebes {\"u}ber einen l{\"a}ngeren Zeitraum eine chemotherapeutische Unterst{\"u}tzung. Damit sind die jetzigen Behandlungsm{\"o}glichkeiten inad{\"a}quat und ben{\"o}tigen Alternativen. In der vorliegenden Arbeit wurden die Signalwege von Plattw{\"u}rmern analysiert, um potentielle Targets f{\"u}r neue therapeutische Ans{\"a}tze zu identifizieren. Dabei konzentrierte ich mich unter Anwendung von molekularbiologischer, biochemischer und zellbiologischer Methoden auf Faktoren, die an Entwicklung und Proliferation von E. multilocularis beteiligt sind. Darunter waren die drei MAP kinases des Parasiten EmMPK1, ein Erk1/2-Ortholog, EmMPK2, ein p38-Ortholog und EmMPK3, ein Erk7/8-Ortholog. Des Weiteren identifizierte und charakterisierte ich EmMKK2, ein MEK1/2-Ortholog des Parasiten, welches zusammen mit den bekannten Kinasen EmRaf und EmMPK1 ein Erk1/2-{\"a}hnliches MAPK Modul bildet. Ich konnte zudem verschiedene Einfl{\"u}sse von Wirtswachstumsfaktoren wie EGF (epidermal growth factor) und Insulin auf die Signalmechanismen des Parasiten und das Larvenwachstum zeigen, darunter die Phosphorylierung von Elp, ein Ezrin-Radixin-Moesin {\"a}hnliches Protein, die Aktivierung von EmMPK1 und EmMPK3 und eine gesteigerte mitotische Aktivit{\"a}t der Echinokokkenzellen. Zus{\"a}tzlich wurden verschiedene Substanzen auf ihre letale Wirkung auf den Parasiten untersucht, darunter befanden sich (1.) generelle Inhibitoren von Tyrosinkinasen (PP2, Leflunamid), (2.) gegen die Aktivit{\"a}t von Rezeptor-Tyrosin-Kinasen gerichtete Pr{\"a}parate, (3.) urspr{\"u}nglich anti-neoplastische Wirkstoffe wie Miltefosin und Perifosin, (4.) Inhibitoren von Serin/ Threonin-Kinasen, die die Erk1/2 MAPK Kaskade blockieren und (5.) Inhibitoren der p38 MAPK. In diesen Untersuchungen hat sich EmMPK2 aus den folgenden Gr{\"u}nden als vielversprechendes Target erwiesen. Aminos{\"a}uresequenz-Analysen offenbarten einige Unterschiede zu menschlichen p38 MAP Kinasen, welche sehr wahrscheinlich die beobachtete gesteigerte basale Aktivit{\"a}t des rekombinanten EmMPK2 verursachen, verglichen mit der Aktivit{\"a}t humaner p38 MAPK-\&\#945;. Zus{\"a}tzlich suggerieren die prominente Autophosphorylierungsaktivit{\"a}t von rekombinantem EmMPK2 und das Ausbleiben einer Interaktion mit den Echinococcus MKKs einen unterschiedlichen Regulierungsmechanismus im Vergleich zu den humanen Proteinen. Die Aktivit{\"a}t von EmMPK2 konnte sowohl in vitro als auch in kultivierten Metazestodenvesikeln durch die Behandlung mit SB202190 und ML3403, zwei ATP kompetitiven Pyridinylimidazolinhibitoren der p38 MAPK, in Konzentrations-abh{\"a}ngiger Weise inhibiert werden. Zudem verursachten beide Substanzen, insbesondere ML3403 die Inaktivierung von Parasitenvesikeln bei Konzentrationen, die kultivierte S{\"a}ugerzellen nicht beeintr{\"a}chtigten. Ebenso verhinderte die Anwesenheit von ML3403 die Generation von neuen Vesikeln w{\"a}hrend der Kultivierung von Echinococcus Prim{\"a}rzellen. Das Targeting von Mitgliedern des EGF-Signalwegs, insbesondere der Erk1/2-{\"a}hnlichen MAPK Kaskade mit Raf- und MEK- Inhibitoren verhinderte die Phosphorylierung von EmMPK1 in in vitro kultivierten Metazestoden. Obwohl das Parasitenwachstum unter diesen Konditionen verhindert wurde, blieb die strukturelle Integrit{\"a}t der Metazestodenvesikeln w{\"a}hrend der Langzeitkultivierung in Anwesenheit der MAPK Kaskade-Inhibitoren erhalten. {\"A}hnliche Effekte wurden beobachtet nach Behandlung mit den anderen zuvor aufgef{\"u}hrten Inhibitoren. Zusammenfassend l{\"a}sst sich festhalten, dass verschiedene Targets identifiziert werden konnten, die hoch sensibel auf die Anwesenheit der inhibitorischen Substanzen reagierten, aber nicht zum Absterben des Parasiten f{\"u}hrten, mit Ausnahme der Pyridinylimidazolen. Die vorliegenden Daten zeigen, dass EmMPK2 ein {\"U}berlebendsignal vermittelnden Faktor darstellt und dessen Inhibierung zur Behandlung der AE benutzt werden k{\"o}nnte. Dabei erwiesen sich p38 MAPK Inhibitoren der Pyridinylimidazolklasse als potentielle neue Substanzklasse gegen Echinokokken.}, subject = {Fuchsbandwurm}, language = {en} } @phdthesis{Kesetovic2016, author = {Kesetovic, Diana}, title = {Synthesis and biological testing of potential anti-tuberculosis drugs targeting the β-ketoacyl ACP synthase}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131301}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {With 9.6 million new cases and 1.5 million deaths in 2014, tuberculosis (TB) is alongside with AIDS the most deadly infection.‎ Foremost, the increased prevalence of resistant strains of M. tuberculosis among the TB-infected population represents a serious thread. Hence, in the last decades, novel drug targets have been investigated worldwide. So far a relatively unexplored target is the cell wall enzyme β-ketoacyl-ACP-synthase "KasA", which plays a crucial role in maintaining the membrane impermeability and hence the cell ability to resist to the immune response and drug therapy. KasA is a key enzyme in the fatty acid synthase "FAS-II" elongation cycle, responsible for the extension of the growing acyl chain within the biosynthesis of precursors for the most hydrophobic constituents of the cell wall - mycolic acids. Design of the novel KasA inhibitors, performed in the research group of Prof. Sotriffer by C. Topf and B. Schaefer, was based on the recently published crystal structure of KasA‎ in complex with its known inhibitor thiolactomycin (TLM). Considering the essential ligand-enzyme interactions, a pharmacophore model was built and applied in the virtual screening of a modified ZINC database. Selected hits with the best in silico affinity data have been reported by Topf‎ and Schaefer‎. In this work, two of the obtained hits were synthesized and their structure was systematically varied. First, a virtual screening hit, chromone-2-carboxamide derivative GS-71, was modified in the amide part. Since the most of the products possessed a very low solubility in the aqueous buffer medium used in biological assays, polar groups (nitro, succinamidyl and trimethyl-amino substituent in position 6 of the chromone ring or hydroxyl group on the benzene ring in the amide part have been inserted to the molecule. Further variations yielded diaryl ketones, diaryl ketone bearing a succinamidyl substituent, carboxamide bearing a methylpiperazinyl-4-oxobutanamido group and methyl-malonyl ester amides. Basically, the essential structural features necessary for the ligand-enzyme interactions have been maintained. The latter virtual screening hit, a pyrimidinone derivative VS-8‎ was synthesized and the structure was modified by substitution in positions 2, 4, 5 and 6 of the pyrimidine ring. Due to autofluorescence, detected in most of the products, this model structure was not further varied. Simultaneously, experiments on solubilization of the first chromone-2-carboxamides with cyclodextrins, cyclic oligosacharides known to form water-soluble inclusion complexes, were performed. Although the assessed solubility of the chromone 3b/DIMEB (1:3) mixture exceeded 14-fold the intrinsic one, the achieved 100 µM solubility was still not sufficient to be used as a stock solution in the binding assay. The experiments with cyclodextrin in combination with DMSO were ineffective. Owing to high material costs necessary for the appropriate cyclodextrin amounts, the aim focused on structural modification of the hydrophobic products. Precise structural data have been obtained from the solved crystal structures of three chromone derivatives: the screening hit GS-71 (3b), its trimethylammonium salt (18) and 6-nitro-substituted N-benzyl-N-methyl-chromone-2-carboxamide (9i). The first two compounds are nearly planar with an anti-/trans-rotamer configuration. In the latter structure, the carboxamide bridge is bent out of the chromone plane, showing an anti-rotamer, too. Considering the relatively low partition coefficient of compound 3b (cLogP = 2.32), the compound planarity and correlating tight molecular packing might be the factors significantly affecting its poor solubility. Regarding the biological results of the chromone-based compounds, similar structure-activity correlations could be drawn from the binding assay and the whole cell activity testing on M. tuberculosis. In both cases, the introduction of a nitro group to position 6 of the chromone ring and the presence of a flexible substituent in the amide part showed a positive effect. In the binding study, the nitro group at position 4 on the N-benzyl residue was of advantage, too. The highest enzyme affinity was observed for N-(4-nitrobenzyl)-chromone-2-carboxamide 4c (KD = 34 µM), 6-nitro substituted N-benzyl-chromone-2-carboxamide 9g (KD = 40 µM) and 6‑nitro-substituted N-(4-nitrobenzyl)-chromone-2-carboxamide 9j (KD = 31 µM), which could not be attributed to the fluorescence quenching potential of the nitro group. The assay interference potential of chromones, due to a covalent binding on the enzyme sulfhydryl groups, was found to be negligible at the assay conditions. Moderate in vivo activity was detected for 6‑nitro-substituted N-benzyl-chromone-2-carboxamide 9g and its N-benzyl-N-methyl-, N‑furylmethyl-, N-cyclohexyl- and N-cyclohexylmethyl derivatives 9i, 9d, 9e, 9f, for which MIC values 20 - 40 µM were assessed. Cytotoxicity was increased in the N‑cyclohexylmethyl derivative only. None of the pyrimidine-based compounds showed activity in vivo. The affinity of the model structure, VS-8, surpassed with KD = 97 µM the assessed affinity of TLM (KD = 142 µM). Since for the model chromone compound GS-71 no reliable KasA binding data could be obtained, a newly synthesized chromone derivative 9i was docked into the KasA binding site, in order to derive correlation between the in silico and in vitro assessed affinity. For the 6‑nitro-derivative 9i a moderate in vivo activity on M. tuberculosis was obtained. The in silico predicted pKi values for TLM and 9i were higher than the corresponding in vitro results, maintaining though a similar tendency, i.e., the both affinity values for compound 9i (pKi predicted = 6.64, pKD experimental = 4.02) surpassed those obtained for TLM (pKi predicted = 5.27, pKD experimental = 3.84). Nevertheless, the experimental pKD values are considered preliminary results. The binding assay method has been improved in order to acquire more accurate data. Owing to the method development, limited enzyme batches and solubility issues, only selected compounds could be evaluated. The best hits, together with the compounds active on the whole cells of M. tuberculosis, will be submitted to the kinetic enzyme assay, in order to confirm the TLM-like binding mechanism. Regarding the in vivo testing results, no correlations could be drawn between the predicted membrane permeability values and the experimental data, as for the most active compounds 9e and 9f, a very low permeability was anticipated (0.4 and 0.7 \%, respectively). Further biological tests would be required to investigate the action- or transport mode.}, subject = {Tuberkelbakterium}, language = {en} } @phdthesis{Engelmann2023, author = {Engelmann, Daria Marie}, title = {Regulation of Mammalian Phosphoglycolate Phosphatase}, doi = {10.25972/OPUS-19957}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199577}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Mammalian phoshoglycolate phosphatase (PGP, also known as AUM) belongs to the ubiquitous HAD superfamily of phosphatases. As several other members of HAD phosphatases, the Mg2+-dependent dephosphorylation is conducted via a nucleophilic attack from a conserved aspartate residue in the catalytic cleft. The protein structure of PGP could not yet be solved entirely. Only a hybrid consisting of the PGP cap and the PDXP core (pyridoxal phosphatase, closest enzyme paralog) was crystallizable so far. PGP is able to efficiently dephosphorylate 2-phosphoglycolate, 2-phospho-L-lactate, 4-phospho-D-erythronate, and glycerol-3-phosphate in vitro which makes them likely physiological substrates. The first three substrates can be derived from metabolic side reactions (during glycolysis) and inhibit key enzymes in glycolysis and pentose phosphate pathway, the latter is situated at the intersection between glycolysis and lipogenesis. 2-phosphoglycolate can also be released in the context of repair of oxidative DNA damage. The activity of purified PGP can be reversibly inhibited by oxidation - physiologically likely in association with epidermal growth factor (EGF) signal transduction. In fact, an association between persistently lacking PGP activity (via downregulation) and the presence of hyperphosphorylated proteins after EGF stimulation has been identified. Reversible oxidation and transient inactivation of PGP may be particularly important for short-term and feedback regulatory mechanisms (as part of the EGF signaling). Furthermore, cellular proliferation in PGP downregulated cells is constantly reduced. Whole-body PGP inactivation in mice is embryonically lethal. Despite the many well-known features and functions, the knowledge about PGP is still incomplete. In the present work the influence of reactive oxygen species (ROS) on PGP activity in cells und a possible connection between oxidative stress and the proliferation deficit of PGP downregulated cells was investigated. For the experiments, a spermatogonial cell line was used (due to the high PGP expression in testis). PGP activity can be reversibly inhibited in cellular lysates by H2O2 (as a ROS representative). Reversible oxidation could thus indeed be physiologically important. More oxidative DNA damage (by bleomycin) showed no PGP-dependent effects here. EGF stimulation (as an inducer of transient and well-controlled ROS production), low concentrations of menadione (as an oxidant) and N-acetylcysteine (as an antioxidant) were able to approximate the proliferation rate in PGP downregulated cells to that of control cells. The redox regulation of PGP could thus have an influence on cellular proliferation as a feedback mechanism - a mechanism that could not take place in PGP downregulated cells. However, the connections are probably even more complex and cannot be elucidated by a sole examination of the proliferation rate. The present results can thus only be regarded as preliminary experiments. For a better understanding of the features and functions of PGP, this work then focused on specific regulation of enzyme activity by pharmacologically applicable small molecules. Four potent inhibitors had previously been identified in a screening campaign. In this work, three of these four inhibiting compounds could be further characterized in experiments with highly purified, recombinant murine and human PGP. Compounds \#2 and \#9 showed competitive inhibition properties with a markedly rising KM value with little or no change in vmax. The results were consistent for all tested protein variants: the murine and the human PGP as well as a PGP/PDXP hybrid protein. Compound \#1 was the most potent and interesting PGP-inhibitory molecule: less change in KM and a constant decrease in vmax as well as a lower impact on the PGP/PDXP hybrid hint at a mixed mode of inhibition as a combination of competitive and non-competitive inhibition. The characterization of the potential inhibitors can serve as a basis for further structural analysis and studies on the complex physiological role of PGP.}, subject = {Phosphoglykolatphosphatase}, language = {en} } @phdthesis{Kumari2014, author = {Kumari, Geeta}, title = {Molecular Characterization of the Induction of Cell Cycle Inhibitor p21 in Response to Inhibition of the Mitotic Kinase Aurora B}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-101327}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {Aurora B ist eine mitotische Kinase, die entscheidende Funktionen in der Zellteilung aus{\"u}bt. Aurora B ist außerdem in einer Vielzahl von Krebsarten mutiert oder {\"u}berexprimiert. Daher ist die Aurora B Kinase ein attraktives Ziel f{\"u}r die Tumortherapie. Gegenw{\"a}rtig werden Aurora B-Inhibitoren zur Behandlung von soliden Tumoren und Leuk{\"a}mien in verschiedenen klinischen Studien getestet. Es fehlen jedoch Informationen, welche molekularen Mechanismen den beschriebenen Ph{\"a}notypen wie Zellzyklusarrest, Aktivierung des Tumorsuppressors p53 und seines Zielgens p21 nach Aurora B-Hemmung zugrunde liegen. Hauptziel dieser Arbeit war es die Mechanismen der p21-Induktion nach Hemmung von Aurora B zu untersuchen. Es konnte gezeigt werden, dass nach Hemmung von Aurora B die p38 MAPK phosphoryliert und somit aktiviert wird. Experimente mit p38-Inhbitoren belegen, dass p38 f{\"u}r die Induktion von p21 und den Zellzyklusarrest ben{\"o}tigt wird. Die Stabilisierung von p53 nach Aurora B-Inhibition und die Rekrutierung von p53 an den p21-Genpromotor erfolgen jedoch unabh{\"a}ngig vom p38-Signalweg. Stattdessen ist p38 f{\"u}r die Anreicherung der elongierenden RNA-Polymerase II in der kodierenden Region des p21-Gens und f{\"u}r die Bildung des p21 mRNA Transkripts notwendig. Diese Daten zeigen, dass p38 transkriptionelle Elongation des p21-Gens nach Aurora B Hemmung f{\"o}rdert. In weiteren Untersuchungen konnte ich zeigen, dass die Aurora B-Hemmung zu einer Dephosphorylierung des Retinoblastoma-Proteins f{\"u}hrt und dadurch eine Abnahme der E2F-abh{\"a}ngigen Transkription bewirkt. Dies l{\"o}st indirekt einen Zellzyklusarrest aus. Weiterhin konnte mit Hilfe von synchronisierten Zellen gezeigt werden, dass p21 nach Durchlaufen einer abnormalen Mitose induziert wird, jedoch nicht nach Aurora B-Hemmung in der Interphase. Interessanterweise werden p38, p53 und p21 schon bei partieller Inhibition von Aurora B aktiviert. Die partielle Inhibition von Aurora B f{\"u}hrt zu chromosomaler Instabilit{\"a}t aber nicht zum Versagen der Zytokinese und zur Bildung polyploider Zellen. Damit korreliert die Aktivierung des p38-p53-p21-Signalweges nicht mit Tetraploidie sondern mit vermehrter Aneuploidie. Die partielle Hemmung von Aurora B f{\"u}hrt außerdem zur vermehrten Entstehung von reaktive Sauerstoffspezies (ROS), welche f{\"u}r die Aktivierung von p38, p21 und f{\"u}r den Zellzyklusarrest ben{\"o}tigt werden. Basierend auf diesen Beobachtungen kann folgendes Modell postuliert werden: Die Hemmung von Aurora B f{\"u}hrt zu Fehlern in der Chromosomenverteilung in der Mitose und damit zu Aneuploidie. Dies f{\"u}hrt zu vermehrter Produktion von ROS, m{\"o}glicherweise durch proteotoxischer Stress, hervorgerufen durch die Imbalanz der Proteinbiosynthese in aneuploiden Zellen. ROS bewirkt eine Aktivierung der p38 MAPK und tr{\"a}gt damit zur Induktion von p21 und dem resultierenden Zellzyklusarrest bei. Aneuploidie, proteotoxischer und oxidativer Stress stellen Schl{\"u}sselmerkmale von Tumorkrankungen dar. Anhand der Ergebnisse dieser Arbeit k{\"o}nnte die Kombination von Aurora B-Hemmstoffen mit Medikamenten, die gezielt aneuploide Zellen angreifen, in Tumorerkrankungen therapeutisch wirksam sein.}, subject = {Zellzyklus}, language = {en} } @phdthesis{Heilos2019, author = {Heilos, Anna}, title = {Mechanistic Insights into the Inhibition of Cathepsin B and Rhodesain with Low-Molecular Inhibitors}, doi = {10.25972/OPUS-17822}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-178228}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Cysteine proteases play a crucial role in medical chemistry concerning various fields reaching from more common ailments like cancer and hepatitis to less noted tropical diseases, namely the so-called African Sleeping Sickness (Human Arfican Trypanosomiasis). Detailed knowledge about the catalytic function of these systems is highly desirable for drug research in the respective areas. In this work, the inhibition mechanisms of the two cysteine proteases cathepsin B and rhodesain with respectively one low-molecular inhibitor class were investigated in detail, using computational methods. In order to sufficiently describe macromolecular systems, molecular mechanics based methods (MM) and quantum mechanical based method (QM), as well as hybrid methods (QM/MM) combining those two approaches, were applied. For Cathespin B, carbamate-based molecules were investigated as potential inhibitors for the cysteine protease. The results indicate, that water-bridged proton-transfer reactions play a crucial role for the inhibition. The energetically most favoured pathway (according to the calculations) includes an elimination reaction following an E1cB mechanism with a subsequent carbamylation of the active site amino acid cysteine. Nitroalkene derivatives were investigated as inhibitors for rhodesain. The investigation of structurally similar inhibitors showed, that even small steric differences can crucially influence the inhibition potential of the components. Furthermore, the impact of a fluorination of the nitroalkene inhibitors on the inhibition mechanism was investigated. According to experimental data measured from the working group of professor Schirmeister in Mainz, fluorinated nitroalkenes show - in contrast to the unfluorinated compounds - a time dependent inhibition efficiency. The calculations of the systems indicate, that the fluorination impacts the non-covalent interactions of the inhibitors with the enzymatic environment of the enzyme which results in a different inhibition behaviour.}, subject = {Cysteinproteasen}, language = {en} } @phdthesis{Paasche2013, author = {Paasche, Alexander}, title = {Mechanistic Insights into SARS Coronavirus Main Protease by Computational Chemistry Methods}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-79029}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {The SARS virus is the etiological agent of the severe acute respiratory syndrome, a deadly disease that caused more than 700 causalities in 2003. One of its viral proteins, the SARS coronavirus main protease, is considered as a potential drug target and represents an important model system for other coronaviruses. Despite extensive knowledge about this enzyme, it still lacks an effective anti-viral drug. Furthermore, it possesses some unusual features related to its active-site region. This work gives atomistic insights into the SARS coronavirus main protease and tries to reveal mechanistic aspects that control catalysis and inhibition. Thereby, it applies state-of-the-art computational methods to develop models for this enzyme that are capable to reproduce and interpreting the experimental observations. The theoretical investigations are elaborated over four main fields that assess the accuracy of the used methods, and employ them to understand the function of the active-site region, the inhibition mechanism, and the ligand binding. The testing of different quantum chemical methods reveals that their performance depends partly on the employed model. This can be a gas phase description, a continuum solvent model, or a hybrid QM/MM approach. The latter represents the preferred method for the atomistic modeling of biochemical reactions. A benchmarking uncovers some serious problems for semi-empirical methods when applied in proton transfer reactions. To understand substrate cleavage and inhibition of SARS coronavirus main protease, proton transfer reactions between the Cys/His catalytic dyad are calculated. Results show that the switching between neutral and zwitterionic state plays a central role for both mechanisms. It is demonstrated that this electrostatic trigger is remarkably influenced by substrate binding. Whereas the occupation of the active-site by the substrate leads to a fostered zwitterion formation, the inhibitor binding does not mimic this effect for the employed example. The underlying reason is related to the coverage of the active-site by the ligand, which gives new implications for rational improvements of inhibitors. More detailed insights into reversible and irreversible inhibition are derived from in silico screenings for the class of Michael acceptors that follow a conjugated addition reaction. From the comparison of several substitution patterns it becomes obvious that different inhibitor warheads follow different mechanisms. Nevertheless, the initial formation of a zwitterionic catalytic dyad is found as a common precondition for all inhibition reactions. Finally, non-covalent inhibitor binding is investigated for the case of SARS coranavirus main protease in complex with the inhibitor TS174. A novel workflow is developed that includes an interplay between theory and experiment in terms of molecular dynamic simulation, tabu search, and X-ray structure refinement. The results show that inhibitor binding is possible for multiple poses and stereoisomers of TS174.}, subject = {SARS}, language = {en} } @phdthesis{Winkler2015, author = {Winkler, Ann-Cathrin Nicole}, title = {Identification of human host cell factors involved in \(Staphylococcus\) \(aureus\) 6850 infection}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114300}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {Staphylococcus aureus is both a human commensal and a pathogen. 20\%-30\% of all individuals are permanently or occasionally carriers of S. aureus without any symptoms. In contrast to this, S. aureus can cause life-threatening diseases e.g. endocarditis, osteomyelitis or sepsis. Here, the increase in antibiotic resistances makes it more and more difficult to treat these infections and hence the number of fatalities rises constantly. Since the pharmaceutical industry has no fundamentally new antibiotics in their pipeline, it is essential to better understand the interplay between S. aureus and the human host cell in order to find new, innovative treatment options. In this study, a RNA interference based whole genome pool screen was performed to identify human proteins, which play a role during S. aureus infections. Since 1,600 invasion and 2,271 cell death linked factors were enriched at least 2 fold, the big challenge was to filter out the important ones. Here, a STRING pathway analysis proved to be the best option. Subsequently, the identified hits were validated with the help of inhibitors and a second, individualised small interfering RNA-based screen. In the course of this work two important steps were identified, that are critical for host cell death: the first is bacterial invasion, the second phagosomal escape. The second step is obligatory for intracellular bacterial replication and subsequent host cell death. Invasion in turn is determining for all following events. Accordingly, the effect of the identified factors towards these two crucial steps was determined. Under screening conditions, escape was indirectly measured via intracellular replication. Three inhibitors (JNKII, Methyl-beta-cyclodeytrin, 9-Phenantrol) could be identified for the invasion process. In addition, siRNAs targeted against 16 different genes (including CAPN2, CAPN4 and PIK3CG), could significantly reduce bacterial invasion. Seven siRNAs (FPR2, CAPN4, JUN, LYN, HRAS, AKT1, ITGAM) were able to inhibit intracellular replication significantly. Further studies showed that the IP3 receptor inhibitor 2-APB, the calpain inhibitor calpeptin and the proteasome inhibitor MG-132 are able to prevent phagosomal escape and as a consequence intracellular replication and host cell death. In this context the role of calpains, calcium, the proteasome and the mitochondrial membrane potential was further investigated in cell culture. Here, an antagonistic behaviour of calpain 1 and 2 during bacterial invasion was observed. Intracellular calcium signalling plays a major role, since its inhibition protects host cells from death. Beside this, the loss of mitochondrial membrane potential is characteristic for S. aureus infection but not responsible for host cell death. The reduction of membrane potential can be significantly diminished by the inhibition of the mitochondrial Na+/Ca2+ exchanger. All together, this work shows that human host cells massively contribute to different steps in S. aureus infection rather than being simply killed by bacterial pore-forming toxins. Various individual host cell factors were identified, which contribute either to invasion or to phagosomal escape and therefore to S. aureus induced cytotoxicity. Finally, several inhibitors of S. aureus infection were identified. One of them, 2-APB, was already tested in a sepsis mouse model and reduced bacterial load of kidneys. Thus, this study shows valuable evidence for novel treatment options against S. aureus infections, based on the manipulation of host cell signalling cascades.}, subject = {Staphylococcus aureus}, language = {en} } @article{GentschevMuellerAdelfingeretal.2011, author = {Gentschev, Ivaylo and M{\"u}ller, Meike and Adelfinger, Marion and Weibel, Stephanie and Grummt, Friedrich and Zimmermann, Martina and Bitzer, Michael and Heisig, Martin and Zhang, Qian and Yu, Yong A. and Chen, Nanhai G. and Stritzker, Jochen and Lauer, Ulrich M. and Szalay, Aladar A.}, title = {Efficient Colonization and Therapy of Human Hepatocellular Carcinoma (HCC) Using the Oncolytic Vaccinia Virus Strain GLV-1h68}, series = {PLOS ONE}, volume = {6}, journal = {PLOS ONE}, number = {7}, doi = {10.1371/journal.pone.0022069}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135319}, pages = {e22069}, year = {2011}, abstract = {Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In this study, we analyzed for the first time the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 in two human hepatocellular carcinoma cell lines HuH7 and PLC/PRF/5 (PLC) in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 efficiently colonized, replicated in, and did lyse these cancer cells in culture. Experiments with HuH7 and PLC xenografts have revealed that a single intravenous injection (i.v.) of mice with GLV-1h68 resulted in a significant reduction of primary tumor sizes compared to uninjected controls. In addition, replication of GLV-1h68 in tumor cells led to strong inflammatory and oncolytic effects resulting in intense infiltration of MHC class II-positive cells like neutrophils, macrophages, B cells and dendritic cells and in up-regulation of 13 pro-inflammatory cytokines. Furthermore, GLV-1h68 infection of PLC tumors inhibited the formation of hemorrhagic structures which occur naturally in PLC tumors. Interestingly, we found a strongly reduced vascular density in infected PLC tumors only, but not in the non-hemorrhagic HuH7 tumor model. These data demonstrate that the GLV-1h68 vaccinia virus may have an enormous potential for treatment of human hepatocellular carcinoma in man.}, language = {en} }