@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{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} } @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} } @phdthesis{Grimm2019, author = {Grimm, Johannes}, title = {Autocrine and paracrine effects of BRAF inhibitor induced senescence in melanoma}, doi = {10.25972/OPUS-18116}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-181161}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {The FDA approval of targeted therapy with BRAFV600E inhibitors like vemurafenib and dabrafenib in 2011 has been the first major breakthrough in the treatment of metastatic melanoma since almost three decades. Despite increased progression free survival and elevated overall survival rates, complete responses are scarce due to resistance development approximately six months after the initial drug treatment. It was previously shown in our group that melanoma cells under vemurafenib pressure in vitro and in vivo exhibit features of drug-induced senescence. It is known that some cell types, which undergo this cell cycle arrest, develop a so-called senescence associated secretome and it has been reported that melanoma cell lines also upregulate the expression of different factors after senescence induction. This work describes the effect of the vemurafenib-induced secretome on cells. Conditioned supernatants of vemurafenib-treated cells increased the viability of naive fibroblast and melanoma cell lines. RNA analysis of donor melanoma cells revealed elevated transcriptional levels of FGF1, MMP2 and CCL2 in the majority of tested cell lines under vemurafenib pressure, and I could confirm the secretion of functional proteins. Similar observations were also done after MEK inhibition as well as in a combined BRAF and MEK inhibitor treatment situation. Interestingly, the transcription of other FGF ligands (FGF7, FGF17) was also elevated after MEK/ERK1/2 inhibition. As FGF receptors are therapeutically relevant, I focused on the analysis of FGFR-dependent processes in response to BRAF inhibition. Recombinant FGF1 increased the survival rate of melanoma cells under vemurafenib pressure, while inhibition of the FGFR pathway diminished the viability of melanoma cells in combination with vemurafenib and blocked the stimulatory effect of vemurafenib conditioned medium. The BRAF inhibitor induced secretome is regulated by active PI3K/AKT signaling, and the joint inhibition of mTor and BRAFV600E led to decreased senescence induction and to a diminished induction of the secretome-associated genes. In parallel, combined inhibition of MEK and PI3K also drastically decreased mRNA levels of the relevant secretome components back to basal levels. In summary, I could demonstrate that BRAF inhibitor treated melanoma cell lines acquire a specific PI3K/AKT dependent secretome, which is characterized by FGF1, CCL2 and MMP2. This secretome is able to stimulate other cells such as naive melanoma cells and fibroblasts and contributes to a better survival under drug pressure. These data are therapeutically highly relevant, as they imply the usage of novel drug combinations, especially specific FGFR inhibitors, with BRAF inhibitors in the clinic.}, subject = {Inhibitor}, 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{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{Knobloch2014, author = {Knobloch, Gunnar}, title = {Biochemical and structural characterization of chronophin}, doi = {10.25972/OPUS-11008}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-110088}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {The haloacid dehalogenase (HAD) family of phosphatases is an ancient, ubiquitous group of enzymes, and their emerging role in human health and disease make them attractive targets for detailed analyses. This thesis comprises the biochemical and structural characterization of chronophin, an HAD-type phosphatase, which has been shown to act on Ser3-phosphorylated cofiln-1, a key regulator of actin dynamics, and on the Ser/Thr-phosphorylated steroid receptor co-activator 3 (SRC-3). Besides being a specific phosphoprotein phosphatase, chronophin also acts on the small molecule pyridoxal 5'-phosphate (PLP, vitamin B6), implying that chronophin serves as a regulator of a variety important physiological pathways. The analysis of chronophin was performed on different levels, ranging from intrinsic regulatory mechanisms, such as the allosteric regulation via dimerization or the characterization of specificity determinants, to modes of extrinsic modulation, including the association with putative interacting proteins or the generation of chronophin-specific inhibitors. The association of the previously identified putative chronophin interactors calcium- and integrinbinding protein 1 (CIB1) and calmodulin was investigated using recombinantly expressed and purified proteins. These studies revealed that the interaction of chronophin with CIB1 or calmodulin is mutually exclusive and regulated by calcium. Neither CIB1 nor calmodulin had an effect on the in vitro chronophin phosphatase activity towards PLP or phospho-cofilin-1, but might regulate other functions of this important phosphatase. The role of chronophin dimerization was studied by generating a constitutively monomeric variant, which showed reduced PLP hydrolyzing activity. X-ray crystallographic studies revealed that dimerization is essential for the positioning of the substrate specificity loop in chronophin, unraveling a previously unknown mechanism of allosteric regulation through a homophilic interaction. This mechanism potentially applies to other enzymes of the C2a subfamily of HAD-type phosphatases, as all structurally characterized members show a conserved mode of dimerization. The general determinants of substrate specificity in the C2a subfamily of HAD phosphatases were investigated by performing domain swapping experiments with chronophin and its paralog AUM and subsequent biochemical analyses of the hybrid proteins. The X-ray crystallographic structure determination of the chronophin catalytic domain equipped with the AUM capping domain revealed the first partial structure of AUM. This structural information was then used in subsequent studies that analyzed the divergent substrate specificities of AUM and chronophin in an evolutionary context. Finally, a set of four chronophin inhibitors were generated based on the structure of PLP and characterized biochemically, showing moderate inhibitory effects with IC50-values in the micromolar range. These compounds nevertheless constitute valuable tools for future in vitro experiments, such as studies concerning the structure-function relationship of chronophin as a PLP phosphatase. In addition, the crystal structure of one inhibitor bound to chronophin could be solved. These results provide the basis for the further development of competitive chronophin inhibitors with increased specificity and potency.}, subject = {Phosphatasen}, 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{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{Ramirez2024, author = {Ramirez, Yesid A.}, title = {Structural basis of ubiquitin recognition and rational design of novel covalent inhibitors targeting Cdu1 from \(Chlamydia\) \(Trachomatis\)}, doi = {10.25972/OPUS-19168}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-191683}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {The WHO-designated neglected-disease pathogen Chlamydia trachomatis (CT) is a gram-negative bacterium responsible for the most frequently diagnosed sexually transmitted infection worldwide. CT infections can lead to infertility, blindness and reactive arthritis, among others. CT acts as an infectious agent by its ability to evade the immune response of its host, which includes the impairment of the NF-κB mediated inflammatory response and the Mcl1 pro-apoptotic pathway through its deubiquitylating, deneddylating and transacetylating enzyme ChlaDUB1 (Cdu1). Expression of Cdu1 is also connected to host cell Golgi apparatus fragmentation, a key process in CT infections. Cdu1 may this be an attractive drug target for the treatment of CT infections. However, a lead molecule for the development of novel potent inhibitors has been unknown so far. Sequence alignments and phylogenetic searches allocate Cdu1 in the CE clan of cysteine proteases. The adenovirus protease (adenain) also belongs to this clan and shares a high degree of structural similarity with Cdu1. Taking advantage of topological similarities between the active sites of Cdu1 and adenain, a target-hopping approach on a focused set of adenain inhibitors, developed at Novartis, has been pursued. The thereby identified cyano-pyrimidines represent the first active-site directed covalent reversible inhibitors for Cdu1. High-resolution crystal structures of Cdu1 in complex with the covalently bound cyano-pyrimidines as well as with its substrate ubiquitin have been elucidated. The structural data of this thesis, combined with enzymatic assays and covalent docking studies, provide valuable insights into Cdu1s activity, substrate recognition, active site pocket flexibility and potential hotspots for ligand interaction. Structure-informed drug design permitted the optimization of this cyano-pyrimidine based scaffold towards HJR108, the first molecule of its kind specifically designed to disrupt the function of Cdu1. The structures of potentially more potent and selective Cdu1 inhibitors are herein proposed. This thesis provides important insights towards our understanding of the structural basis of ubiquitin recognition by Cdu1, and the basis to design highly specific Cdu1 covalent inhibitors.}, subject = {Ubiquitin}, language = {en} }