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TNFR1 and TNFR2 regulate the extrinsic apoptotic pathway in myeloma cells by multiple mechanisms
(2011)
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
Identification of human host cell factors involved in \(Staphylococcus\) \(aureus\) 6850 infection
(2015)
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.
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.
Although the physiological roles of BChE are not yet determined to date, the importance of this enzyme is continuously increasing as it was found to be associated with several disorders like diabetes mellitus type 2, cardiovascular diseases, obesity and especially with Alzheimer’s disease (AD). In consequence, for investigations of BChE’s pathological role in these diseases and to find new medication strategies, the development of selective and potent inhibitors is necessary.
For this purpose, the current work progresses in five chapters on the exploration of the chemical, physical and biochemical properties of tetrahydroquinazoline based carbamates which were previously reported to be selective BChE inhibitors with potency in the low nanomolar range.
1) A Novel Way to Radiolabel Human Butyrylcholinesterase for PET through Irreversible Transfer of the Radiolabeled Moiety:
PET-radiotracers represent an innovative tool to determine the distribution and the expression of a biological target in vivo. BChE lacks to a large degree of such tracers with a few exceptions. In this work, methods were developed to incorporate the radioisotopes 11C and 18F into the carbamate moiety of an tetrahydroquinazoline based inhibitor. In contrast to reversibly acting PET-probes, the described radiotracers were proven by kinetic studies to transfer the radioisotope covalently onto the active site of BChE, thus labeling the enzyme directly and permanently.
2) Discovery of Highly Selective and Nanomolar Carbamate-Based Butyrylcholinesterase Inhibitors by Rational Investigation into Their Inhibition Mode:
To investigate the role of the tetrahydroquinazoline carrier scaffold on BChE inhibition, carbamate based inhibitors were synthesized. These compounds were successively used to perform kinetic investigations to determine their inhibition mode. Based on these data, a plausible binding model was postulated explaining the influence of the tetrahydroquinazoline carrier scaffold for binding at BChE’s active site just before carbamate transfer takes place. Additionally, these compounds feature neuroprotective properties and prevent oxidative stress induced cell death in their carbamate form as well as after the release of the tetrahydroquinazoline carrier scaffold.
3) Dual Addressing of Butyrylcholinesterase by Targeting the Catalytic Active Site (CAS) and the Peripheral Anionic Site (PAS):
Compounds which are dual-targeting the CAS and the PAS of BChE are the most potent and selective BChE inhibitors to date with inhibition values in the picomolar range. In this work, a strategy is described how to turn tetrahydroquinazoline based carbamates into dual binding BChE inhibitors. These inhibitors feature a carbamate moiety which is covalently transferred onto the CAS of BChE, and in addition provide a second pharmacophore connected via a linker to the carbamate moiety which is proposed to target the PAS. Preliminary results reveal a high tolerance of BChE towards different linker lengths without decrease in affinity.
4) Investigation into Selective Debenzylation and Ring Cleavage of Quinazoline based Heterocycles:
The tetrahydroquinazoline system is well investigated in terms of its synthesis and its selective oxidation. To explore the reactivity of this system, a tetracyclic tetrahydroquinazoline was exposed to common reduction agents. These experiments revealed a high sensitivity of the tetrahydroquinazoline core towards several reduction conditions
5) Experimental and Theoretical Investigation into the Stability of Cyclic Aminals:
Tetrahydroquinazolines are known to degrade in acidic media through hydrolysis of their aminal system; but literature is lacking of a systematic investigation into this behavior. Therefore, different tetrahydroquinazolines were synthesized and exposed to phosphate buffered systems with defined pH-values. A clear increase of the hydrolysis rate of the aminal system was determined in dependency of an increasing acidic media. Computational studies predicted and experimental studies proved that hydrolysis takes place in an acidic environment while the condensation of this system is preferred in neutral or basic aqueous media.
Die TGF-β-Proteinfamilie umfasst eine Vielzahl von zumeist homodimeren sezernierten Liganden in höheren Tieren, die viele Vorgänge und Entwicklungen im Embryo wie im adulten Lebewesen über absolute oder graduelle Einflussnahme steuern. Die Signalweiterleitung ins Zytoplasma und den Nukleus erfolgt über promisk paarig rekrutierte Typ-I- und Typ-II-Rezeptoren, ehe vorwiegend rezeptorabhängig verschiedene SMAD-Proteine von Typ-I-Kinasen der Rezeptoren aktiviert werden, in den Kern translozieren und die Transkription induzieren. Zu jedem Zeitpunkt dieser Signalweiterleitung kann mittels verschiedener endogener Inhibitoren regulatorisch eingegriffen werden. Dem bisher einzig bekannten membranständigen Pseudorezeptor Bambi (BMP and Activine membrane bound inhibitor) wurde in vorangegangenen Arbeiten inhibitorisches Potential gegenüber dem BMP- und Activin-vermittelten Signalweg über Bindung an distinkte ligandenadressierte Rezeptoren zugeschrieben, wobei die genaue Wirkweise bislang noch vollkommen unklar war.
In der vorliegenden Arbeit wurde initial ein Homologiemodell der extrazellulären Domäne von hBambi anhand der gelösten Kristallstruktur der extrazellulären Domäne von BR1A im gebundenen Zustand (PDB-ID: 1REW) erstellt. Anhand dieses Modells wurde eine Arbeitshypothese entwickelt und es gelang in der Folge, biologisch aktives rekombinantes Protein zum einen aus transfizierten Insektenzellen sowie aus der Renaturierung aus bakteriellen Einschlusskörpern in hinreichender Menge herzustellen und chromatographisch aufzureinigen. Nach einer vergleichenden Qualitätskontrolle beider Exprimate wurden mittels CD-Spektroskopie und analytischer Gelfiltration der Anteil der Sekundärstrukturelemente sowie der Oligomerisierungsgrad erfolgreich bestimmt. In SPR-Bindestudien wurde der Beweis erbracht, dass hBambi-ECD Affinität zu annähernd allen getesteten Liganden der BMP-/GDF-Gruppe, die den SMAD-1/-5-/-8-Signalweg aktivieren, zeigt. Bekannte Typ-I- und Typ-II-Bindungsmutanten von BMP-2 wurden ebenfalls von hBambi-ECD quasi wildtypisch gebunden. Verschiedene Rezeptorektodomänen sowie ActivinA wurden, wie bisher in der Literatur fälschlich angenommen wurde, hingegen nicht gebunden. Die propagierte Homooligomerisierung von Bambi wird überdies nicht über die extrazelluläre Domäne vermittelt. Eingesetzt in Stimulationsversuche mit BMP-responsiven Zellen wurde eine konzentrationsabhängige inhibierende Wirkung von freier hBambi-ECD auf die BMP-2-vermittelte Signalweiterleitung mit unterschiedlichen Nachweismethoden ermittelt, welche die Ergebnisse aus den SPR-Versuchen erfolgreich bestätigten.
In einem weiteren Teil dieser Arbeit wurden verschiedene chimäre Konstrukte aus für Bambi- und BR1A-Domänen kodierenden Sequenzen kloniert, in HEK Ad293-Zellen zusammen mit BMP- und Activin-responsiven Reportergenkonstrukten transient transfiziert und Stimulationsversuche mit BMP-2 und ActivinA durchgeführt. Wildtypisches Bambi zeigte hierbei ein ambivalentes Verhalten in Bezug auf die Regulation des BMP-2-Signals: geringe Mengen wirken agonistisch, höhere Mengen antagonistisch auf die Ausbildung des Reporters. Im Fall von ActivinA zeigte sich hingegen kein antagonistischer Einfluss von Bambi. In den Experimenten mit chimären Varianten erfolgte durch die erhaltenen Daten die Eingrenzung der Bindestelle von hBambi-ECD an BMP-2 auf den Bereich der Typ-I-Bindestelle. Ein direkter Einfluss der intrazellulären Domäne auf den BMP-2-Signalweg wurde ausgeschlossen. Weiterhin konnte gerade in Versuchen mit einem Antikörper gegen BR1A-ECD eine weitere Eigenheit der Bindung von Bambi an den Liganden offenbart werden: so bildet das Konstrukt aus hBambi-ECD und der intrazellulären BR1A-Domäne mit zugehöriger GS-Box und Typ-I-Kinase einen korrekt in den signalaktiven heterohexameren Komplex rekrutierten funktionellen Typ-I-Rezeptor.
Mit den in dieser Arbeit erzielten Ergebnissen, nämlich der gelungenen Erstellung eines Herstellungsprotokolls der ECD, deren erfolgreich identifizierten Bindepartnern sowie der Charakterisierung der Bindung an BMP-2 ist der Grundstein für die Strukturaufklärung von hBambi-ECD gelegt, welche weitere Klarheit in die Funktionalität dieses Modulators der BMP-/GDF-vermittelten Signalweiterleitung bringen wird. Ebenso sind erste das Verständnis der ICD aufklärende Ergebnisse erzielt worden, die das Fundament für weitere Experimente und darauf folgende Kenntnisgewinne darstellen werden.
Das Ziel der Arbeit war zu untersuchen, ob der Stoffwechsel kolorektaler Karzi-nomzellen geeignete Targetstrukturen für mögliche therapeutische Ansätze aufweist. In Krebszellen induziert sowohl der Warburg-Effekt bei Normoxie als auch die anaerobe Glykolyse bei Hypoxie eine massive Bildung von Laktat. Wird die Krebszelle dauerhaft daran gehindert, die für die Glykolyse notwendi-gen Reduktionsäquivalente NADH+H+ mit Hilfe der Laktatdehydrogenase zu reoxidieren und/oder Laktat über die Transporter MCT1 und MCT4 nach außen zu schleusen, dann löst diese Kombination aus Mangelsituation und intrazellulärer Ansäuerung den apoptotischen Zelltod aus. Für die Situation in vivo ist entscheidend, dass auch Zellen von Normalgeweben zwar Laktat in Hypoxie bilden, dies jedoch keine vorherrschende physiologische Situation darstellt.
Die Hemmstoffe Natriumoxamat (NaOx) für die Laktatdehydrogenase und α-Cyano-4-Hydroxycinnamat (αCHC) für MCT1 und MCT4 wurden an den sechs humanen kolorektalen Karzinomzelllinien Colo741, HCT116, HT29, LS174T, SW620 und WiDr untersucht. Zusätzlich wurde der Glukoseverbrauch und die Laktatbildung bestimmt und die Funktion der Atmungskette überprüft. Die IC50-Werte für 5-FU, NaOx und αCHC wurden bestimmt und danach NaOx in einer Konzentration von 40x10-3 mol/L, αCHC in einer Konzentration von 2x10-3 mol/L und 5-FU in einer Konzentration von 5x10-6 mol/L eingesetzt. Die Zellen wurden bei tumorphysiologischen Sauerstoffkonzentrationen von 5 % und 1 % Sauerstoff für bis zu 120 Stunden inkubiert.
Die Funktion der Atmungskette in den Mitochondrien der kolorektalen Karzi-nomzellen wurde u. a. durch Bestimmung wichtiger Kenngrößen wie dem P:O Quotienten und des respiratorischen Kontrollindex (RKI) nachgewiesen. Fünf der sechs Karzinomzelllinien wiesen im Vergleich zur Kontrollzelllinie J774 einen verringerten P:O-Quotienten und respiratorischen Kontrollindex (RKI) auf, was darauf hindeutet, dass die Funktion der Mitochondrien dieser Zellen im Vergleich zu Kontrollzellen zwar verringert war, aber nicht vollständig aufgehoben. Dieses Ergebnis stützt die allgemein akzeptierte Auffassung, dass die meisten Tumore über funktionelle Mitochondrien verfügen.
Durch die Analyse des Glukosestoffwechsels wurden die sechs kolorektalen Zelllinien, die einen unterschiedlich stark ausgeprägten glykolytischen Phänotyp aufwiesen, nach der Stärke der Laktatbildung bei 5 % Sauerstoff in drei Kategorien eingeordnet. Zudem wurde für jede der sechs Zelllinien die Expression von LDH-A, LDH-B sowie MCT-1 und MCT-4 auf Proteinebene nachgewiesen.
Wesentliches Ziel der Untersuchungen war die Überprüfung des antiprolife-rativen Potentials der beiden Inhibitoren NaOx und αCHC einzeln oder in Kombination mit 5-FU bei den tumorspezifischen Sauerstoffkonzentrationen von 5 % und 1 %. Die Kombination aus NaOx und αCHC induzierte bei 1 % Sauerstoff nach 9 Tagen in Kultur zytotoxische Effekte und war damit so wirksam wie 5x10-6 mol/L 5-FU. Die Zugabe von 5-FU zur Kombination aus NaOx und αCHC führte zu keiner Steigerung des zelltoxischen Effektes. Die beiden Inhibitoren NaOx und αCHC waren für SW620 Zellen weniger wirksam als für Zellen der anderen fünf Zelllinien. Das mehr „oxidative“ Profil von SW620 Zellen (bester P:O-Quotient, geringste Laktatbildung bei 5 % und 1 % Sauerstoff; zudem die höchsten IC50-Werte für NaOx und αCHC) könnte erklären, warum die beiden Stoffwechselinhibitoren, die einen glykolytischen Phänotyp (starke Bildung von Laktat) erfordern, für SW620 Zellen von geringerer Wirksamkeit waren.
Für die Hemmstoffe NaOx und αCHC wurden zytostatische bzw. zytotoxische Effekte in kolorektalen Karzinomzellen gezeigt. Dies deutet darauf hin, dass Krebszellen auf einen ungehinderten glykolytischen Stoffwechsel angewiesen sind. Für beide Hemmstoffe wurde ebenfalls gezeigt, dass sie auch bei tumorre-levanten Sauerstoffkonzentrationen von 5 % und 1 % wirksam sind.
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.
Theoretical Investigations on the Interactions of Small Compounds with their Molecular Environments
(2015)
In the first part of this work, a combination of theoretical methods for the rational design of covalent inhibitor is presented. Starting from the crystal structure of the covalent complex of a lead compound, quantum mechanical and QM/MM calculations were used to derive the exact geometry of the preceeding non-covalent enzyme inhibitor complex. The geometry of the latter mainly determines the reactivity of the inhibitor against its target enzyme concerning the formation of the covalent bond towards an active site residue. Therefore, this geometry was used as starting point for the optimization of the substitution pattern of the inhibitor such as to increase its binding affinity without loosing its ability to covalently bind to the target protein. The optimization of the chemical structure was supported by using docking procedures, which are best suited to estimate binding affinities that arise from the introduced changes. A screening of the novel substitution patterns resulted in a first generation of model compounds which were further tested for their reactivity against the target. Dynamic simulations on the novel compounds revealed that the orientation that compounds adopt within the active site are such that a covalent interaction with the enzyme is no longer possible. Hence, the chemical structure was further modified, including not only changes in the substituents but also within the core of the molecule. Docking experiments have been conducted to assure sufficiently high binding affinities and to obtain the most favored binding poses. Those have then again been used for dynamic simulations which resulted in structures, for which the bond formation process appeared feasible. A final series of QM/MM calculations considering various protonation states was computed to estimate the reaction energies for the covalent attachment of the inhibitor to the enzyme. The theoretical results indicate a reasonable high inhibition potency of the novel compounds.
The second part concentrates on the environmental influences on the electron density of an inhibitor molecule. Therefore, a vinylsulfone-based model compound was selected for which an experimental crystal structure for the pure compound as well as a theoretically determined enzyme-inhibitor complex have been available. To provide reference data for the larger systems, the conformational space of the isolated molecule was screened for favorable geometries which were later compared to those within the crystal and protein surrounding. The geometry of the crystal structure could readily be taken from the experimental data whereas calculations on the protein complex revealed four potential non-covalent complexes exhibiting different arrangements of the molecule within the active site of the protein as well as two possible protonation states of the catalytic dyad. Hence, all four protein complexes have been compared to the crystal structure of the molecule as well as against the more favorable geometries of the isolated molecule being determined within vacuum or aqueous surrounding. Whereas the molecule itself was found to adopt comparable geometries within all investigated environments, the interactions pattern between the crystal surrounding and the protein differed largely from each other. The favorable formation of dimers within the crystal has a strong stabilizing effect and explains the extraordinarily good quality of the crystal. Within the protein however, repulsive forces have been found between the protein and the inhibitor. The origin of the repulsion could be traced back to effect of on of the substituents to the vinyl scaffold. The difference in the chemical structure in comparison to a well known inhibitor might also explain the experimentally found loss of activity for the model compound in comparison to K11777.
Weltweit zählt die Tuberkulose zu den tödlichsten und am weitesten verbreiteten Infektionskrankheiten. Missstände in der ohnehin komplexen Therapie einerseits und fehlende Entwicklung neuartiger adäquater Wirkstoffe andererseits, führten zur Entstehung von multi- und sogar total-resistenten Keimen. Der Haupterreger ist das Mycobacterium tuberculosis. Charakteristisch für Mykobakterien ist eine dicke und undurchlässige wachsartige Zellwand mit einem großen Anteil an bestimmten Fettsäuren. Die mykobakterielle Biosynthese dieser Fettsäuren unterscheidet sich stark von eukaryotischen Zellen. Die selektive Beeinflussung dieses Systems führt zu nicht überlebensfähigen Mykobakterien und stellt somit ein idealer Angriffspunkt für Arzneistoffe dar.
Die vorliegende Arbeit befasst sich mit der Entwicklung neuartiger direkter Hemmstoffe von InhA, einem für den Zellwandaufbau des Mycobacterium tuberculosis essenziellem Enzym.
Es wurden zwei photometrische gekoppelt-enzymatische Assay-Systeme im 96-Well-Format entwickelt, die sich das Absorptions- bzw. Fluoreszenzverhalten des Coenzyms NADH zu Nutze machen.
Das hierzu benötigte Enzym InhA wurde überexprimiert und aufgereinigt. Mehrere Synthesemethoden für das im Testverfahren verwendete Substrat 2-trans-Octenoyl-CoA (2toCoA) wurden etabliert.
Die etablierten Assay-Systeme wurden mit Hilfe von Positivkontrollen validiert. Grundlegende Experimente zur Errichtung einer substratunabhängigen orthogonalen Methode mittels MST wurden getätigt.
Basierend auf den Ergebnissen eines in Vorarbeiten durchgeführten virtuellen Screenings wurden erste potenzielle Inhibitoren kommerziell erworben und getestet. Nachfolgend wurde mit der Synthese von Derivaten begonnen, welche auf iterativem Wege optimiert wurden (Testung – Docking – Synthese neuer Derivate). Hierdurch wurde eine umfassende Substanzbibliothek bestehend aus insgesamt 254 Verbindungen aufgebaut. Diese setzte sich aus unterschiedlich substituierten Thiazolidin-2,4-dionen- und Thiazolin-2-on-Derivaten, Derivaten der ähnlich strukturierten Fünfring-Heterozyklen Rhodanine, Thiohydantoine und Hydantoine und weiteren Strukturklassen bestehend aus Biphenylether-, Pyrrolidoncarboxamid-, Pyridon- und Sulfonamid-Derivaten zusammen. Die Verbindungen wurden entweder selbst synthetisiert, kommerziell erworben oder von Kooperationspartnern bezogen. Neben der Etablierung zuverlässiger und effizienter Syntheserouten stand hierbei ebenso die strukturelle Aufklärung der stereochemischen Verhältnisse der Produkte im Mittelpunkt.
Die Verbindungen der aufgebauten Substanzbibliothek wurden mit dem etablierten InhA-Testsystem auf ihre inhibitorischen Eigenschaften gegenüber InhA untersucht. Soweit möglich wurden Struktur-Aktivitätsbeziehungen abgeleitet. Insbesondere einige disubstituierte Thiazolidindione zeigten eine schwache Hemmung von bis zu 25 %. Die zur Aufklärung des Inhibitionsmechanismus durchgeführten Experimente deuten auf eine unkompetitive Hemmung hin. Bei den direkten Testungen an Mykobakterien konnten die inhibitorischen Eigenschaften hingegen nicht bestätigt werden.
Weiterhin wurden Testungen an Cystein- und Serin-Proteasen von Erregern anderer Infektionskrankheiten durchgeführt. Das Thiazolinon SV102 wurde hierbei als nicht-kompetitiver Hemmstoff von Cathepsin B mit einem Ki-Wert von 1.3 µM identifiziert. Die Synthese und Testung weiterer Thiazolin-2-on-Derivate sowie Cokristallisationsversuche mit Cathepsin B sind somit in Betracht zu ziehen. Die getesteten Thiazolidindion-Derivate der Substanzbibliothek zeigten hierbei mittelstarke bis gute Hemmeigenschaften, die ebenfalls an den Erregern beobachtbar waren. Relativiert werden diese vielversprechenden Ergebnisse allerdings durch eine ebenfalls zu beobachtende Zytotoxizität. Weiterhin konnte eine antibakterielle Wirkung der untersuchten Verbindungen in zellulären Assay-Systemen nicht gezeigt werden.
Abschließend wurde die Eignung der Thiazolidindione und verwandter Fünfringheterozyklen als Leitstruktur für potenzielle InhA-Inhibitoren, aber auch die Eignung dieser Verbindungsklasse als potenzielle Leitstruktur per se diskutiert.
Aurora B is a mitotic kinase that is essential for cell division. Because it is mutated or overexpressed in a range of cancer types, it has been suggested as a novel therapeutic target. Currently chemical inhibitors against Aurora B are in various phases of clinical trials for treatment of solid tumors and leukemia. Information regarding the molecular requirements for the reported phenotypes of Aurora B inhibition such as cell cycle arrest, activation of the tumor suppressor p53 and its target p21 are not well understood.
In this study, I investigated the requirements for p21 induction after Aurora B inhibition. I found that p38 is phosphorylated and activated when Aurora B is inhibited. Experiments with chemical inhibitors against p38 indicate that p38 is required for p21 induction and cell cycle arrest in response to Aurora B inhibition. p53 induction after impairment of Aurora B function and the recruitment of p53 to its binding site in the p21 gene promoter occur independently of p38 signaling. Instead, I found that p38 is required for the enrichment of the elongating RNA Polymerase II in the coding region of the p21 gene. Furthermore, p38 is required for formation of the full-length p21 mRNA transcript. These data indicate that p38 promotes the transcriptional elongation of p21 gene in response to Aurora B inhibition. In further experiments I could show that the p21 causes cell cycle arrest due to a decrease in E2F-dependent transcription by promoting the dephosphorylation of the retinoblastoma protein.
Using synchronized cells I could show that the induction of p21 in response to Aurora B inhibition requires transition through an aberrant mitosis and does not occur in cells that are arrested in interphase. Interestingly, p38, p53 and p21 are already induced by partial inhibition of Aurora B, which results in aneuploidy but not in cytokinesis failure and in tetraploidy. This supports the notion that activation of p38-p53-p21 signaling correlates with aneuploidy but not with tetraploidy or binucleation. Partial inhibition of Aurora B also leads to increased generation of reactive oxygen species (ROS), which are required for the activation of p38, p21 and cell cycle arrest. Based on these observations I propose the following model: Inhibition of Aurora B leads to chromosome missegregation resulting in aneuploidy. This results in increased generation of ROS (reactive oxygen species) possibly through proteotoxic stress caused by an imbalance of protein synthesis in aneuploid cells. ROS triggers the activation of p38, which then stimulates the transcriptional elongation of p21 resulting in cell cycle arrest.
Aneuploidy, proteotoxic stress and oxidative stress are hallmarks of cancer cells. Based on my results reported in this study, I suggest that the combination of Aurora B inhibitors with drugs that specifically target aneuploid cells might be a novel strategy for cancer therapy, as this is a lethal combination for proliferation of cancer cells.