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CD69 is a transmembrane lectin that can be expressed on most hematopoietic cells. In monocytes, it has been functionally linked to the 5-lipoxygenase pathway in which the leukotrienes, a class of highly potent inflammatory mediators, are produced. However, regarding CD69 gene expression and its regulatory mechanisms in monocytes, only scarce data are available. Here, we report that CD69 mRNA expression, analogous to that of 5-lipoxygenase, is induced by the physiologic stimuli transforming growth factor-β (TGF-β) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in monocytic cells. Comparison with T- and B-cell lines showed that the effect was specific for monocytes. CD69 expression levels were increased in a concentration-dependent manner, and kinetic analysis revealed a rapid onset of mRNA expression, indicating that CD69 is a primary TGF-β/1α,25(OH)2D3 target gene. PCR analysis of different regions of the CD69 mRNA revealed that de novo transcription was initiated and proximal and distal parts were induced concomitantly. In common with 5-lipoxygenase, no activation of 0.7 kb or ~2.3 kb promoter fragments by TGF-β and 1α,25(OH)2D3 could be observed in transient reporter assays for CD69. Analysis of mRNA stability using a transcription inhibitor and a 3′UTR reporter construct showed that TGF-β and 1α,25(OH)2D3 do not influence CD69 mRNA stability. Functional knockdown of Smad3 clearly demonstrated that upregulation of CD69 mRNA, in contrast to 5-LO, depends on Smad3. Comparative studies with different inhibitors for mitogen activated protein kinases (MAPKs) revealed that MAPK signalling is involved in CD69 gene regulation, whereas 5-lipoxygenase gene expression was only partly affected. Mechanistically, we found evidence that CD69 gene upregulation depends on TAK1-mediated p38 activation. In summary, our data indicate that CD69 gene expression, conforming with 5-lipoxygenase, is regulated monocyte-specifically by the physiologic stimuli TGF-β and 1α,25(OH)2D3 on mRNA level, although different mechanisms account for the upregulation of each gene.
Nep1 (Emg1) is a highly conserved nucleolar protein with an essential function in ribosome biogenesis. A mutation in the human Nep1 homolog causes Bowen–Conradi syndrome—a severe developmental disorder. Structures of Nep1 revealed a dimer with a fold similar to the SPOUT-class of RNA-methyltransferases suggesting that Nep1 acts as a methyltransferase in ribosome biogenesis. The target for this putative methyltransferase activity has not been identified yet. We characterized the RNA-binding specificity of Methanocaldococcus jannaschii Nep1 by fluorescence- and NMR-spectroscopy as well as by yeast three-hybrid screening. Nep1 binds with high affinity to short RNA oligonucleotides corresponding to nt 910–921 of M. jannaschii 16S rRNA through a highly conserved basic surface cleft along the dimer interface. Nep1 only methylates RNAs containing a pseudouridine at a position corresponding to a previously identified hypermodified N1-methyl-N3-(3-amino-3-carboxypropyl) pseudouridine (m1acp3-Psi) in eukaryotic 18S rRNAs. Analysis of the methylated nucleoside by MALDI-mass spectrometry, HPLC and NMR shows that the methyl group is transferred to the N1 of the pseudouridine. Thus, Nep1 is the first identified example of an N1-specific pseudouridine methyltransferase. This enzymatic activity is also conserved in human Nep1 suggesting that Nep1 is the methyltransferase in the biosynthesis of m1acp3-Psi in eukaryotic 18S rRNAs.
To overcome poor treatment response of pediatric high-risk acute lymphoblastic leukemia (ALL), novel treatment strategies are required to reactivate programmed cell death in this malignancy. Therefore, we take advantage of using small-molecule antagonists of Inhibitor of apoptosis (IAP) proteins, so called Smac mimetics such as BV6, which are described to overcome apoptosis resistance and thereby sensitize tumor cells for several apoptotic stimuli. To address the question whether redox alterations can sensitize leukemic cells for Smac mimetic-mediated cell death, we interfered with the cellular redox status in different ALL cell lines. Here, we show for the first time that redox alterations, mediated by the glutathione depleting agent Buthioninesulfoximine (BSO), prime ALL cells for BV6-induced apoptosis. Besides ALL cell lines, BV6/BSO cotreatment similarly synergizes in cell death induction in patient-derived primary leukemic samples. In contrast, the combination treatment does not exert any cytotoxicity against peripheral blood lymphocytes (PBLs) or mesenchymal stroma cells (MSCs) from healthy donors, suggesting some tumor selectivity of this treatment. We also identify the underlying molecular mechanism of the novel synergistic drug interaction of BSO and BV6. We demonstrate that both agents act in concert to increase reactive oxygen species (ROS) production, lipid peroxidation and finally apoptotic cell death. Enhanced ROS levels in the combination treatment account for cell death induction, since several ROS scavengers, like NAC, MnTBAP and Trolox attenuate BSO/BV6-induced apoptosis. BSO/BV6-induced ROS can be mainly classified as lipid peroxides, since the vitamin E derivate α-Tocopherol as well as Glutathione peroxidase 4 (GPX4), which both specifically reduce lipid-membrane peroxides, prevent lipid peroxidation, caspase activation and cell death induction. Vice versa, GPX4 knockdown and pharmacological inhibition of GPX4 by RSL3 or Erastin enhance BV6-induced cell death. Importantly, cell death induction critically depends on the formation of a complex consisting of RIP1/FADD/Caspase-8, since all complex components are required for ROS production, lipid peroxidation and cell death induction. Taken together, we demonstrate that BSO and BV6 cooperate to induce ROS production and lipid peroxidation which are eventually required for caspase activation and cell death execution. Collectively, findings of this study indicate that BV6-induced apoptosis is mediated via redox alterations offering promising new treatment strategy to overcome apoptosis resistance in ALL.
Poster presentation at 5th German Conference on Cheminformatics: 23. CIC-Workshop Goslar, Germany. 8-10 November 2009 We demonstrate the theoretical and practical application of modern kernel-based machine learning methods to ligand-based virtual screening by successful prospective screening for novel agonists of the peroxisome proliferator-activated receptor gamma (PPARgamma) [1]. PPARgamma is a nuclear receptor involved in lipid and glucose metabolism, and related to type-2 diabetes and dyslipidemia. Applied methods included a graph kernel designed for molecular similarity analysis [2], kernel principle component analysis [3], multiple kernel learning [4], and, Gaussian process regression [5]. In the machine learning approach to ligand-based virtual screening, one uses the similarity principle [6] to identify potentially active compounds based on their similarity to known reference ligands. Kernel-based machine learning [7] uses the "kernel trick", a systematic approach to the derivation of non-linear versions of linear algorithms like separating hyperplanes and regression. Prerequisites for kernel learning are similarity measures with the mathematical property of positive semidefiniteness (kernels). The iterative similarity optimal assignment graph kernel (ISOAK) [2] is defined directly on the annotated structure graph, and was designed specifically for the comparison of small molecules. In our virtual screening study, its use improved results, e.g., in principle component analysis-based visualization and Gaussian process regression. Following a thorough retrospective validation using a data set of 176 published PPARgamma agonists [8], we screened a vendor library for novel agonists. Subsequent testing of 15 compounds in a cell-based transactivation assay [9] yielded four active compounds. The most interesting hit, a natural product derivative with cyclobutane scaffold, is a full selective PPARgamma agonist (EC50 = 10 ± 0.2 microM, inactive on PPARalpha and PPARbeta/delta at 10 microM). We demonstrate how the interplay of several modern kernel-based machine learning approaches can successfully improve ligand-based virtual screening results.
Introduction: Despite the excellent anti-inflammatory and immunosuppressive action of glucocorticoids (GCs), their use for the treatment of inflammatory bowel disease (IBD) still carries significant risks in terms of frequently occurring severe side effects, such as the impairment of intestinal tissue repair. The recently-introduced selective glucocorticoid receptor (GR) agonists (SEGRAs) offer anti-inflammatory action comparable to that of common GCs, but with a reduced side effect profile.
Methods: The in vitro effects of the non-steroidal SEGRAs Compound A (CpdA) and ZK216348, were investigated in intestinal epithelial cells and compared to those of Dexamethasone (Dex). GR translocation was shown by immunfluorescence and Western blot analysis. Trans-repressive effects were studied by means of NF-κB/p65 activity and IL-8 levels, trans-activation potency by reporter gene assay. Flow cytometry was used to assess apoptosis of cells exposed to SEGRAs. The effects on IEC-6 and HaCaT cell restitution were determined using an in vitro wound healing model, cell proliferation by BrdU assay. In addition, influences on the TGF-β- or EGF/ERK1/2/MAPK-pathway were evaluated by reporter gene assay, Western blot and qPCR analysis.
Results: Dex, CpdA and ZK216348 were found to be functional GR agonists. In terms of trans-repression, CpdA and ZK216348 effectively inhibited NF-κB activity and IL-8 secretion, but showed less trans-activation potency. Furthermore, unlike SEGRAs, Dex caused a dose-dependent inhibition of cell restitution with no effect on cell proliferation. These differences in epithelial restitution were TGF-β-independent but Dex inhibited the EGF/ERK1/2/MAPK-pathway important for intestinal epithelial wound healing by induction of MKP-1 and Annexin-1 which was not affected by CpdA or ZK216348.
Conclusion: Collectively, our results indicate that, while their anti-inflammatory activity is comparable to Dex, SEGRAs show fewer side effects with respect to wound healing. The fact that SEGRAs did not have a similar effect on cell restitution might be due to a different modulation of EGF/ERK1/2 MAPK signalling.
Hepatitis C virus (HCV) assembly and production is closely linked to lipid metabolism. Indeed, lipid droplets (LD) have been shown to serve as a platform for HCV assembly. To investigate the effect of HCV on the host cell proteome, 2D-gelelectrophoresis with subsequent MALDI-TOF mass spectrometry of HCV replicating and the corresponding control cells were done. Based on this analysis, it was found out that HCV-replicating Huh7.5 cells revealed lower amounts of TIP47 (tail interacting protein of 47kD) compared to HCV-negative cells. TIP47, a cytoplasmic sorting factor, has been shown to be associated with lipid droplets. As it is known that HCV-replication and assembly takes place at the so called ”membranous web” that is composed of LDs and rearranged ER-derived membranes, it was tempting to investigate the role of TIP47 in HCV life-cycle. Western blot analysis did reveal that overexpression of TIP47 in HCV replicating Huh7.5 cells leads to decreased amounts of the HCV core protein while the levels of non-structural protein (NS)5A and intracellular HCVgenomes are increased. Moreover, in TIP47 overproducing cells higher amounts of infectious HCV particles are secreted. Vice versa, inhibition of TIP47 expression by siRNA results in a decreased level of intracellular NS5A, increased amounts of intracellular core and less infectious viral particles in the supernatant. In addition, complete silencing of TIP47 by lentiviral transduction abolishes HCV replication that can be restored by transfection of these cells with a TIP47 expression construct. It has been shown recently that apoE binds to NS5A and that this interaction plays an important role for the HCV life cycle (Benga et al., 2010). The C-terminal part of TIP47 harbours a 4 helix bundle motif and displays high homology to the N-terminus of apoE. Therefore, we investigated the interaction of NS5A and TIP47. Confocal double immunofluorescence microscopy revealed that a fraction of NS5A colocalizes with TIP47. Coimmunoprecipitation experiments and a yeast-two-hybrid screening confirmed the interaction between NS5A and TIP47 and deletion of the N-terminal-TIP47-PAT domain abolishes this interaction. From this we conclude that the TIP47-NS5A interaction is required for virus morphogenesis. Moreover, TIP47 can bind to Rab9 and this is relevant for targeting the viral particle out of the cell. In accordance to this, TIP47 was identified to be associated to the viral particle. Mutants of TIP47 that fail to bind Rab9 reveal lower amounts and a changed distribution of the HCV core protein. Furthermore, we could see that the core staining colocalizes with subcellular structures that were identified as autophagosomes using a p62-specific antibody which is a specific autophagosome-marker. Based on this, we hypothized that destruction of the Rab9 binding domain misdirects the viral particle towards the lysosomal compartment.
For the first time it could be shown that TIP47 interacts with NS5A and is associated to the viral particle, therefore plays a crucial role for the virus morphogenesis and secretion of the viral article.
Taken together, these results indicate that TIP47 is an essential cellular factor for the life cycle of HCV Abstract and might be used as target for antiviral treatment, e.g. by targeting the NS5A-TIP47 interaction, based on small molecules that mimic the NS5A-specific sequence that binds to TIP47 which might result in a competition of the TIP47/NS5A interaction.
Background: Nitric oxide (NO) is an essential vasodilator. In vascular diseases, oxidative stress attenuates NO signaling by both chemical scavenging of free NO and oxidation and down-regulation of its major intracellular receptor, the alpha/beta heterodimeric heme-containing soluble guanylate cyclase (sGC). Oxidation can also induce loss of sGC's heme and responsiveness to NO.
Results: sGC activators such as BAY 58-2667 bind to oxidized/heme-free sGC and reactivate the enzyme to exert disease-specific vasodilation. Here we show that oxidation-induced down-regulation of sGC protein extends to isolated blood vessels. Mechanistically, degradation was triggered through sGC ubiquitination and proteasomal degradation. The heme-binding site ligand, BAY 58-2667, prevented sGC ubiquitination and stabilized both alpha and beta subunits.
Conclusion: Collectively, our data establish oxidation-ubiquitination of sGC as a modulator of NO/cGMP signaling and point to a new mechanism of action for sGC activating vasodilators by stabilizing their receptor, oxidized/heme-free sGC.
The arachidonic acid cascade is a key player in inflammation, and numerous well-established drugs interfere with this pathway. Previous studies have suggested that simultaneous inhibition of 5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH) results in synergistic anti-inflammatory effects. In this study, a novel prototype of a dual 5-LO/sEH inhibitor KM55 was rationally designed and synthesized. KM55 was evaluated in enzyme activity assays with recombinant enzymes. Furthermore, activity of KM55 in human whole blood and endothelial cells was investigated. KM55 potently inhibited both enzymes in vitro and attenuated the formation of leukotrienes in human whole blood. KM55 was also tested in a cell function-based assay. The compound significantly inhibited the LPS-induced adhesion of leukocytes to endothelial cells by blocking leukocyte activation.
Die Beteiligung an Schlüsselfunktionen in zellulären Signalwegen macht Kinasen zu einem vielversprechenden Ansatzpunkt in der Wirkstoffentwicklung bei verschiedenen menschlichen Erkrankungen wie z.B. Krebs oder auch Autoimmun- und Entzündungskrankheiten. Die Prävention von post-translationalen Modifikationen durch Phosphorylierung und somit die Regulierung der nachgeschalteten Signalwege ist das Ziel von Kinaseinhibitoren. Die katalytische Aktivität von Kinasen ist abhängig von ATP, welches im hochkonservierten aktiven Zentrum bindet. Bedingt durch diese kinomweite hohe Konservierung stellt die Entwicklung von hoch selektiven ATP-mimetischen Inhibitoren eine Herausforderung dar. Typische ATP-Mimetika sind flach und die oft hydrophoben Moleküle weisen meist eine große Zahl an frei rotierbaren Bindungen auf. Um das aus dieser Flexibilität hervorgehende Problem der teils mangelnden Selektivität zu umgehen, kann eine bioaktive Konformation des Inhibitors durch Makrozyklisierung fixiert werden. Als Konsequenz dieser konformationellen Einschränkung können die entropischen Kosten während des Bindens reduziert werden und folglich zu einer gesteigerten Affinität gegenüber der Kinase führen.
Der Grundstein dieser Arbeit war der makrozyklische Pyrazolo[1,5-a]pyrimidin basierte FLT3 Kinaseinhibitor ODS2004070 (37). Im Rahmen eines kinomweiten Screenings konnten hohe Affinitäten zu verschiedensten Kinasen detektiert werden, was 37 zu einer guten Leitstruktur für das Design von potenten und selektiven Kinaseinhibitoren machte. Im Rahmen dieser Arbeit blieb das literaturbekannte Pyrazolo[1,5-a]pyrimidin basierte ATP-mimetische Bindemotiv sowie das makrozyklische Grundgerüst 37 bis auf einige wenige Variation unverändert.
Strukturelle Optimierungen zur Fokussierung der Selektivität wurden am sekundären Amin zwischen Bindemotiv und Linker als auch über die freie Carbonsäure durchgeführt. Mit einer Anzahl von mehr als 430 identifizierten Phosphorylierungsstellen ist die pleiotropisch und konstitutiv aktive Casein Kinase 2 (CK2) an verschiedensten zellulären Prozessen wie dem Verlauf des Zellzyklus, der Apoptose oder der Transkription regulatorisch beteiligt. Die Fehlregulation von CK2 wird häufig mit der Pathologie von Krankheiten wie zum Beispiel Krebs assoziiert, was CK2 zu einem vielversprechenden Ziel klinischer Untersuchungen macht.
Im Rahmen des CK2-Projekts war es möglich, durch spezifische Modifikationen an 37, die hoch selektiven und potenten CK2-Inhibitoren 47 und 60 zu entwickeln. Ebenfalls gezeigt wurde, dass kleine strukturelle Veränderungen, wie z.B. Makrozyklisierung, einen signifikanten Effekt auf Selektivität und Potenz des Inhibitors haben kann.
Weiter Untersuchungen der Verbindungen lenkten den Fokus weiterer Arbeiten u.a. auf die Serin/Threonin Kinase 17A (STK17A) oder auch death-associated protein kinase-related apoptosis-inducing protein kinase 1 (DRAK1) genannt. Sie ist Teil der DAPK Familie und gehört zusammen mit anderen Kinasen zu den weniger erforschten Kinasen. Bis heute ist nicht viel über ihre zellulären Funktionen und die Beteiligung an pathophysiologischen Prozessen bekannt. Berichtet wurde jedoch eine Überexpression in verschiedenen Formen von Hirntumoren des zentralen Nervensystems (Gliom). Strukturelle Modifikationen, unter Erhalt des makrozyklischen Grundgerüsts 37, führten zu dem hoch selektiven und potenten DRAK1 Inhibitor 121, der alle Kriterien für eine chemical probe Verbindung erfüllt.
Ein weiteres Ziel dieser Arbeit war die AP-2-assoziierte Protein Kinase 1 (AAK1) aus der NAK Familie, bestehend aus AAK1, BIKE und GAK. Sie ist als potenzielles therapeutisches Ziel für viele verschieden Krankheiten wie z.B. neuropathische Schmerzen, Schizophrenie und Parkinson identifiziert. Durch die Regulierung der Clathrin-mediierten Endozytose ist AAK1 an intrazellulären Bewegungen verschiedener nicht zusammenhängenden RNS- und DNSViren, wie beispielsweise HCV, DENV oder EBOV, beteiligt. Ebenfalls berichtet wurde eine mögliche Assoziation mit dem SARS-CoV-2 Virus, was das Interesse an neuen selektiven AAK1 Inhibitoren verstärkte. Die Entwicklung der hochpotenten und selektiven AAK1 Inhibitoren 61 und 63 basierte ebenfalls auf dem makrozyklischen Grundgerüst 37, das bereits im CK2- und DRAK1-Projekt verwendet wurde.
Zusammenfassend lässt sich sagen, dass es im Rahmen dieser Arbeit gelungen ist, ausgehend von einem höchst unselektiven makrozyklischen Grundgerüst, hochpotente und selektive Kinaseinhibitoren für CK2, DRAK1 und AAK1 zu entwickeln und zu charakterisieren. Im Zuge von Untersuchungen verschiedener Struktur-Wirkungsbeziehungen wurde gezeigt, dass es durch geringfügige strukturelle Modifikationen möglich ist, die kinomweite Selektivität zu variieren und auf eine Kinase zu fokussieren. Diese Arbeit brachte nicht nur die erwähnten Inhibitoren hervor, sondern bildet auch die Grundlage für weitere Projekte zur Entwicklung von hoch potenten und selektiven Verbindungen als potenzielle chemische Werkzeuge für den Einsatz in der Forschung.