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11.
Valter Bergant Shintaro Yamada Vincent Grass Yuta Tsukamoto Teresa Lavacca Karsten Krey MariaTeresa Mühlhofer Sabine Wittmann Armin Ensser Alexandra Herrmann Anja vom Hemdt Yuriko Tomita Shutoku Matsuyama Takatsugu Hirokawa Yiqi Huang Antonio Piras Constanze A Jakwerth Madlen Oelsner Susanne Thieme Alexander Graf Stefan Krebs Helmut Blum Beate M Kümmerer Alexey Stukalov Carsten B SchmidtWeber Manabu Igarashi Thomas Gramberg Andreas Pichlmair Hiroki Kato 《The EMBO journal》2022,41(17)
The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19. 相似文献
12.
Evangelia Stergiakouli Romy Gaillard Jeremy M. Tavaré Nina Balthasar Ruth J. Loos Hendrik R. Taal David M. Evans Fernando Rivadeneira Beate St Pourcain André G. Uitterlinden John P. Kemp Albert Hofman Susan M. Ring Tim J. Cole Vincent W.V. Jaddoe George Davey Smith Nicholas J. Timpson 《Obesity (Silver Spring, Md.)》2014,22(10):2252-2259
13.
Adalbert Krawczyk Miriam Dirks Maren Kasper Anna Buch Ulf Dittmer Bernd Giebel Lena Wildschütz Martin Busch Andre Goergens Karl E. Schneweis Anna M. Eis-Hübinger Beate Sodeik Arnd Heiligenhaus Michael Roggendorf Dirk Bauer 《PloS one》2015,10(1)
The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans. 相似文献
14.
Fast J Håkansson M Muranyi A Gippert GP Thulin E Evenäs J Svensson LA Linse S 《Biochemistry》2001,40(33):9887-9895
Water molecules are found to complete the Ca2+ coordination sphere when a protein fails to provide enough ligating oxygens. Hydrogen bonding of these water molecules to the protein backbone or side chains may contribute favorably to the Ca2+ affinity, as suggested in an earlier study of two calbindin D(9k) mutants [E60D and E60Q; Linse et al. (1994) Biochemistry 33, 12478-12486]. To investigate the generality of this conclusion, another side chain, Gln 22, which hydrogen bonds to a Ca2+-coordinating water molecule in calbindin D(9k), was mutated. Two calbindin D(9k) mutants, (Q22E+P43M) and (Q22N+P43M), were constructed to examine the interaction between Gln 22 and the water molecule in the C-terminal calcium binding site II. Shortening of the side chain, as in (Q22N+P43M), reduces the affinity of binding two calcium ions by a factor of 18 at low ionic strength, whereas introduction of a negative charge, as in (Q22E+P43M), leads to a 12-fold reduction. In 0.15 M KCl, a 7-fold reduction in affinity was observed for both mutants. The cooperativity of Ca2+ binding increases for (Q22E+P43M), while it decreases for (Q22N+P43M). The rates of Ca2+ dissociation are 5.5-fold higher for the double mutants than for P43M at low ionic strength. For both mutants, reduced strength of hydrogen bonding to calcium-coordinating water molecules is a likely explanation for the observed effects on Ca2+ affinity and dissociation. In the apo forms, the (Q22E+P43M) mutant has lower stability toward urea denaturation than (Q22N+P43M) and P43M. 2D (1)H NMR and crystallographic experiments suggest that the structure of (Q22E+P43M) and (Q22N+P43M) is unchanged relative to P43M, except for local perturbations in the loop regions. 相似文献
15.
Pei W. Thomas Timothy Spicer Michael Cammarata Jennifer S. Brodbelt Peter Hodder Walter Fast 《Bioorganic & medicinal chemistry》2013,21(11):3138-3146
Due to the global threat of antibiotic resistance mediated by New Delhi metallo-beta-lactamase-1 (NDM-1) and the lack of structurally diverse inhibitors reported for this enzyme, we developed screening and counter-screening assays for manual and automated formats. The manual assay is a trans-well absorbance-based endpoint assay in 96-well plates and has a Z′ factor of 0.8. The automated assay is an epi-absorbance endpoint assay in 384-well plates, has a Z′ factor of ?0.8, good signal/baseline ratios (>3.8), and is likely scalable for high-throughput screening (HTS). A TEM-1-based counter-screen is also presented to eliminate false positives due to assay interference or off-target activities. A pilot screen of a pharmacologically characterized compound library identified two thiol-modifying compounds as authentic NDM-1 inhibitors: p-chloromecuribenzoate (p-CMB) and nitroprusside. Recombinant NDM-1 has one Cys residue that serves as a conserved active-site primary zinc ligand and is selectively modified by p-CMB as confirmed by LC–MS/MS. However a C208D mutation results in an enzyme that maintains almost full lactamase activity, yet is completely resistant to the inhibitor. These results predict that covalent targeting of the conserved active-site Cys residue may have drawbacks as a drug design strategy. 相似文献
16.
Williams NM Williams H Majounie E Norton N Glaser B Morris HR Owen MJ O'Donovan MC 《Nucleic acids research》2008,36(17):e112
Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility. Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts. We have developed a simple competitive PCR based method to determine DNA copy number which uses the entire genome of a single chimpanzee as a competitor thus eliminating the requirement for competitive sequences to be synthesized for each assay. This results in the requirement for only a single reference sample for all assays and dramatically increases the potential for large numbers of loci to be analysed in multiplex. In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls. 相似文献
17.
Malawski GA Hillig RC Monteclaro F Eberspaecher U Schmitz AA Crusius K Huber M Egner U Donner P Müller-Tiemann B 《Protein science : a publication of the Protein Society》2006,15(12):2718-2728
This study describes an efficient multiparallel automated workflow of cloning, expression, purification, and crystallization of a large set of construct variants for isolated protein domains aimed at structure determination by X-ray crystallography. This methodology is applied to MAPKAP kinase 2, a key enzyme in the inflammation pathway and thus an attractive drug target. The study reveals a distinct subset of truncation variants with improved crystallization properties. These constructs distinguish themselves by increased solubility and stability during a parallel automated multistep purification process including removal of the recombinant tag. High-throughput protein melting point analysis characterizes this subset of constructs as particularly thermostable. Both parallel purification screening and melting point determination clearly identify residue 364 as the optimal C terminus for the kinase domain. Moreover, all three constructs that ultimately crystallized feature this C terminus. At the N terminus, only three amino acids differentiate a noncrystallizing from a crystallizing construct. This study addresses the very common issues associated with difficult to crystallize proteins, those of solubility and stability, and the crucial importance of particular residues in the formation of crystal contacts. A methodology is suggested that includes biophysical measurements to efficiently identify and produce construct variants of isolated protein domains which exhibit higher crystallization propensity. 相似文献
18.
F. Ekkehardt Hahn Beate Heidrich Alexander Hepp Eduardo Sola 《Inorganica chimica acta》2006,359(15):4840-4846
The reaction of 1-methyl-3-(2-propenyl)imidazolium bromide (1) or 1,3-bis(2-propenyl)-imidazolium bromide (2) with [Ir(μ-OMe)(cod)]2 afforded the five coordinated iridium(I) carbene complexes [IrBr(L)(cod)] (3) (L=1-methyl-3-(2-propenyl)imidazolin-2-ylidene) and (4) (L=1,3-bis(2-propenyl)imidazolin-2-ylidene). The reaction proceeds via an in situ deprotonation of the imidazolium salt. Molecular structure determinations on 3 and 4 confirmed the coordination of the carbene ligands via the carbene carbon atom and one allyl group in both complexes. Treatment of complex 3 with an excess of AgBF4 gave the dinuclear bromo bridged complex [(Ir(μ-Br)(L)(cod)]2BF4 (5) (L=1-methyl-3-(2-propenyl)imidazolin-2-ylidene). The reaction of complex 4 with an excess of AgBF4 led to the mononuclear complex [Ir(L)(cod)]BF4 (6) (L=1,3-bis(2-propenyl)imidazolin-2-ylidene) where both N-allyl substituents are coordinated to the iridium(I) center. 相似文献
19.
Soni Pullamsetti Stefanie Krick Hüseyin Yilmaz Hossein Ardeschir Ghofrani Christian Schudt Norbert Weissmann Beate Fuchs Werner Seeger Friedrich Grimminger Ralph Theo Schermuly 《Respiratory research》2005,6(1):128
Background
The aim of the study was to assess the chronic effects of combined phosphodiesterase 3/4 inhibitor tolafentrine, administered by inhalation, during monocrotaline-induced pulmonary arterial hypertension (PAH) in rats.Methods
CD rats were given a single subcutaneous injection of monocrotaline to induce PAH. Four weeks after, rats were subjected to inhalation of tolafentrine or sham nebulization in an unrestrained, whole body aerosol exposure system. In these animals (i) the acute pulmonary vasodilatory efficacy of inhaled tolafentrine (ii) the anti-remodeling effect of long-term inhalation of tolafentrine (iii) the effects of tolafentrine on the expression profile of 96 genes encoding cell adhesion and extracellular matrix regulation were examined. In addition, the inhibitory effect of tolafentrine on ex vivo isolated pulmonary artery SMC cell migration was also investigated.Results
Monocrotaline injection provoked severe PAH (right ventricular systolic pressure increased from 25.9 ± 4.0 to 68.9 ± 3.2 after 4 weeks and 74.9 ± 5.1 mmHg after 6 weeks), cardiac output depression and right heart hypertrophy. The media thickness of the pulmonary arteries and the proportion of muscularization of small precapillary resistance vessels increased dramatically, and the migratory response of ex-vivo isolated pulmonary artery smooth muscle cells (PASMC) was increased. Micro-arrays and subsequent confirmation with real time PCR demonstrated upregulation of several extracellular matrix regulation and adhesion genes, such as matrixmetalloproteases (MMP) 2, 8, 9, 10, 11, 12, 20, Icam, Itgax, Plat and serpinb2. When chronically nebulized from day 28 to 42 (12 daily aerosol maneuvers), after full establishment of severe pulmonary hypertension, tolafentrine reversed about 60% of all hemodynamic abnormalities, right heart hypertrophy and monocrotaline-induced structural lung vascular changes, including the proportion of pulmonary artery muscularization. The upregulation of extracellular matrix regulation and adhesion genes was reduced by nearly 80% by inhalation of the tolafentrine. When assessed in vitro, tolafentrine blocked the enhanced PASMC migratory response.Conclusion
In conclusion, we demonstrate for the first time that inhalation of combined PDE3/4 inhibitor reverses pulmonary hypertension fully developed in response to monocrotaline in rats. This "reverse-remodeling" effect includes structural changes in the lung vascular wall and key molecular pathways of matrix regulation, concomitant with 60% normalization of hemodynamics. 相似文献20.
Oxidative stress caused by pyocyanin impairs CFTR Cl(-) transport in human bronchial epithelial cells 总被引:1,自引:0,他引:1
Schwarzer C Fischer H Kim EJ Barber KJ Mills AD Kurth MJ Gruenert DC Suh JH Machen TE Illek B 《Free radical biology & medicine》2008,45(12):1653-1662
Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H(2)O(2) threefold above the endogenous H(2)O(2) production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 microM) oxidized the cytosol from a resting value of -318+/-5 mV by 48.0+/-4.6 mV within 2 h; a comparable oxidation was induced by 100 microM H(2)O(2). Whereas resting Cl(-) secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl(-) secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for DeltaF508 CFTR failed to secrete Cl(-) in response to pyocyanin or H(2)O(2), indicating that these oxidants specifically target the CFTR and not other Cl(-) conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H(2)O(2), depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs. 相似文献