Small-molecule pyrimidine inhibitors of the cdc2-like (Clk) and dual specificity tyrosine phosphorylation-regulated (Dyrk) kinases: Development of chemical probe ML315

Substituted pyrimidine inhibitors of the Clk and Dyrk kinases have been developed, exploring structure-activity relationships around four different chemotypes. The most potent compounds have low-nanomolar inhibitory activity against Clk1, Clk2, Clk4, Dyrk1A and Dyrk1B. Kinome scans with 442 kinases using agents representing three of the chemotypes show these inhibitors to be highly selective for the Clk and Dyrk families. Further off-target pharmacological evaluation with ML315, the most selective agent, supports this conclusion

Teaching old compounds new tricks: efficient N2 fixation by simple Fe(N2)(diphosphine)2 complexes

The Fe(0) species Fe(N2)(dmpe)2 exists in equilibrium with the previously unreported dimer, [Fe(dmpe2)2(μ-N2)]. For the first time these complexes, alongside Fe(N2)(depe)2, are shown unambiguously to produce N2H4 and/or NH3 upon addition of triflic acid; for Fe(N2)(depe)2 this represents one of the highest electron conversion efficiencies for Fe complexes to date

Nucleozin Targets Cytoplasmic Trafficking of Viral Ribonucleoprotein-Rab11 Complexes in Influenza A Virus Infection

Novel antivirals are needed to supplement existing control strategies for influenza A virus (IAV). A promising new class of drug, exemplified by the compound nucleozin, has recently been identified that targets the viral nucleoprotein (NP). These inhibitors are thought to act as "molecular staples" that stabilize interactions between NP monomers, promoting the formation of nonfunctional aggregates. Here we detail the inhibitory mechanism of nucleozin, finding that the drug has both early- and late-acting effects on the IAV life cycle. When present at the start of infection, it inhibited viral RNA and protein synthesis. However, when added at later time points, it still potently blocked the production of infectious progeny but without affecting viral macromolecular synthesis. Instead, nucleozin blocked the cytoplasmic trafficking of ribonucleoproteins (RNPs) that had undergone nuclear export, promoting the formation of large perinuclear aggregates of RNPs along with cellular Rab11. This effect led to the production of much reduced amounts of often markedly smaller virus particles. We conclude that the primary target of nucleozin is the viral RNP, not NP, and this work also provides proof of the principle that IAV replication can be effectively inhibited by blocking cytoplasmic trafficking of the viral genome.MRC grant: (G0700815), University of Cambridge/Trinity College grant: (Newton Trust), RGC Hong Kong grant: (GRF 768010 M)

Molecular Dynamics Studies of the Nucleoprotein of Influenza A Virus: Role of the Protein Flexibility in RNA Binding

The influenza viruses contain a segmented, negative stranded RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP). X-ray structures have shown that NP contains well-structured domains juxtaposed with regions of missing electron densities corresponding to loops. In this study, we tested if these flexible loops gated or promoted RNA binding and RNA-induced oligomerization of NP. We first performed molecular dynamics simulations of wt NP monomer and trimer in comparison with the R361A protein mutated in the RNA binding groove, using the H1N1 NP as the initial structure. Calculation of the root-mean-square fluctuations highlighted the presence of two flexible loops in NP trimer: loop 1 (73–90), loop 2 (200–214). In NP, loops 1 and 2 formed a 10–15 Å-wide pinch giving access to the RNA binding groove. Loop 1 was stabilized by interactions with K113 of the adjacent β-sheet 1 (91–112) that interacted with the RNA grove (linker 360–373) via multiple hydrophobic contacts. In R361A, a salt bridge formed between E80 of loop 1 and R208 of loop 2 driven by hydrophobic contacts between L79 and W207, due to a decreased flexibility of loop 2 and loop 1 unfolding. Thus, RNA could not access its binding groove in R361A; accordingly, R361A had a much lower affinity for RNA than NP. Disruption of the E80-R208 interaction in the triple mutant R361A-E80A-E81A increased its RNA binding affinity and restored its oligomerization back to wt levels in contrast with impaired levels of R361A. Our data suggest that the flexibility of loops 1 and 2 is required for RNA sampling and binding which likely involve conformational change(s) of the nucleoprotein

A Novel Small Molecule Inhibitor of Hepatitis C Virus Entry

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development

Auswirkung von körperlicher Belastung auf die Konzentration von 3-Nitrotyrosin im Atemkondensat

Due to its function, the lung is more likely exposed to environmental influences than other organs of the human body. Frequently the occupational medicine and health care have to deal with complaints due to pulmonary reasons, which cannot be detected by established diagnostic methods (i.e. spirometry), as those methods do only examine existing structural and functional pulmonary damages. The analysis of breath condensate is a new non-invasive method to examine processes in the air passages. The breath condensate contains non-gaseous compounds of exhaled breath, which reflects the composition of the bronchoalveolar extracellular lining fluid. The marker 3-nitrotyrosine, which can be detected in breath condensate, helps to examine processes of oxidative stress in the lower airways. At the present time the analysis of breath condensate is not established in the diagnostic routine. This study claims to improve this matter. 20 probands (aged 20-35 years, equally male and female genders) were examined due to the changes of 3-nitrotyrosine concentration in exhaled breath condensate (EBC) caused by physical exercise testing. They were separated in 10 atopic and 10 non-atopic probands. Each group contained 5 smokers and 5 non-smokers. Eventually we got four groups of probands: 1. smoker/atopics; 2. smoker/ non-atopics; 3. non-smoker/atopics; 4. non-smoker/non-atopics.A standardized questionnaire documented the current health status including the atopic status and smoking habitsof each proband. After a physical examination and lung function testing (spirometry respectively bodyplethysmography), the first frequency-controlled breath condensate donation took place (point of time T0), which lasted 15 minutes. Following a cardiopulmonary exercise testing took place until the proband reached his anaerobic threshold. The second breath condensate donation (length of time 15 minutes; point of time T1) was executed 15 minutes after reaching the anaerobic threshold, which was verified by an interpretation of capillary blood gas testing. The third breath condensate donation (point of time T2) was performed 90 minutes after reaching the anaerobic threshold. The nitrotyrosine concentration was determined by LC-MS/MS after SPE-accumulation. The mean concentration of nitrotyrosine (in pg/ml) before and after exercise testing were 50,83 pg/ml (SD: ± 31,27), 51,94 pg/ml (SD: ± 27,81), 25,57 pg/ml (SD: ± 20,33). The median nitrotyrosine concentration (in pg/ml) before and after exercise testing were 39,1 pg/ml (IQR: 47,55), 50,1 pg/ml (IQR:45,15), 18,8 pg/ml (IQR: 24,05). CONCLUSION: Physical exercise shows no significant effect on the 3-nitrotyrosine concentration in exhaled breath condensate (EBC)