Chemical structure of Tripolin A and Tripolin B.

<p>Chemical structure of Tripolin A and Tripolin B.</p

Selectivity of Tripolins against a panel of kinases.

<p>IC₅₀ values of Tripolin A and Tripolin B against Aurora A, Aurora B and a panel of other selected kinases.</p

Tripolin A selectively inhibits Aurora A over Aurora B in cultured tumor cells.

<p>(A) Representative immunofluorescence images of HeLa cells in metaphase treated with solvent control (DMSO), 20 µM Tripolin A or Tripolin B for 5 h and 24 h. In the merged images Aurora A is pseudocolored red, pAurora T288 green, DNA blue. (Scale bars, 5 µm). (B) Fluorescence intensity (% percentage) of pAurora A T288 on centrosomes and total Aurora A on spindles were quantified in control metaphase cells or cells treated with Tripolin A or Tripolin B (n≥20 cells for each group, from at least two independent experiments). **: 0.0010.05; (Mann-Whitney test, two-tailed). Error bars represent SEM. (C) Western Blot analysis for Aurora A, Aurora B and pHistone H3 Ser10 in Tripolin A and Tripolin B-treated mitotic cells. α-tubulin was used as a loading control. (D) Representative immunofluorescence images of bipolar metaphase HeLa cells treated with solvent control (DMSO), 20 µM Tripolin A or Tripolin B for 24 h. In the merged images pHistone H3 Ser10 is pseudocolored red, Aurora B green, DNA blue. (Scale bars, 5 µm).</p

New 7‑Methylguanine Derivatives Targeting the Influenza Polymerase PB2 Cap-Binding Domain

The heterotrimeric influenza virus polymerase performs replication and transcription of viral RNA in the nucleus of infected cells. Transcription by “cap-snatching” requires that host-cell pre-mRNAs are bound via their 5′ cap to the PB2 subunit. Thus, the PB2 cap-binding site is potentially a good target for new antiviral drugs that will directly inhibit viral replication. Docking studies using the structure of the PB2 cap-binding domain suggested that 7-alkylguanine derivatives substituted at position <i>N</i>-9 and <i>N</i>-2 could be good candidates. Four series of 7,9-di- and 2,7,9-trialkyl guanine derivatives were synthesized and evaluated by an AlphaScreen assay in competition with a biotinylated cap analogue. Three synthesized compounds display potent in vitro activity with IC₅₀ values lower than 10 μM. High-resolution X-ray structures of three inhibitors in complex with the H5N1 PB2 cap-binding domain confirmed the binding mode and provide detailed information for further compound optimization

Tripolin A treatment results in spindle and centrosomal defects.

<p>(A) Representative immunofluorescence images of mitotic HeLa cells treated with DMSO, 20 µM Tripolin A for 24 h, 100 nM MLN8237 for 24 h or Aurora A siRNAs. In the merged images α-tubulin is pseudocolored red, DNA blue. (Scale bars, 5 µm). (B) Graph showing the percentage of normal, multipolar, misaligned, disorganized and monopolar figures in control mitotic cells (DMSO or control siRNAs) and mitotic cells treated with Tripolin A, MLN8237 or Aurora A siRNA (n = 300 cells for each group, from three independent experiments). (C) Western Blot analysis for Aurora A levels in Aurora A siRNA treated cells. α-tubulin was used as a loading control. (D) Images of mitotic HeLa cells treated with DMSO, 20 µM Tripolin A for 5 h and 24 h or Aurora A siRNA. In the merged images Aurora A is pseudocolored red, pericentrin green, DNA blue. (Scale bar 5 µm). (E) Graph showing the percentage of mitotic cells with fragmented centrosomes (up), or acentrosomal poles (down) in control mitotic cells (DMSO or control siRNA) and mitotic cells treated with Tripolin A, or Aurora A siRNA (n = 150 cells for each group, from three independent experiments).</p

Tripolins inhibit Aurora kinase activity <i>in vitro</i>.

<p><i>(</i>A) Chemical structure of Tripolin A and Tripolin B. (B) Graph showing IC₅₀ values (in µM) of Tripolin A (red) and Tripolin B (green) in the presence of different ATP concentrations, using an <i>in vitro</i> kinase assay. (C) Differential Scanning Fluorimetry results for Aurora A in the presence and absence of the inhibitors. Blue curve determines the melting temperature of Aurora A alone (45°C), red in the presence of Tripolin A (47°C) and green in the presence of Tripolin B (53°C).</p

Tripolin A alters pole-to-pole distance and MT stability in mitotic cells and influences interphase MT array.

<p>(A) Maximum projections from z-stacks of a representative control cell and representative cells treated with Tripolin A. In the merged images α-tubulin is pseudocolored red; pericentrin is green, DNA is blue. Yellow arrows indicate interpolar distance. (B) Interpolar distances were measured based on pericentrin staining in HeLa cells (n≥100 cells for each group, from at least three independent experiments). ***: p<0.0001; (Student's t-test, two-tailed). Error bars indicate SD. (C) Longitudinal line scans of tubulin intensity from metaphase spindles of control and Tripolin A treated HeLa cells (n = 5 for each group). Intensities were normalized to the maximum value of the control curve, and spindle size was interpolated. Curves indicate mean values. (D) Representative immunofluorescence images of HeLa cells in interphase treated with DMSO, 100 nM MLN8237 for 1 h or 20 µM Tripolin A for 1 h and 24 h. In the merged images α-tubulin is pseudocolored red, DNA blue. (Scale bar 10 µm). (E) Graph showing the percentages of interphase cells with altered MT array, classified in the indicated arbitrary categories in control cells (DMSO) and cells treated with MLN8237 or Tripolin A (n = 150 cells for each group, from three independent experiments).</p

Overview of Motifs Found in the Fly

<p>Significant predictions from the yeast two-hybrid set for the fly. Blue dots in the center of each cluster represent proteins with four or more interaction partners (red and white dots) containing at least one confidently predicted motif (<i>p-</i>value < 0.001; <i>S_cons</i> ≤ 8 × 10⁻¹⁵). Partner proteins containing the motif are represented by red dots, whereas proteins lacking the motif are indicated by white dots. Clusters are labelled as gene name→detected motif. Yellow circles enclose known motifs: SH3→PxxP [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-b38" target="_blank">38</a>], PP1→RVxF [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-b22" target="_blank">22</a>], C-terminal binding protein (CtBP)→PxDLS [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-b52" target="_blank">52</a>], SR splicing factors RS-rich segments [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-b53" target="_blank">53</a>], and CG6843→SxKSKxxK, a likely nuclear localization signal. The Translin→VxxxRxYS motif was experimentally tested (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-g003" target="_blank">Figure 3</a>). The grey circles enclose clusters with low-complexity patterns. Two additional known motifs were also found in the fly using more relaxed criteria than those used for the other motifs in the figure: Groucho→WRPW [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-b07" target="_blank">7</a>] and Dynein light chain→TQT [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-b26" target="_blank">26</a>] as the variant A(TI)QT(DE). The latter was also identified as significant in the domain sets. Proteins are denoted either by their FlyBase accession codes or protein names when available.</p

A Lit-1 MAP Kinase SxPxxxS Motif

<p>The MAP kinase lit-1 surrounded by its interaction partners containing the SxPxxxS motif. Details are as for <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-g003" target="_blank">Figure 3</a>. Yellow boxes show the location of deletion mutants known to affect the interaction. Cbr, <i>C. briggsae;</i> Cel, <i>C. elegans</i>.</p

Specific Inhibitors of HIV Capsid Assembly Binding to the C‑Terminal Domain of the Capsid Protein: Evaluation of 2‑Arylquinazolines as Potential Antiviral Compounds

Assembly of human immunodeficiency virus (HIV-1) represents an attractive target for antiretroviral therapy which is not exploited by currently available drugs. We established high-throughput screening for assembly inhibitors based on competition of small molecules for the binding of a known dodecapeptide assembly inhibitor to the C-terminal domain of HIV-1 CA (capsid). Screening of >70000 compounds from different libraries identified 2-arylquinazolines as low micromolecular inhibitors of HIV-1 capsid assembly. We prepared focused libraries of modified 2-arylquinazolines and tested their capacity to bind HIV-1 CA to compete with the known peptide inhibitor and to prevent the replication of HIV-1 in tissue culture. Some of the compounds showed potent binding to the C-terminal domain of CA and were found to block viral replication at low micromolar concentrations