13 research outputs found
Small molecule inhibitors of yeast sporulation.
<p>(A) A scatterplot of sensitivity scores (calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042853#s4" target="_blank">Methods</a>) representing the impact of compounds from the NIH clinical collection in the sporulation screening assays. Compounds that inhibit growth or that interfered with the fluorescence-based assay because of auto-fluorescence were not included in this representation. Compounds with a score of greater than 5 are shown in green. DMSO controls are shown in red. (B) A Venn diagram representing the overlap of inhibitors in the growth assay and the two sporulation assays (colored in yellow and green, respectively). 200 compounds had no effect in any of the three assays, 231 inhibited vegetative growth (of BY4741 and/or AD1-9 strains), and 64 inhibited sporulation. The overlap of between growth and sporulation inhibitors was 49. (C) Chemical structures of 12 sporulation-specific inhibitors that were confirmed to inhibit sporulation by microscopy analysis.</p
Tripelennamine sensitizes autophagy-deficient yeast mutants.
<p>(A) Heatmap and dendrogram depicting hierarchical clustering of homozygous deletion pool data from three independent experiments. Quantile normalized and log2-transformed microarray fluorescence signals were analyzed using the Significance Analysis of Microarrays (SAM) software (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042853#s4" target="_blank">Methods</a>) and identified 49 genes (indicated on the right-hand side) that were significantly depleted in the presence of tripelennamine (TA) compared to a “no drug” control. Darker shades of red indicate higher fluorescence signals and therefore a higher abundance of that strain in the pool (see legend). (B) Growth of the <i>ho</i>Δ<i>/HO</i> (control) and <i>neo1</i>Δ<i>/NEO1</i> heterozygous deletion strains (see legend) were determined in the presence of various concentrations of (TA) (indicated on the x-axis). Growth rates relative to the “no drug” control (calculated as a ratio of AvgG, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042853#s4" target="_blank">Methods</a>) were determined for each concentration and are plotted on the y-axis. (C) Sporulation efficiency of the <i>ho</i>Δ<i>/HO</i> control strain (black curve) and the <i>neo1</i>Δ<i>/NEO1</i> heterozygous deletion strain (red curve) sporulated in the presence or absence of TA is indicated as percent spores on the y-axis. Both strains were incubated in sporulation media for 48 hours at various concentrations of TA (indicated on the x-axis) and percentage of spores was determined by microscopy. A total of 100 cells were counted for every condition.</p
Two screening assays for sporulation efficiency.
<p>(A) Schematic of the fluorescence-based assay used to identify sporulation inhibitors. Meiotic landmark events indicated as Ent (entry), DS (pre-meiotic DNA replication), Rec (recombination), MI and MII (first and second meiotic division), and Spo (spore formation). The normal final product is depicted as an ascus with four spores (green). Compounds that inhibit sporulation or that are cytotoxic in sporulating yeast cells will suppress the expression of the sporulation-specific gene <i>CDA2</i> and therefore also <i>CDA2</i> promotor-driven <i>GFP</i> expression. (B) Real-time measurement of fluorescence intensities in cells harboring the <i>GFP</i>-reporter, which were sporulated in the presence of varying concentrations of ammonium sulfate (AS) as indicated. Fluorescence of the sporulation culture was measured every 15 min over the course of 20 hours (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042853#s4" target="_blank">Methods</a>). (C) Schematic of the post-recombination growth assay. The two defective alleles (<i>his4x</i> and <i>his4B</i>) can give rise to a functional <i>HIS4</i> allele upon meiotic recombination. The histidine-auxotrophic diploid mother cells can therefore produce histidine-prototrophic spores (indicated in blue). If this event is suppressed by a compound, either because it directly inhibits meiotic recombination or because it is cytotoxic to sporulating yeast cells, no histidine-prototrophs are formed. (D) Proof-of-concept of the <i>his4x</i>/<i>his4B</i> assay. Cells were sporulated for 5 hours in the presence of varying concentrations of ammonium sulfate (AS) as indicated. Aliquots of cells were then transferred to agar plates that lack histidine (-his) or leucine (-leu) and incubated for two days at 30°C, prior to measuring colony density (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042853#s4" target="_blank">Methods</a>) of each spot on the -his agar. Agar plates lacking leucine served as a loading control. The barplot in the upper panel shows mean and standard deviation data from 4 independent experiments.</p
Tripelennamine strongly inhibits viability and meiotic M-phase in sporulating yeast.
<p>(A) Images of Nomarski microscopy of cells sporulated in the absence of drug (control), in the presence of 2 mM ammonium sulfate (AS), or 100 µM tripelennamine (TA) for 24 hours. Part of the TA image was magnified; arrows indicate granular bodies of unknown origin. (B) 5-fold serial dilution of yeast sporulated for 24 hours in the presence of three different concentrations of TA (indicated on the right) and then transferred to rich media in order to determine the rate of cell survival. Pictures were taken after incubating the rich media agar plates for 2 days at 30°C. (C) FACS analysis of pre-meiotic DNA synthesis of a “no drug” control, and in cells treated with ammonium sulfate (2 mM), or tripelennamine (100 µM). Samples were taken at 2, 3, 4, 6, and 8 hours after induction of sporulation. Staggered histograms show the frequencies (plotted on the y-axis) of relative DNA content, measured as propidium iodide intensity (plotted on the x-axis). (D) Percentage of cells that have completed meiotic M-phase (cells with 2 DAPI foci and cells with 3 or 4 DAPI foci) and those that have formed mature asci over time in sporulation medium (given in hours, x-axis) in the absence (control, circles and solid lines), or the presence of 100 µM tripelennamine (treatment, squares and dashed lines). (E) Expression patterns of representative genes involved in meiotic development (left column), nitrogen catabolite repression (<i>GAT1</i> and <i>GAP1</i>), glycolysis/gluconeogenesis (<i>CDC19</i> and <i>PGK1</i>), and stress response (<i>AZR1</i>). Log2-transformed fluorescence signals of RMA-normalized microarray data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042853#s4" target="_blank">Methods</a>) are plotted on the y-axis and are graphed versus samples taken in rich media and pre-sporulation media (in the absence of tripelennamine), or total time (4 and 8 hours) the cultures spent in sporulation media in the absence (black curve) or presence (red curve) of TA.</p
A generic HTS assay for kinase screening: Validation for the isolation of an engineered malate kinase
<div><p>An end-point ADP/NAD<sup>+</sup> acid/alkali assay procedure, directly applicable to library screening of any type of ATP-utilising/ADP producing enzyme activity, was implemented. Typically, ADP production is coupled to NAD<sup>+</sup> co-enzyme formation by the conventional addition of pyruvate kinase and lactate dehydrogenase. Transformation of enzymatically generated NAD<sup>+</sup> into a photometrically active alkali derivative product is then achieved through the successive application of acidic/alkali treatment steps. The assay was successfully miniaturized to search for malate kinase activity in a structurally-guided library of LysC aspartate kinase variants comprising 6,700 clones. The screening procedure enabled the isolation of nine positive variants showing novel kinase activity on (L)-malate, the best mutant, LysC V115A:E119S:E434V exhibited strong substrate selectivity for (L)-malate compared to (L)-aspartate with a (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>)<sub>malate</sub>/(<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>)<sub>aspartate</sub> ratio of 86. Double mutants V115A:E119S, V115A:E119C and E119S:E434V were constructed to further probe the origins of stabilising substrate binding energy gains for (L)-malate due to mutation. The introduction of less sterically hindering side-chains in engineered enzymes carrying E119S and V115A mutations increases the effective volume available for substrate binding in the catalytic pocket. Improved binding of the (L)-malate substrate may be assisted by less hindered movement of the Phe184 aromatic side-chain. Additional favourable long-range electostatic effects on binding arising from the E434V surface mutation are conditionally dependent upon the presence of the V115A mutation close to Phe184 in the active-site.</p></div
Calculated (L)-malate electrostatic binding interaction energies in the V115A:E119S and V115A:E119S:E434V Ec-LysC mutant ternary complexes with Mg-ADP.
<p>Calculated (L)-malate electrostatic binding interaction energies in the V115A:E119S and V115A:E119S:E434V Ec-LysC mutant ternary complexes with Mg-ADP.</p
Utilisation of additional (L)-malate binding energy gained by LysC mutation in stabilising enzyme-substrate and enzyme transition-state complexes.
<p>Utilisation of additional (L)-malate binding energy gained by LysC mutation in stabilising enzyme-substrate and enzyme transition-state complexes.</p
Principle of the end-point alkali assay method for the high-throughput screening of kinase activity.
<p>Principle of the end-point alkali assay method for the high-throughput screening of kinase activity.</p
Enzyme binding site interactions in a modelled complex of the LysC E119S:V115A double mutant with (L)-malate and Mg-ADP.
<p>A network of direct and water-mediated interactions between (L)-malate and enzyme residues and Mg-ADP is depicted as dashed line orange vectors connecting donor and acceptor heavy-atom co-ordinate positions. The Mg<sup>2+</sup> ion is shown as an ochre-coloured space filling sphere, and water molecules mediating substrate binding and metal ion co-ordination interactions as cyan-coloured spheres. Atoms in (thick) stick representations of (L)-malate, ADP and labelled mutant enzyme side-chains are coloured according to element type: carbon, grey; nitrogen, blue; oxygen, red; and phosphorus, orange. The oxygen atom of the 2-OH hydroxyl group of (L)-malate that replaces the charged α-NH3 group in (L)-aspartate is indicated. Carbon atoms in (thin) stick side-chains representations in an overlay of the X-ray structure of the R-state <i>holo</i> complex of the wild-type enzyme with (L)-aspartate and Mg-ADP (PDB code 2j0w) are shown in green. For comparison an alternative E119 side-chain conformation observed in the inactive T-state <i>apo</i> form of the wild type enzyme (PDB code 2j0x), re-constructed on the 2j0w backbone, is depicted in (thin) stick representation with yellow-coloured carbon atoms.</p