Observation of J/ψpJ/\psi p Resonances Consistent with Pentaquark States

<p>Transiently transfected HeLa cells containing a total of 100 ng cDNA / 0.125 ml of hWT V2R or the L83Q mutant were treated with different pharmacoperones (10 μM or #50 was 1 μM) to show if the rescued L83Q mutant is coupled to Gs (cAMP). The cells were incubated with the pharmacoperones for 16 h, then washed and stimulated with 1 μM vasopressin containing 0.2 mM IBMX for 30 minutes and the cAMP response was measured. DMSO (vehicle) is a negative control that does not rescue the mutant and SR121463B is a known pharmacoperone for the L83Q mutant. The results shown in the figure are from at least 3 independent experiments performed in quadruplicate (n = 3 ± SEM).</p

IP production following transient transfection of HeLa cells for selected GPCRs and V2R mutants.

<p>Transiently transfected HeLa cells containing a total of 100 ng cDNA/ 0.125 ml of hWT V2R or the mutant hL83Q were used to compare Gq (IP) coupling with the vasopressin receptor, endogenous muscarinic M3 subtype and histamine H1 receptors with or without 10 μM SR121463B pharmacoperone (rescues the L83Q mutant), DMSO (vehicle) is a negative control. Cells were stimulated with various doses of vasopressin for 2 h, acetylcholine and histamine dihydrochloride for 30 minutes. The results shown in the figure are from at least 3 independent experiments performed in quadruplicate (n = 3 ± SEM), p value < 0.05 is considered significant.</p

IP production from HeLa cells stably expressing WT V2R, mutant L83Q or tTA.

<p>Stably transfected HeLa cells containing tTA + hWTV2R or tTA + L83Q mutant were used to compare Gq (IP) coupling (a) with the vasopressin receptor with or without 10 μM SR121463B pharmacoperone (rescues the L83Q mutant), DMSO (vehicle) is a negative control. (b) Stably transfected HeLa cells with the WT V2R or tTA alone, were used to show if the oxytocin, V1a or V1b receptors are being stimulated with vasopressin to produce IP. Cells were stimulated with various doses of vasopressin for 2 h because the HeLa cells produce low amounts of total IP with the addition of 5 mM LiCl. The results shown in the figure are from at least 3 independent experiments performed in quadruplicate (n = 3 ± SEM), p value < 0.05 is considered significant.</p

Constitutive or stimulated activity of V2R mutants with or without rescue.

<p>Transiently transfected HeLa cells containing a total of 100ng cDNA / 0.125 ml of hWT V2R, L83Q mutant and empty vector were used to assess if there is constitutive activity (CA) with the IP pathway, 9a, or with the cAMP pathway, 9b. Figure 9c shows that Y128S, is coupled to Gq/11 in the presence of agonist (1 μM vasopressin for 2 hours), but does not show constitutive activity (no agonist). Figure 9d shows constitutive activity of Y128S for the cAMP pathway. Cells were stimulated for 30 minutes with 1 μM vasopressin. The results shown in the figure are from at least 3 independent experiments performed in quadruplicate (n = 3 ± SEM), p values < 0.05 is considered significant.</p

Pharmacoperones used in this study, showing that compounds in many different structural classes rescue the mutant L83Q.

<p>The 14 compounds shown have been grouped into 7 structure classes.</p

DS_775055 – Supplemental material for A Cytotoxic Three-Dimensional-Spheroid, High-Throughput Assay Using Patient-Derived Glioma Stem Cells

<p>Supplemental material, DS_775055 for A Cytotoxic Three-Dimensional-Spheroid, High-Throughput Assay Using Patient-Derived Glioma Stem Cells by Victor Quereda, Shurong Hou, Franck Madoux, Louis Scampavia, Timothy P. Spicer and Derek Duckett in SLAS Discovery</p

Map of the human arginine-vasopressin 2 receptor showing mutants associated with nephrogenic diabetes insipidus in black.

<p>The location of L83Q and Y128S, the mutants used in the present study, are shown. The amino acid sequence of the naturally-occurring ligand, vasopressin, is shown at the top. When there are multiple mutations at a single site, the number of mutations is noted in a triangle. The mutants in this study are a blue circle so they stand out. Adapted and modified from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181830#pone.0181830.ref036" target="_blank">36</a>].</p

First-in-Class Inhibitors of Sulfur Metabolism with Bactericidal Activity against Non-Replicating <i>M. tuberculosis</i>

Development of effective therapies to eradicate persistent, slowly replicating <i>M. tuberculosis</i> (<i>Mtb</i>) represents a significant challenge to controlling the global TB epidemic. To develop such therapies, it is imperative to translate information from metabolome and proteome adaptations of persistent <i>Mtb</i> into the drug discovery screening platforms. To this end, reductive sulfur metabolism is genetically and pharmacologically implicated in survival, pathogenesis, and redox homeostasis of persistent <i>Mtb</i>. Therefore, inhibitors of this pathway are expected to serve as powerful tools in its preclinical and clinical validation as a therapeutic target for eradicating persisters. Here, we establish a first functional HTS platform for identification of APS reductase (APSR) inhibitors, a critical enzyme in the assimilation of sulfate for the biosynthesis of cysteine and other essential sulfur-containing molecules. Our HTS campaign involving 38 350 compounds led to the discovery of three distinct structural classes of APSR inhibitors. A class of bioactive compounds with known pharmacology displayed potent bactericidal activity in wild-type <i>Mtb</i> as well as MDR and XDR clinical isolates. Top compounds showed markedly diminished potency in a conditional ΔAPSR mutant, which could be restored by complementation with <i>Mtb</i> APSR. Furthermore, ITC studies on representative compounds provided evidence for direct engagement of the APSR target. Finally, potent APSR inhibitors significantly decreased the cellular levels of key reduced sulfur-containing metabolites and also induced an oxidative shift in mycothiol redox potential of live <i>Mtb</i>, thus providing functional validation of our screening data. In summary, we have identified first-in-class inhibitors of APSR that can serve as molecular probes in unraveling the links between <i>Mtb</i> persistence, antibiotic tolerance, and sulfate assimilation, in addition to their potential therapeutic value

Five compounds significantly decrease MAPK1 phosphorylation in K562 cells.

<p>DMSO or compound was added to K562 cells at a final concentration of 1% or 10 μM, respectively, and incubated for 4 hours at 37°C before being lysed and analyzed by immunoblot with anti-pMAPK1/3 and anti-MAPK1/3. Each compound was tested with six biological replicates. Band intensity was quantified using LI-COR Odyssey, and the ratio of pMAPK/MAPK signal intensity is graphed for each compound and its DMSO control. Compounds are identified by their PubChem CID number.</p

Identification of Small Molecules that Disrupt Signaling between ABL and Its Positive Regulator RIN1

<div><p>Constitutively active BCR-ABL kinase fusions are causative mutations in the pathogenesis of hematopoietic neoplasias including chronic myelogenous leukemia (CML). Although these fusions have been successfully targeted with kinase inhibitors, drug-resistance and relapse continue to limit long-term survival, highlighting the need for continued innovative drug discovery. We developed a time-resolved Förster resonance energy transfer (TR-FRET) -based assay to identify compounds that disrupt stimulation of the ABL kinase by blocking its ability to bind the positive regulator RIN1. This assay was used in a high throughput screen (HTS) of two small molecule libraries totaling 444,743 compounds. 708 confirmed hits were counter-screened to eliminate off-target inhibitors and reanalyzed to prioritize compounds with IC₅₀ values below 10 μM. The CML cell line K562 was then used to identify five compounds that decrease MAPK1/3 phosphorylation, which we determined to be an indicator of RIN1-dependent ABL signaling. One of these compounds is a thiadiazole, and the other four are structurally related acyl piperidine amides. Notably, these five compounds lower cellular BCR-ABL1 kinase activity by blocking a positive regulatory interaction rather than directly inhibiting ABL catalytic function.</p></div