Recovery of <i>M. smegmatis</i> after Vortexing and Filtration.

<p>Mean ±Standard Deviation; n = 4.</p

Comparison of unfiltered and filtered <i>M. smegmatis</i>.

<p>Unfiltered <i>M. smegmatis</i> under 40x magnification (A) and plated onto agar (B); <i>M. smegmatis</i> filtered through 5-µm pore filter under 40 x magnification (C) and plated onto agar (D), scale bar applies to both A and C.</p

Histogram of unfiltered, vortexed and filtered <i>M. smegmatis</i>.

<p>Distributions of resorufin fluorescence signals from 384 wells of a 384-well plate contained unfiltered (A), vortexed (B) or filtered (C) <i>M. smegmatis</i>. After the treatment, the bacteria were distributed into the 384-well plates followed by the addition of resazurin, which was converted to resorufin by the living bacteria.</p

Agreement analysis of duplicate plates from unfiltered, vortexed and filtered bacteria.

<p>Correlation of two duplicate assay plates tested against LOPAC compounds using unfiltered bacteria (A), vortexed bacteria (B) and filtered bacteria (C).</p

Supplemental_Material_for_Hull-Ryde_et_al – Supplemental material for Identification of Cosalane as an Inhibitor of Human and Murine CC–Chemokine Receptor 7 Signaling via a High-Throughput Screen

<p>Supplemental material, Supplemental_Material_for_Hull-Ryde_et_al for Identification of Cosalane as an Inhibitor of Human and Murine CC–Chemokine Receptor 7 Signaling via a High-Throughput Screen by Emily A. Hull-Ryde, Melissa A. Porter, Kenneth A. Fowler, Dmitri Kireev, Kelin Li, Catherine D. Simpson, Maria F. Sassano, Mark J. Suto, Kenneth H. Pearce, William Janzen and James M. Coghill in SLAS Discovery</p

Identification of Small Molecule Inhibitors That Block the <i>Toxoplasma gondii</i> Rhoptry Kinase ROP18

The protozoan parasite <i>Toxoplasma gondii</i> secretes a family of serine-threonine protein kinases into its host cell in order to disrupt signaling and alter immune responses. One prominent secretory effector is the rhoptry protein 18 (ROP18), a serine-threonine kinase that phosphorylates immunity related GTPases (IRGs) and hence blocks interferon gamma-mediated responses in rodent cells. Previous genetic studies show that ROP18 is a major virulence component of <i>T. gondii</i> strains from North and South America. Here, we implemented a high throughput screen to identify small molecule inhibitors of ROP18 <i>in vitro</i> and subsequently validated their specificity within infected cells. Although ROP18 was not susceptible to many kinase-directed inhibitors that affect mammalian kinases, the screen identified several sub-micromolar inhibitors that belong to three chemical scaffolds: oxindoles, 6-azaquinazolines, and pyrazolopyridines. Treatment of interferon γ-activated cells with one of these inhibitors enhanced immunity related GTPase recruitment to wild type parasites, recapitulating the defect of Δ<i>rop18</i> mutant parasites, consistent with targeting ROP18 within infected cells. These compounds provide useful starting points for chemical biology experiments or as leads for therapeutic interventions designed to reduce parasite virulence