<i>Candida albicans</i> strains used in this study.

<p><i>Candida albicans</i> strains used in this study.</p

Number of initial hits identified in the PCL screen.

<p>Number of initial hits identified in the PCL screen.</p

Secondary microbiologic and virulence assays further eliminate false-positive hits.

<p>To further eliminate false-positive hits, the effects of benzbromarone (BENZ, at a concentration of 18.4 µM) on wild-type <i>C. albicans</i> were tested and the results were compared to the phenotypic characteristics of the <i>sur7</i>Δ null mutant strain. In most cases, in the presence of benzbromarone, wild-type strains SC5314 and DAY185 mimicked the phenotypic profile of the <i>sur7</i>Δ mutant strain. Only results for SC5314 are shown in each panel. (A) The defects in secretion of degradative lipases, as indicated by the lack of the zone of precipitation surrounding the colony grown on Tween 80 agar, and in the inability to form mature hyphae on filamentation-inducing media (M199 agar and RPMI-1640) suggest that benzbromarone may act to reduce levels of Sur7p. (B) The ability of benzbromarone to inhibit biofilm formation of wild-type <i>C. albicans</i> was also tested and compared to the defective biofilm formed by the <i>sur7</i>Δ null mutant. Since metabolic activity can vary between strains of <i>C. albicans</i>, the resulting biofilm mass was measured using the crystal violet staining method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110354#pone.0110354-Ramage2" target="_blank">[49]</a>. When grown in the presence of benzbromarone, biofilm formation was reduced compared to wild-type growth. However, the reduction in biofilm formation of the benzbromarone-treated cells was not comparable to the biofilm formed by the <i>sur7</i>Δ strain. Statistical significance is indicated with asterisks, with p<0.0001. (C) The ability to kill macrophages by <i>C. albicans</i> was assessed by co-incubation of wild-type yeast cells with murine macrophage cells, in the presence or absence of benzbromarone. Virulent, wild-type <i>C. albicans</i> kill a significant proportion of macrophage cells when co-incubated for a period of 24 hours. When benzbromarone is present, a significant number of macrophage cells survive the co-incubation with wild-type <i>C. albicans</i>, similar to the <i>sur7</i>Δ null mutant strain. Statistical significance is indicated with an asterisk, with p<0.0001. (D) Cell wall morphology of the yeast form was characterized by staining yeast cells grown in the absence or presence of benzbromarone. Loss of function of <i>SUR7</i> results in plasma membrane invaginations and distinct cell wall-derived structures within the cell, which stain with calcofluor white as highlighted with arrows <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110354#pone.0110354-Bernardo1" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110354#pone.0110354-Alvarez1" target="_blank">[31]</a>. At the concentration of benzbromarone tested, the distribution and staining of cell wall material in wild-type cells grown in the presence of benzbromarone was indistinguishable from wild-type grown cells.</p

List of PCL compounds confirmed in dose-response assays.

<p>*The arrow indicates whether a compound elicited an increase (inducer) or decrease (inhibitor) in expression levels, as measured by fold-response of the GFP-tagged protein.</p><p>EC₅₀ (in µM) values are reported for each respective compound confirmed as a hit, as determined in subsequent dose-response assays.</p><p>“n.d.” signifies that the hit was not confirmed in dose-response assays and therefore, no EC₅₀ values have been determined.</p><p>List of PCL compounds confirmed in dose-response assays.</p

Changes in levels of GFP-tagged protein are readily detected.

<p>(A) Cells were grown in varying concentrations of fluconazole (FLU) and the response was measured using the HyperCyt system. Compared to the untagged control strain, a significant increase in fluorescence levels was detected for CDR1-GFP. Similarly, we queried the response of the FTR1-GFP strain to changes in iron content of the growth medium (B). These results demonstrate that we can detect changes to protein levels of our target proteins. Error bars indicate standard deviation values of the gated population of cells sampled and not of replicates of biological samples.</p

Measured green fluorescence (median channel fluorescence, MCF) of each strain used in the Primary Screen.

<p>*Calculated with untagged control (DAY185) as the potential positive control for Inhibitor screens.</p><p>Measured green fluorescence (median channel fluorescence, MCF) of each strain used in the Primary Screen.</p

Novel Activities of Select NSAID R-Enantiomers against Rac1 and Cdc42 GTPases

<div><p>Rho family GTPases (including Rac, Rho and Cdc42) collectively control cell proliferation, adhesion and migration and are of interest as functional therapeutic targets in numerous epithelial cancers. Based on high throughput screening of the Prestwick Chemical Library^® and cheminformatics we identified the R-enantiomers of two approved drugs (naproxen and ketorolac) as inhibitors of Rac1 and Cdc42. The corresponding S-enantiomers are considered the active component in racemic drug formulations, acting as non-steroidal anti-inflammatory drugs (NSAIDs) with selective activity against cyclooxygenases. Here, we show that the S-enantiomers of naproxen and ketorolac are inactive against the GTPases. Additionally, more than twenty other NSAIDs lacked inhibitory action against the GTPases, establishing the selectivity of the two identified NSAIDs. R-naproxen was first identified as a lead compound and tested in parallel with its S-enantiomer and the non-chiral 6-methoxy-naphthalene acetic acid (active metabolite of nabumetone, another NSAID) as a structural series. Cheminformatics-based substructure analyses—using the rotationally constrained carboxylate in R-naproxen—led to identification of racemic [R/S] ketorolac as a suitable FDA-approved candidate. Cell based measurement of GTPase activity (in animal and human cell lines) demonstrated that the R-enantiomers specifically inhibit epidermal growth factor stimulated Rac1 and Cdc42 activation. The GTPase inhibitory effects of the R-enantiomers in cells largely mimic those of established Rac1 (NSC23766) and Cdc42 (CID2950007/ML141) specific inhibitors. Docking predicts that rotational constraints position the carboxylate moieties of the R-enantiomers to preferentially coordinate the magnesium ion, thereby destabilizing nucleotide binding to Rac1 and Cdc42. The S-enantiomers can be docked but are less favorably positioned in proximity to the magnesium. R-naproxen and R-ketorolac have potential for rapid translation and efficacy in the treatment of several epithelial cancer types on account of established human toxicity profiles and novel activities against Rho-family GTPases.</p></div

R-naproxen inhibits migration of immortalized human ovarian cancer cells.

<p>Cell migration was measured using a Boyden chamber assay. (<b>A-B</b>) Ovarian cancer (OvCa429 and OvCa433) cells were plated on filters and treated with 0–300 μM R-naproxen, S-naproxen, 6-MNA or the Rac1 inhibitor NSC23766 and then allowed to migrate for 48 h. Migrated cells were photographed and counted in three independent fields per chamber. N = 3 One-way ANOVA and Tukey's multiple comparison test shows values significantly (**P<0.01, ***P<0.001) different from untreated controls.</p

Compound docking to Cdc42 based on DOCK9 magnesium exclusion model.

<p>The docking model is based on the work of Yang et al., 2009 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142182#pone.0142182.ref077" target="_blank">77</a>] wherein Val1951 in the sensing domain of the DOCK9 GEF (dark green ribbon) was suggested to reduce nucleotide interaction with Mg²⁺ via steric hinderance or ‘exclusion’ and thereby destabilize nucleotide binding to cause release from the GTPase active site. <b>(A-B)</b> The crystal structure of Cdc42-GDP in complex with the DOCK9 GEF (PDB ID 2WMN) was used to predict the active site docking of (<b>C-D</b>) 6-MNA, (<b>E-F</b>) R-, S-naproxen and (<b>G-H</b>) R-, S-ketorolac. (<b>B, D, F, H</b>) The carboxyl moiety in all compounds is proposed to interact with the Mg²⁺, thereby reducing interaction with GDP and reducing binding affinity analogous to Val1951 (teal). R-naproxen and R-ketorolac are shown in red. 6-MNA rust, S-naproxen and S-ketorolac are shown in green. R-enantiomers show more favorable interaction with Mg²⁺ than S-enantiomers due to rotational constraints imposed on the carboxylate by the stereocenter. For quantification of free energy of ligand binding and distances see Table G in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142182#pone.0142182.s001" target="_blank">S1 File</a>.</p

R-naproxen inhibits Rac and Cdc42 activation in response to growth factor stimulus of cells.

<p>(<b>A-B</b>) GLISA effector binding assays were used to quantify Rac1 and Cdc42 GTPase activities in Swiss 3T3 fibroblast cell lysates following preincubation for varying times (15 min or 1 h) or with varying concentrations of R-naproxen, S-naproxen or 6MNA, with or without (+/-) 100 ng/ml EGF stimulation. In panel A, R-naproxen and 6-MNA were used at 300 μM and time of exposure varied as indicated. One-way ANOVA and Dunnett’s multiple comparison test shows select R-Naproxen samples significantly (*p<0.05, ***p<0.001) different from EGF stimulated controls. In panel B, drug doses ranged from 10–300 μM as indicated. CID2950007 is a selective inhibitor of Cdc42 and served as a positive control. N = 3–8. One-way ANOVA and Dunnett’s multiple comparison test did not identify significant differences relative to EGF-stimulated controls. (<b>C-D</b>) Flow cytometric G-trap assay was used to quantitatively assess dose dependent inhibition of Rac1 and Cdc42 activation in HeLa cells following 2 h pre-treatment with R-naproxen, S-naproxen or 6MNA (30–1000 μM) and 2 min EGF stimulation (100 ng/ml). The inhibition curves were fitted to the sigmoidal dose-response equation in GraphPad Prism 5. Quantification of three independent measurements are plotted ± SEM.</p