Activity of compounds 13 and 33 against <i>C. albicans</i> biofilms <i>in vitro</i>.

<p>(A) Six-hour <i>in vitro C. albicans</i> biofilms were incubated overnight at 30°C in the presence of compound 13 or 33 (1.56–50 µg/ml) or fluconazole (62.5–500 µg/ml) as a control. After drug exposure, <i>C. albicans</i> biofilm viability was assessed using XTT and growth reduction was determined relative to medium control biofilm. An unpaired t-test revealed that <i>C. albicans</i> biofilm was significantly more sensitive to compounds 13 (†) and 33 (‡) than to fluconazole (1000 µg/ml) (p = <0.05). The data are the mean ± SD of three samples per treatment group, and are representative of two independent experiments. (B) Synergistic activity of compounds with fluconazole in <i>C. albicans</i> biofilm <i>in vitro</i>. Six-hour <i>in vitro C. albicans</i> biofilms were incubated overnight at 30°C with compound 13 (1.6 µg/ml) or 33 (6.3 µg/ml) and fluconazole (1000 µg/ml) alone and in combination. After drug exposure, the XTT assay was used to assess growth reduction of the drug-treated wells relative to media control. The data are representative of a trend of synergy observed over a range of concentrations of compounds 13 and 33 (0.4–25 µg/ml) with fluconazole (62.5–1000 µg/ml), and are displayed as the mean ± SD of two independent experiments.</p

Spectrum of activity of compounds 13 and 33 against pathogenic fungi.^*

*<p>Broth microdilution quantification of compound MIC (µg/ml) against yeast and filamentous fungal pathogens. The number of fungal strain isolates tested is in parentheses. The values are representative of at least two independent experiments.</p>+<p>NT = not tested.</p

Hik1 <i>Saccharomyces cerevisiae</i> small molecule screen results.

†<p>18,432 compounds from Maybridge HitFinder Collection; 1,150 compounds from NIH Clinical Collection; 880 compounds from Prestwick Chemical Library.</p

Microarray analysis of <i>C. albicans</i> exposed to compounds 13 and 33.

<p>Changes in transcript expression of oxidative (A) and heavy metal (B) stress response gene clusters during a time course of <i>C. albicans</i> exposed to compounds 13 or 33. The fluorescence intensity (i.e. transcript abundance) relative to the given transcript value at time 0 is shown for <i>C. albicans</i> exposed to compound for 20, 40 and 60 minutes.</p

Compounds 13 and 33 exert synergy with fluconazole against <i>in vitro Candida albicans</i> biofilm^*.

*<p>FIC index = [EC25(A in combination)]/[EC25(A alone)]+[EC25(B in combination)]/[EC25(B alone)], where A is compound 13 or 33 and B is fluconazole. An index of <0.5 indicates synergism. The results are averages ± SD of two independent experiments.</p

Small molecule structures.

<p>The chemical names of compounds 13 and 33 are 2-nitro-5-(4-nitroStyryl)furan (MW = 260.20) and 2-(methylthio)-5-[(5-nitro-1,3-thiazol-2-yl)thio]-1,3,4-thiadiazole (MW = 292.39) respectively.</p

Hik1 <i>Saccharomyces</i> bioassay small molecule screen schematic.

<p>The small molecule screen was performed in three steps. (I) The Hik1-expressing <i>Saccharomyces</i> strain was seeded into 96-well plates containing 10 µM of small molecules. Media and fludioxonil containing wells served as negative and positive controls respectively. Hit compounds were defined as molecules that inhibited growth by at least 50% after overnight incubation at 30°C (black circle). (II) Hit compounds were assayed against parental and Hik1-expressing <i>Saccharomyces</i> to identify only molecules with Hik1-dependent activity. Compounds that inhibited the growth of both strains were discarded (black crosses). Molecules that inhibited Hik1-Saccharomyces growth by at least 50%, but did not hinder growth of the parental strain by more than 10% were considered hits (black circle). (III) Activity against the fungal pathogens <i>C. albicans</i> and <i>A. fumigatus</i> was assessed by disc diffusion. Top agar was seeded with yeast or spores, compound containing discs were then placed on the solidified agar, and the plates were incubated overnight at 37°C. Compounds that generated zones of inhibition (white circle) against both organisms were subjected to further analysis.</p

Toxicity of compounds 13 and 33.

<p>The hemolytic activity of compounds 13, 33, and amphotericin B was measured (OD405 nm) by the release of hemoglobin from red blood cells after a one-hour exposure to compound at 37°C. 0.1% Triton X-100 was used as a control. The data are represented as the mean ± SD of three samples per treatment group from three independent experiments.</p

Compounds 13 and 33 are not Group III HHK-dependent in <i>Aspergillus nidulans</i>^*.

*<p>Broth microdilution quantification of compound MIC (µg/ml) against parental and ΔNikA strains of <i>A. nidulans</i>. The values are representative of three independent experiments.</p

Pretreatment with heavy metal stress increases the sensitivity of <i>Candida albicans</i> to compound 33.

<p><i>C. albicans</i> cultures were incubated overnight at 30°C in the presence of cadmium concentrations shown. After exposure to this heavy metal, <i>C. albicans</i> was exposed to compound 33 at concentrations shown and incubated at 30°C overnight. Growth was assessed using the XTT assay and growth reduction was measured relative to the medium control. The data are the mean ± SD of at least three samples per treatment group and are representative of three independent experiments. There was a significant interaction for the two conditions (F[12, 60] = 2.59, p = 0.008; ANOVA test) indicating that pre-exposure of <i>C. albicans</i> to heavy metal increased sensitivity to compound 33.</p