Data_Sheet_1.docx

<p>Zinc is an essential micronutrient, required for a range of zinc-dependent enzymes and transcription factors. In mammalian cells, zinc serves as a second messenger molecule. However, a role for zinc in signaling has not yet been established in the fungal kingdom. Here, we used the intracellular zinc reporter, zinbo-5, which allowed visualization of zinc in the endoplasmic reticulum and other components of the internal membrane system in Candida albicans. We provide evidence for a link between cyclic AMP/PKA- and zinc-signaling in this major human fungal pathogen. Glucose stimulation, which triggers a cyclic AMP spike in this fungus resulted in rapid intracellular zinc mobilization and this “zinc flux” could be stimulated with phosphodiesterase inhibitors and blocked via inhibition of adenylate cyclase or PKA. A similar mobilization of intracellular zinc was generated by stimulation of cells with extracellular zinc and this effect could be reversed with the chelator EDTA. However, zinc-induced zinc flux was found to be cyclic AMP independent. In summary, we show that activation of the cyclic AMP/PKA pathway triggers intracellular zinc mobilization in a fungus. To our knowledge, this is the first described link between cyclic AMP signaling and zinc homeostasis in a human fungal pathogen.</p

High-Resolution Screening Combined with HPLC-HRMS-SPE-NMR for Identification of Fungal Plasma Membrane H⁺‑ATPase Inhibitors from Plants

Crude extracts of 33 plant species were assessed for fungal plasma membrane (PM) H⁺-ATPase inhibition. This led to identification of 18 extracts showing more than 95% inhibition at a concentration of 7.5 mg/mL and/or a concentration-dependent activity profile. These extracts were selected for semi-high-resolution fungal PM H⁺-ATPase inhibition screening, and, on the basis of these results, Haplocoelum foliolosum (Hiern) Bullock and Sauvagesia erecta L. were selected for investigation by high-resolution fungal PM H⁺-ATPase inhibition screening. Structural analysis performed by high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HPLC-HRMS-SPE-NMR) led to identification of chebulagic acid (<b>1</b>) and tellimagrandin II (<b>2</b>) from H. foliolosum. Preparative-scale isolation of the two metabolites allowed determination of IC₅₀ values for PM H⁺-ATPase, and growth inhibition of Saccharomyces cerevisiae and Candida albicans. Chebulagic acid and tellimagrandin II are both potent inhibitors of the PM H⁺-ATPase with inhibitory effect on the growth of S. cerevisiae

IC₅₀ values of compounds 4–12 against ATP hydrolysis activity of fungal and mammalian P-type ATPases.

<p><i>n</i> = 3.</p

Docking poses of compounds 4–12.

<p>Oxygen, nitrogen, bromine, chlorine and fluorine atoms in red, blue, dark red, green, and light blue, respectively. A) Docking (green) and experimental (yellow) binding mode of <b>7</b> to SERCA. SERCA is shown as cartoon with carbon in blue, and amino acids D59, D254 and N101 are shown as sticks. Compound <b>7</b> is shown as sticks with carbon in yellow. B-F) Docking of <b>4</b>–<b>12</b> into the <i>C</i>. <i>albicans</i> Pma1 homology model. The Pma1 model is shown as cartoon with carbon in gray, and amino acids Q101, N130 and N267 are shown as sticks. B) Docking of <b>7</b> (S)(green) and <b>7</b> (R) (orange) C) Docking of <b>4</b> (magenta), <b>5</b> (yellow), <b>6</b> (blue), <b>7</b> (green), <b>8</b> (pink) (all S). D) Docking of <b>9</b> (S) (magenta), and <b>10</b> (S) (yellow). E) Docking of <b>11</b> (S)(yellow), <b>12</b> (S) (blue), and <b>12</b> (R) (orange). F) Docking of <b>9</b> (brown), <b>10</b> (cyan), and <b>11</b> (yellow)(all R).</p

ATP hydrolysis and growth inhibition by library hit compounds.

<p>MIC is defined as 50% growth inhibition, <i>n</i> = 3.</p

Chemical structure of the tetrahydrocarbazole scaffold (top), three initial hits from the library screening (1–3) and nine rationally designed tetrahydrocarbazole analogues used in this study (4–12).

<p>The chiral center is indicated by an asterisk.</p

Fungal growth inhibition by compounds 4–12.

<p>MIC is defined as 50% growth inhibition, <i>n</i> = 3–6. FLC: fluconazole; VRC: voriconazole; AMB: amphotericin B.</p

Antifungal activity of tetrahydrocarbazoles against <i>C</i>. <i>albicans</i>.

<p>A) Time-kill analysis and B) Post-antifungal effect of tetrahydrocarbazoles: ● = DMSO-treated control cells, ○ = cells treated with AMB (0.54 μM), * = cells treated with VRC (2.8 μM), □ = cells treated with <b>6</b> (20 μM) and ◊ = cells treated with <b>8</b> (20 μM). Values in A) and B) are the mean (±SEM) for three independent experiments. Samples withdrawn at the indicated times in A) and B) were evaluated for CFU.</p

The binding pocket of compound 7.

<p>A) Compound <b>7</b> with polar and hydrophobic interactions and <i>m</i>F_o-<i>D</i>F_c difference electron density map before the ligand was modeled (green, contoured at 3 σ) and anomalous difference map (orange, contoured at 4.5 σ, calculated using phases of the final refined model and anomalous difference data from 56–6 Å resolution). B) Slightly rotated view of the ligand binding site, revealing the position of the functionally important residue E309 in SERCA. C) Close-up view of the mostly uncharged surface of the ligand-binding site and its extension towards the P-domain (asterisk). Carbon atoms are colored blue in SERCA and yellow in <b>7</b>, oxygen red, nitrogen blue, bromine dark red, and fluorine in pale cyan.</p

Crystal structure of the SERCA•7•TNPATP complex.

<p>A) Cartoon representation of the SERCA•<b>7</b>•TNPATP complex. Domains and ligand-binding sites are indicated. Compound <b>7</b> (carbon in yellow, oxygen in red, bromine in dark red, fluorine in pale cyan, nitrogen in blue) and TNPATP (carbon in marine, oxygen in red, phosphorous in orange, nitrogen in blue) are shown in stick representation. B) Sliced surface representation revealing the position of the ligand-binding pocket at the cytosolic membrane interface (TNPATP omitted for clarity, colors as in A).</p