Rottlerin

Bryostatin-1 and prostratin were purchased from BioMol Research Laboratories, Inc. (PA, USA). SAHA was purchased from Cayman chemicals (MI, USA). GF109203X (Bisindolylmaleimide-1), rottlerin and H7 dihydrochloride were purchased from Calbiochem (CA, USA). Compound-C and 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) were purchased from Toronto Research Chemicals, Inc. (ON, Canada). All other chemicals were purchased from Sigma Chemical Co. (MO, USA). The stock solutions of rottlerin (10 mM), H7 dihydrochloride and bryostatin (25 ng/ml) were prepared in dimethylsulfoxide (DMSO), and GF109203X (5 mM) was prepared in 50% DMSO in distilled water. AICAR (100 mM), metformin (100 mM), minocycline (10 mM), AZT (10 mM) and sodium salicylate (100 mM) were prepared in PBS followed by filter sterilization and aliquoted for one time use and stored at −20°C until used. Human anti-CD4, CXCR4, CD25, CD69 antibodies were purchased from BD-Biosciences (CA, USA). Anti-PKC isotype, phospho- and total-AMPK antibodies were purchased from Cell Signaling (MA, USA), anti-β actin antibody was purchased from Sigma, anti-rabbit-HRP and anti-mouse-HRP conjugate antibodies were purchased from Bio-Rad (CA, USA).

The in vivo toxicity and infection assessment tests were carried out using the mutant strain of Caenorhabditis elegans AU37, according to Breger et al.^{59 (link)}, with some modifications, as described below. The Rottlerin sample was evaluated at concentrations of 3.90 to 1000 μg/mL. DMSO was used as solvent (final concentration ≤ 1%). The mutant strain of C. elegans AU37 was grown on Nematode Growth Medium (NGM) plates seeded with Escherichia coli OP50 and incubated at 16 °C for 3 days. After incubation, the supernatant was washed with the bleaching solution (sodium hypochlorite + NaOH) to synchronize the larvae at the L4 stage. Plates containing larvae synchronized in the L4 phase were washed with M9 buffer and the supernatant was placed in 15-mL conical tubes. Subsequently, 20 µL of the larval suspension was added to each well of a 96-well flat-bottom microplate, along with 80 µL of Brain Heart Infusion (BHI) medium + antibiotics (200 mg/mL Streptomycin, 200 mg/mL ampicillin and 90 μg/mL kanamycin) and 100 µL of Rottlerin, and incubated for 24 h at 25 °C. Counting of live and dead larvae was performed on an EVOS M5000 Imaging System Microscope (Thermo Fisher Scientific, Massachusetts, USA) and the percentage of mortality was calculated.
In vivo infection tests were performed with the same yeasts selected for antibiofilm activity assays (C. albicans—ATCC 90028, C. dubliniensis—ATCC MYA-646 and C. auris—clinical isolate). For so, after the larvae synchronization procedure, 100 µL of L4 stage larvae was pipetted into NGM plates containing an inoculum of the evaluated yeasts and incubated for three hours at 25 °C. Subsequently, the already infected larvae were washed with M9 buffer and transferred to 15-mL falcon tubes, being centrifuged three times to remove excess yeast that may be adhered to the worm cup. Larvae were then added into wells of 96-well plates containing 60% M9 buffer, 40% BHI broth, 10 μg/mL cholesterol in ethanol, 90 μg/mL kanamycin and 200 mg/mL ampicillin. The larvae were divided into three groups: uninfected and untreated larvae, infected and untreated larvae and infected larvae treated with Rottlerin or Amphotericin B (used as control at concentrations of 1 to 16 µg/mL). The plates were incubated for 2 days at 25 °C and the mortality rate was calculated daily. On the first day of infection, worms were stained with SYTOX Green (Invitrogen, CA, USA) at a concentration of 1 μM and were incubated for 15 min at room temperature. Images were captured by EVOS M5000 Imaging System Microscope (Thermo Fisher Scientific, Massachusetts, USA).
Descriptive statistical analyses of numerical variables consisted of sample size, missing observations, arithmetic mean, median, standard deviation, 95% confidence interval of the mean and minimum and maximum values. To compare the survival percentage averages between treatments, concentrations and yeasts, the analysis of variance technique was used with the effect estimated by the partial eta squared statistics. To compare performance over the days, analysis of variance with repeated measures was used with the effect calculated by the partial eta squared statistics. Post-hoc comparisons for significant effects were performed using Tukey’s test with the effect estimated using Cohen’s D statistics. The significance level adopted was 0.05. The computational package used for statistical analyses was JASP version 0.17.3 for MacOS.

NC114 was synthesized as described in our previous study [14 (link)]. Rottlerin was purchased from Sigma‐Aldrich, and CIAP was obtained from Takara Bio Inc. (Shiga, Japan).