Identification of the 4-AQ molecules with anti-virulence activity against C. albicans.a The chemical structures of HHQ (2-heptyl-4-hydroxyquinoline) and selected 4-AQ molecules differing in a group bound to the side chain (in blue) and their anti-virulence activities on P. aeruginosa PAO1. b Heatmap illustrating the hierarchical clustering of 4-AQs, HHQ and clinical antifungals, classified using Euclidean distance and Ward’s method into three distinct groups based on their similarity in inhibitory activity against three Candida strains (average of two independent experiments) and toxicity profiles in zebrafish embryos (n = 20 per concentration, tested in triplicate). c The normally developing zebrafish embryos without the signs of toxicity after 5-day exposure to 4-AQ derivatives 11 (Group II), and 6 and 24 (Group III) are shown. In contrast, the embryos treated with 2 µM nystatin (Group I) and 5 µM HHQ were severely damaged and suffered from liver necrosis (dashed line outlining the dark liver), impaired yolk uptake (asterisk), life-threatening pericardial edema (arrow), jaw deformation (arrowhead) and head deformation (bracket). d Heatmap showing the four groups of molecules within the 4-AQs series that differ in their filamentation inhibitory activity (the number of media in which filament formation was completely blocked). After fungal cells were exposed to 10 µM of each molecule for 4-6 days and colony morphology was examined microscopically, each molecule was scored binary for its effect on filament development (presence or absence of filaments) and hierarchically clustered using Ward’s clustering method with Euclidean distance. e The representative images showing the morphology of the fungal colonies after the 3-day treatment with a dose of 10 µM of the selected 4-AQ derivatives (6, 11 and 24). Robust filaments and/or wrinkled colonies were developed in control (0.02% DMSO) treatment, while active molecules inhibited the formation of filaments and/or caused smooth colonies. Hierarchical clustering was performed using the Euclidian distance and Ward’s method.

The 4-AQ anti-virulence compounds have a synergistic interaction with nystatin in the suppression of Candida albicans filamentation.Inhibition of filamentation of C. albicans SC5314 in combined treatment with nystatin and 4-AQ molecules (6, 11 and 24) compared to the individual agents, evaluated in RPMI medium (a, b) and Spider medium (c, d). Color-based heat maps (a, c) show the categorized filamentation phenotypes after the 3-day treatments, assessed by the morphology of the fungal colonies (filament density and length, b, d) in comparison to the control (DMSO) as follows: no effect (very dense and long filaments developed; black boxes), weak inhibitory effect (lower filamentation density with long filaments; dark gray boxes), moderate inhibitory effect (short filaments developed; light gray boxes), good inhibitory effect (rare filaments developed; light green boxes), and complete inhibition (no filaments developed; green boxes).

Anti-biofilm activity of the 4-AQ molecules 6, 11 and 24.The molecules were administered at different doses and examined for the ability to a inhibit biofilm formation (at 6.25–50 µM) and b disintegrate 24-hour-old biofilm (25-100 µM) of C. albicans SC5314. Values are the average of a representative experiment performed in quadruplicate ± SD. Statistically significant differences between the treated groups and the control (0.1% DMSO) were determined using one-way ANOVA and the Bonferroni test (n.s. P > 0.5, *** P < 0.001). c Confocal microscopy images of the mixed biofilm of C. albicans (RFP-expressing, M137 strain) and P. aeruginosa PAO1 (GFP-expressing) formed in the presence of 0.1% DMSO (control) or 50 µM of 11 showed that the applied.

Hematotoxicity and liver toxicity of the most potent 4-AQ molecules 6, 11, and 24 compared to HHQ and miconazole.a The hemolytic activity tested on sheep red blood cells was compared with that of HHQ and miconazole, a clinical antifungal drug used as a positive control. b The hemolysis rate was determined in relation to the hemolysis of erythrocytes induced by 1% Triton X, which was arbitrarily set to 100%. Values are average of two independent experiment performed in triplicates ± SD. Statistically significant differences between the treated groups and control (0.1% DMSO) were determined using one-way ANOVA (***P < 0.001). c Hepatotoxicity was evaluated in transgenic Tg(-2,8fabp10:EGFP) zebrafish embryos with fluorescently labeled liver (n = 8) using the liver area index. d The morphology of the transgenic zebrafish embryos and their liver fluorescence after different treatments is shown. One-way ANOVA with the Bonferroni test was used to determine statistical significance by comparing fluorescence signal of the control sample with each treated group (*** P ≤ 0.001).

Combination treatment with nystatin and 11 or 24 is more effective than individual treatments in the zebrafish model of disseminated candidiasis.a Filamentation of C. albicans M137 expressing RFP at 24 h post-treatment (hpt). b Proportion of embryos with/without visible hyphae in the hindbrain at 24 hpt, assessed by fluorescence microscopy (n = 20 embryos in each group). c Dose-dependent effect of nystatin, 4-AQ (11 and 24) and their combinations on the progress of C. albicans infection. Each data point represents the integrated red fluorescence intensity of a single alive zebrafish embryo at 24 hpt, and the signal in each group (8 embryos) is expressed as mean ± SD. Statistical significance was determined by χ2 test by comparing the distribution of embryos with/without hyphae in the control (DMSO-treated) and treated groups (*** P ≤ 0.001) as well as in the groups receiving nystatin and combination treatments (## P ≤ 0.01). Also, one-way ANOVA with the Bonferroni test was used to determine statistical significance by comparing fluorescence signal of the control (DMSO-treated) sample with each treated group (*** P ≤ 0.001), as well as between the nystatin-treated and the corresponding combination-treated group (## P ≤ 0.01). d the survival of the infected embryos treated with nystatin, 4-AQ and their combination compared to DMSO-treated embryos (control), as represented by Kaplan-Meier curve. Statistically significant differences in the survival between the treated and untreated groups (## P ≤ 0.01), as well as between the nystatin-treated groups with the corresponding combination treatment groups (## P ≤ 0.01) were determined using a log rank (Mantel-Cox) test.