Cardio-green butyl acetate targets ergosterol to kill Candida albicans

Figures

• 

  Figure 1. Synthesis and structural confirmation of CaGA (A) Two-step synthetic route showing conversion of CaG into its butyl acetate-functionalized derivative (CaGA). Step 1: formation of bromo-butyl acetate intermediate; Step 2: substitution reaction yielding CaGA.

• 

  Figure 2. Antifungal activity of CaGA against C. albicans (A) CFU assay reveals a dose-dependent inhibition of C. albicans, where complete inhibition was observed at 16 μg/mL of CaGA. (B) Intracellular leakage of C. albicans with 4 μg/mL of CaGA for 0, 5, 10, 20, 30, and 60 min. AmB was used as a positive control. (C) SEM micrograph of untreated and treated C. albicans with 4 μg/mL of CaGA. (D) Nile red staining assay of C. albicans at 4 μg/mL of CaGA. Untreated (PBS) C. albicans was used as a negative control. All the experiments were performed three times independently in triplicate, while statistical significance was considered at the level of p < 0.05. Error bars represent standard deviation (SD).

• 

  Figure 3. Biofilm eradication potential of CaGA (A) Crystal violet assay shows efficient eradication of C. albicans biofilms in comparison to the AmB. C. albicans treated with PBS 1% Triton X-100 were used as negative and positive controls, respectively. (B) Fluorescence microscopic image of live/dead biofilm eradication assay of untreated biofilms (upper panel), and treated biofilms (lower panel) for 1 h at 4 μg/mL of CaGA (i) sytox green, (ii) propidium iodide, and (iii) merged. Error bars represent SD, while statistical significance is considered at the level of p < 0.05. All the experiments were performed three times independently in triplicate.

• 

  Figure 4. Biophysical characterization of CaGA-ergosterol interaction (A) UV-visible absorption spectra of CaGA in the presence of increasing ergosterol concentrations showing a decrease and red shift of the ergosterol peak at 282 nm, consistent with complex formation. (B) ITC profile shows an exothermic binding event (ΔH = −3.02 × 103 cal/mol·K, K = 3.2 × 104 M−1). The thermodynamic parameters indicate a spontaneous, enthalpy-driven interaction involving hydrophobic and hydrogen-bonding forces between CaGA and ergosterol.

• 

  Figure 5. Comparative MD simulation of AmB and CaGA interactions with ergosterol-rich membranes (A) Snapshot shows AmB (magenta) inserting deeply into the ergosterol-rich bilayer (ergosterol in orange), forming a transmembrane channel-like arrangement. (B) Snapshot shows CaGA (cyan) remaining near the membrane surface while engaging ergosterol via hydrogen bonding and hydrophobic contacts, indicating a surface-associated, non-pore-forming mode of action. (C) RC profiles from steered MD simulations illustrate the relative depth of membrane penetration over time for AmB and CaGA. (D) Corresponding force profiles show that AmB requires a higher pulling force to cross the bilayer, reflecting stronger lipid disruption, whereas CaGA maintains lower energy perturbations consistent with membrane thinning rather than pore formation. (E) Time evolution of hydrogen bonds, demonstrating transient but frequent interactions for CaGA compared with AmB.

• 

  Figure 6. Representative membranes showing CaGA and AmB embedded in the C. albicans bilayer (A) Molecular system of AmB is inserted above the outer membrane (OT) region. (B) Molecular system of CaGA near the OT region. The ergosterols are shown in orange spheres, and other lipids are indicated as lines. The phosphate head is shown in red spheres, and the water molecules are represented as transparent surfaces in gray color. For SMD, the compounds were steered from the outer membrane (OT) to the inner membrane (IN) region. The starting (C) and final (D) conformations of CaGA while translocating the cell membrane of C. albicans.

• 

  Figure 7. Initial and final conformations of AmB and CaGA in C. albicans during SMD (A) The starting and (B) final conformations of AmB while translocating the C. albicans membrane. (C) The starting and (D) final conformations of CaGA while translocating the C. albicans membrane.