_D(KLAKLAK)₂ has fungicidal activity against Mucorales.

<p>(<b>A</b>) Micrographs of <i>R. oryzae</i> and <i>M. circinelloides</i> spores after six hours of exposure to AMB (4 µg/ml), FLU (128 µg/ml), _D(KLAKLAK)₂ (150 µg/ml, 300 µg/ml) or a negative control peptidomimetic, _D(CVRAC)₂ (300 µg/ml), showing that germination was completely inhibited by _D(KLAKLAK)₂ at MIC (300 µg/ml)and by AMB (4 µg/ml). Scale bar, 200 µm. (<b>B</b> and <b>C</b>) Mycelia were incubated with AMB (4 µg/ml), FLU (128 µg/ml), _D(KLAKLAK)₂ (150 µg/ml, 300 µg/ml) or _D(CVRAC)₂ (300 µg/ml). The extent of hyphal damage was monitored over time by the XTT reduction assay, which indicated _D(KLAKLAK)₂-induced dose-dependent killing relative to control drugs (*p ≤ 0.0001). (<b>D</b> and <b>E</b>) Spores were exposed to AMB (4 µg/ml), FLU (128 µg/ml), _D(KLAKLAK)₂ (150 µg/ml, 300 µg/ml) or the negative control peptidomimetic (300 µg/ml) for one hour. _D(KLAKLAK)₂ created a post-antifungal effect demonstrated by a shift of the growth curve to the right compared to the control drugs. The lag period was increased by approximately three hours (**p ≤ 0.001), followed by a rapid recovery.</p

_D(KLAKLAK)₂ causes Mucorales apoptosis.

<p>(<b>A</b>) Relative quantification indicated significantly higher levels (**p ≤ 0.001) of cytochrome <i>c</i> release from mitochondria of <i>R. oryzae</i> and <i>M. circinelloides</i> into the cytosol in the presence of _D(KLAKLAK)₂ (150 µg/ml) relative to FLU (128 µg/ml) or the negative control peptidomimetic (300 µg/ml). (<b>B</b>) Fluorescence and DIC micrographs of <i>R. oryzae</i> and <i>M. circinelloides</i> germlings stained with the CaspACE FITC-VAD-FMK In Situ Marker, indicating metacaspase activation upon incubation with _D(KLAKLAK)₂ (150 µg/ml) compared to FLU (128 µg/ml) or the negative control peptidomimetic (300 µg/ml). Scale bar, 200 µm. (<b>C</b>) Fluorescence quantitative analysis of <i>R. oryzae</i> and <i>M. circinelloides</i> germlings in the presence of drugs revealed significant levels of metacaspase activation in germlings treated with _D(KLAKLAK)₂ (**p ≤ 0.001).</p

Exposure to _D(KLAKLAK)₂ triggers intracellular ROS accumulation.

<p>(<b>A</b>) Fluorescence and DIC micrographs of <i>R. oryzae</i> and <i>M. circinelloides</i> germlings stained with the oxidation-sensitive dye DHR-123, showing ROS production after incubation with _D(KLAKLAK)₂ (150 µg/ml) or AMB (2 µg/ml). Scale bar, 200 µm. (<b>B</b>) Quantitative analysis recorded with a microplate reader (excitation, 488 nm; emission, 525 nm), demonstrating a significant increase in ROS release from mitochondria in the presence of _D(KLAKLAK)₂ (**p ≤ 0.001) relative to FLU (128 µg/ml) or negative control peptidomimetic (300 µg/ml).</p

_D(KLAKLAK)₂ induces ultrastructural cellular changes in <i>R. oryzae</i>.

<p>TEM of germlings (6,000-fold) incubated with _D(KLAKLAK)₂ (150 µg/ml, 300 µg/ml) revealed extensive vacuolization comparable to that induced by AMB (2 µg/ml). The increase in number and size of vacuoles was visualized with the vacuolar probe FM4-64 (red). staining of intact plasma membrane FM4-64 diffusion into the cytoplasm and staining of fragmented vacuoles in treated _D(KLAKLAK)₂ (150 µg/ml, 300 µg/ml) and AMB (2 µg/ml). MitoTracker staining (green) indicated morphological changes and swelling of mitochondria in _D(KLAKLAK)₂-exposed germlings as opposed to the punctate pattern observed in samples treated with control drugs. TEM scale bar, 2 µm. Fluorescent micrographs scale bar, 200 µm.</p

_D(KLAKLAK)₂ induces mitochondrial membrane depolarization (ΔΨ_m).

<p>(<b>A</b>) Fluorescence and DIC micrographs of <i>R. oryzae</i> and <i>M. circinelloides</i> germlings stained with the oxidation-sensitive dye RH-123, indicating membrane depolarization after incubation with _D(KLAKLAK)₂ (150 µg/ml) or AMB (2 µg/ml). Scale bar, 200 µm. (<b>B</b>) Quantitative analysis recorded with a microplate reader (excitation, 488 nm; emission, 525 nm), demonstrating significant membrane depolarization levels (**p ≤ 0.001), post-exposure to _D(KLAKLAK)₂ compared to FLU (128 µg/ml) or the negative control peptidomimetic (300 µg/ml).</p

_D(KLAKLAK)₂ alters plasma membrane homeostasis.

<p>(<b>A</b>) Fluorescence micrographs of <i>R. oryzae</i> and M. circinelloides mycelia post-exposure to AMB (2 µg/ml), FLU (128 µg/ml), _D(KLAKLAK)₂ (150 µg/ml, 300 µg/ml) or _D(CVRAC)₂ (300 µg/ml). The DiBAC bright green fluorescence is indicative of a loss of viability due increased membrane permeability. Differential interference contrast (DIC) images served as controls for the presence of germlings. Scale bar, 200 µm. (<b>B</b> and <b>C</b>) The dose-dependent cellular ATP release in the medium after <i>R. oryzae</i> and M. circinelloides hyphae treatment with _D(KLAKLAK)₂ (150 µg/ml, 300 µg/ml) was similar to that induced by colistin (32 µg/ml) (***p ≤ 0.0002).</p