Impairing the interaction between Erg11 and cytochrome P450 reductase Ncp1 enhances azoles’ antifungal activities

Li, Wanqian; Whiteway, Malcolm; Hang, Sijin; Yu, Jinhua; Lu, Hui; Jiang, Yuanying

doi:10.1038/s41467-025-62131-z

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Published: 24 July 2025

Impairing the interaction between Erg11 and cytochrome P450 reductase Ncp1 enhances azoles’ antifungal activities

Nature Communications volume 16, Article number: 6821 (2025) Cite this article

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Abstract

Azole effectiveness against candidiasis can be compromised by Candida albicans resistance and tolerance, and unfortunately, few clinically useful compounds can enhance azole antifungal activities. We find that the amino acids V234, F235 and L238 of Erg11 are critical for its interaction with Ncp1, and the Ncp1-Erg11 association is important in azole response. Ellipticine and its analog phiKan 083 block this Erg11-Ncp1 interaction by targeting Ncp1, and boost antifungal effects of fluconazole in vitro and in vivo. A series of steps influencing this process—an initial elevation in reactive oxygen species, leading to protein oxidation and misfolding in the endoplasmic reticulum (ER) that causes ER stress. This stress leads to Ca²⁺ release from the ER, mitochondrial Ca²⁺ accumulation and dysfunction, increased ROS production, and apoptosis of C. albicans cells. Overall, disrupting the Erg11-Ncp1 interaction in C. albicans can serve as a useful approach to enhancing the antifungal properties of azoles.

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Introduction

Candida albicans, a common fungus that can cause serious infections, may lead to fatal disseminated candidemia in patients with weakened immune systems^1,2. Azoles, which inhibit lanosterol 14-α-demethylase (Erg11), are key antifungal agents for treating and preventing candidiasis^3,4. However, long-term use can lead to C. albicans resistance, raising the minimum inhibitory concentration (MIC) of azoles needed to inhibit growth compared to susceptible strains⁵. As well, azoles may not effectively treat infections from non-resistant C. albicans due to the organism’s tolerance, which is linked to the fungistatic effect of azoles⁶. This characteristic allows tolerant strains to grow beyond the MIC⁷, leading to reduced azole efficacy against candidiasis, increased resistance, and persistent candidemia^3,8,9. Therefore, the development of resistance and tolerance to azoles presents a significant clinical challenge in treating candidiasis.

Combination therapy represents a promising strategy for augmenting the antifungal activities of azoles¹⁰. However, translating in vitro efficacy enhancements to clinical use faces challenges^6,11. Some compounds show target protein conservation, limited fungal selectivity, and human toxicity. Calcium ion (Ca²⁺) homeostasis maintains C. albicans azole tolerance, and calcineurin inhibition by cyclosporin A could enhance azole effects, but cyclosporin A’s immune suppression limits its use^6,11. Poor pharmacokinetic parameters of azole adjuvants also pose issues. For example, CZ66 enhances azole efficacy by inhibiting Erg251 but has a short half-life in mice¹². Overcoming these challenges requires exploring different mechanisms to improve azole antifungal efficacy.

Erg11, a cytochrome P450 enzyme, catalyzes the 14-methylation of lanosterol in C. albicans through three cycles^13,14, each requiring two electrons from nicotinamide adenine dinucleotide phosphate (NADPH)-dependent Ncp1 reductase^15,16. This electron transfer forms a high-affinity complex between the proteins¹⁷. In coupled P450 systems, electrons from NADPH are fully used for substrate hydroxylation, but leakage can produce reactive oxygen species (ROS). The ER produces ROS, oxidizing proteins like thioredoxins and causing ER stress¹⁸. Tunicamycin enhances C. albicans’ susceptibility to FLC by inhibiting protein N-glycosylation and inducing ER stress³. Carvacrol also boosts FLC’s anti-C. albicans activity via ER stress¹⁹. In response, C. albicans activates the unfolded protein response (UPR) pathway, involving the protein kinase Ire1 and the transcription factor Hac1²⁰. Deleting the IRE1 gene in Saccharomyces cerevisiae or C. albicans increases sensitivity to FLC and impedes ergosterol synthesis gene expression, respectively^20,21. Removing the HAC1 gene enhances C. albicans’ susceptibility to azole drugs²². Thus, inhibiting the Erg11-Ncp1 interaction may suppress Erg11, elevate ER-derived ROS, augment ER stress, and enhance azole efficacy, but further research is needed to elucidate the mechanism.

This study investigated the role of amino acids V234, F235, and L238 of Erg11 in the Erg11-Ncp1 interaction. Disrupting this interaction increases C. albicans’ susceptibility to azoles by promoting misfolded protein production, leading to ER stress, Ca²⁺ buildup in mitochondria, dysfunction, and apoptosis. Ellipticine, targeting Ncp1, inhibits the Erg11-Ncp1 interaction, hyphal growth, and enhances antifungal activity of FLC in vivo. This study shows that impairing the Erg11-Ncp1 interaction can be a useful strategy for enhancing the antifungal activity of azoles.

Results

The Erg11_200-251 region and the Ncp1_1-63 region are important for the interaction between Erg11 and Ncp1

Previous studies have provided evidence of the physical interaction between Erg11 and Ncp1 in both C. albicans and S. cerevisiae^23,24. However, the specific mechanism of this interaction remains elusive. To investigate this, we employed a membrane yeast two-hybrid (MYTH) system. In this system, Erg11 was fused with the C-terminal half of ubiquitin (Cub) and LexA-VP16 (an artificial transcription factor), while Ncp1 was fused with the mutated N-terminal half of ubiquitin (NubG). The interaction between Erg11 and Ncp1 resulted in the proximity of Cub and NubG, thereby facilitating the reconstitution of split-ubiquitin and the activation of two auxotrophic genes, HIS3 and ADE2, allowing the yeast’s survival on a medium devoid of histidine and adenine (Fig. 1a)²⁵. To prevent self-activation in the MYTH system, it is necessary to add the HIS3 gene product inhibitor 3-aminotriazole (3-AT) to the SD-Leu-Trp-His-Ade medium. Spotting assays demonstrated that co-expression of Erg11 and Ncp1 allowed yeast to grow on the selective medium, providing evidence for the interaction between Erg11 and Ncp1 (Fig. 1b and Supplementary Fig. 1a). To further validate this interaction, fusion proteins Erg11-13Myc and 3HA-Ncp1, were generated with a 13×Myc tag at the C-terminal of Erg11 (Supplementary Fig. 1b, d) and a 3×HA tag at the N-terminal of Ncp1 (Supplementary Fig. 1c, d). Co-immunoprecipitation (co-IP) assays demonstrated that Erg11 was immunoprecipitated using an anti-Myc antibody, and the co-IP of Ncp1 was detected through an anti-HA antibody. Reciprocally, when Ncp1 was immunoprecipitated employing an anti-HA antibody, the presence of Erg11 was detected using an anti-Myc antibody (Fig. 1c).

**Fig. 1: The Erg11_200-251 region and the Ncp1_1-63 region are important for the Erg11-Ncp1 interaction.**

It has been proposed that the interaction between Erg11 and Ncp1 occurs via the electrostatic interaction between the C-terminal heme region of Erg11 and the N-terminal flavin mononucleotide (FMN) region of Ncp1^26,27. Thus, we utilized the MYTH system to examine the interaction between Ncp1 and a series of C-terminal truncated fragments of Erg11, including Erg11_1-490, Erg11_1-458, Erg11_1-428, Erg11_1-378, Erg11_1-293, Erg11_1-279, Erg11_1-251, and Erg11_1-200. Although the interaction between the full length of Erg11 and Ncp1 gives the strongest signal, most of the C-terminal truncated fragments of Erg11 are still capable of interacting with Ncp1. However, the fragment Erg11_1-200 is unable to interact with Ncp1 (Fig. 1d and Supplementary Fig. 1e), suggesting the importance of the Erg11_200-251 region in facilitating the interaction between Erg11 and Ncp1. We further employed a C. albicans mutant in which the N-terminus of Ncp1 was tagged with 3×HA, and subsequently truncated Erg11 from the C-terminus and labeled it with 13×Myc, generating two combinations, Erg11_1-251-13Myc/3HA-Ncp1 and Erg11_1-200-13Myc/3HA-Ncp1 (Supplementary Fig. 1d). Co-IP findings revealed that the Erg11_1-251 fragment exhibited an interaction with Ncp1, whereas the Erg11_1-200 fragment did not (Fig. 1e). This supports the significance of the Erg11_200-251 region in the Erg11-Ncp1 interaction.

Similarly, we employed the MYTH system to assess the interaction between Erg11 and various N-terminal truncated fragments of Ncp1, including Ncp1_63-680, Ncp1_91-680, Ncp1_134-680, and Ncp1_171-680. Our findings demonstrate that only full-length Ncp1 exhibits binding affinity towards Erg11, while all N-terminal truncated fragments of Ncp1 fail to interact with Erg11 (Fig. 1f). We further tagged the C-terminus of Erg11 with 13×Myc, and truncated Ncp1 from the N-terminus and tagged it with 3×HA, creating two mutants, 3HA-Ncp1_63-680 and 3HA-Ncp1_91-680 (Supplementary Fig. 1d). Co-IP results indicated that neither the Ncp1_63-680 fragment nor the Ncp1_91-680 fragment exhibited any interaction with Erg11 (Fig. 1g). In addition, we also truncated the C-terminus of Ncp1 and tagged it with 3HA, generating three mutants, Ncp1-3HA, Ncp1_1-600-3HA and Ncp1_1-500-3HA (Supplementary Fig. 1d). The Co-IP results establish that both the Ncp1_1-600 fragment and the Ncp1_1-500 fragment interacted with Erg11 (Supplementary Fig. 1f); full-length Ncp1 is not required. These findings demonstrated that the Ncp1_1-63 region plays a key role in the Erg11-Ncp1 interaction.

Amino acid residues V234, F235, and L238 in Erg11 contribute to the Erg11-Ncp1 interaction

We further identify the key amino acid residues in the Erg11_200-251 region for the Erg11-Ncp1 interaction using molecular dynamics simulations. After 50 ns of kinetic simulation, the Erg11-Ncp1 complex system reached an equilibrium state with a root mean square deviation value of about 8.5 Å (Supplementary Fig. 2a). The binding free energy (ΔG_bind) of this complex system was −127.21 kcal/mol. To explore key residues at the interface of this interaction, we modeled the contribution of each residue to the binding energy (Supplementary Table 1). Amino acid residues with lower ΔG_bind are considered more critical in the Erg11-Ncp1 interaction. The three residues with the lowest ΔG_bind and largest energy contributions in the Erg11_200-251 region were V234, F235 and L238, with ΔG_bind of −0.85 kcal/mol, −1.95 kcal/mol, and −2.04 kcal/mol, respectively (Fig. 2a). Therefore, we infer that these residues in the Erg11_200-251 region play a key role in mediating this interaction.

To validate these predictions, we synthesized the ERG11 gene de novo, substituting the amino acid residues V234, F235, and L238 with alanine residues A234, A235, and A238, respectively. The co-expression of the mutated Erg11^{V234A, F235A, L238A} (Erg11^m) and Ncp11 showed no association in the MYTH system, indicating the need for these amino acid residues in the Erg11-Ncp1 interaction (Fig. 2b and Supplementary Fig. 2b). We further synthesized a nucleic acid sequence of Erg11^m-13Myc and recombined it at the ADE2 gene locus in the 3HA-Ncp1 mutant, generating the Erg11^m-13Myc/3HA-Ncp1 mutant. The co-IP results indicated that the interaction of Erg11^m with Ncp1 was lost (Fig. 2c, d). We also built the single mutant constructs Erg11^V234A-13Myc/3HA-Ncp1, Erg11^F235A-13Myc/3HA-Ncp1, and Erg11^L238A-13Myc/3HA-Ncp1; the co-IP results showed that all individual amino acid mutations did not affect the interaction between Erg11 and Ncp1 (Supplementary Fig. 2c–f). We additionally selected and mutated three amino acid residues (D214, D223, and D225) within the Erg11_200-251 region to alanine, based on minimal involvement in the Erg11-Ncp1 interaction suggested by molecular docking results. Subsequently, the nucleic acid sequence of Erg11^{D214A, D223A, D225A} (Erg11^m’)-13Myc was integrated into the ADE2 gene locus of the 3HA-Ncp1 mutant, yielding the Erg11^m’-13Myc/3HA-Ncp1 mutant. Co-IP experiments confirmed that the interaction between Erg11^m’ and Ncp1 remained intact (Supplementary Fig. 2g, h). Overall, amino acid residues V234, F235, and L238 in Erg11 play a pivotal role in facilitating the Erg11-Ncp1 interaction.

Impairing the Erg11-Ncp1 interaction increases the susceptibility of C. albicans to azoles

To evaluate the importance of the Erg11-Ncp1 interaction for the susceptibility of C. albicans to azoles, we synthesized a nucleic acid sequence for Erg11^m and then inserted it at the ADE2 gene locus in the heterozygous deleted mutant ERG11/erg11Δ. Subsequently, the remaining allele of the ERG11 gene was eliminated, generating an erg11^m/erg11Δ, in which Erg11^m and Ncp1 are unable to interact (Fig. 3a). To determine whether mutations in the amino acid residues V234, F235, and L238 of Erg11 affect protein expression and localization, we performed western blotting and confocal microscopy. Results showed no difference in Erg11 protein expression between the wild-type (Erg11-13Myc) and mutant (Erg11^m-13Myc) strains (Supplementary Fig. 3a). Additionally, both GFP-tagged Erg11 and Erg11^m localized to the ER (Supplementary Fig. 3b).

**Fig. 3: Impairing the Erg11-Ncp1 interaction enhances the susceptibility of *C. albicans* to azoles.**

Furthermore, we have created mutants of NCP1/ncp1Δ, erg11Δ/erg11Δ and ncp1Δ/ncp1Δ (Supplementary Fig. 3c–e). In comparison to the wild-type strain SN152 and the mutants ERG11/erg11Δ, NCP1/ncp1Δ, erg11Δ/erg11Δ, and ncp1Δ/ncp1Δ, the erg11^m/erg11Δ mutant exhibited increased susceptibility to azoles such as FLC, miconazole, voriconazole, ketoconazole, and itraconazole (Fig. 3b and Supplementary Fig. 3f). Additionally, given C. albicans’ tolerance to FLC, disk diffusion assays revealed significant growth of C. albicans SN152 cells within the inhibition zone of 25 μg FLC when cultured on YPD plates and incubated at 30 °C for 48 h^3,12. FLC paper disk displayed a larger and cleaner inhibition zone on the plate spread with the erg11^m/erg11Δ mutant when compared with the wild-type strain SN152 (Fig. 3c). Furthermore, relative to these strains, the values of MIC and SMG of FLC were both lower against the erg11^m/erg11Δ mutant (Fig. 3d). Time-growth curve assays also indicated that the erg11^m/erg11Δ mutant exhibited increased susceptibility to FLC (Supplementary Fig. 3g). Notably, ergosterol effectively nullified the antifungal activity of FLC against the above strains, except for the erg11^m/erg11Δ mutant, as shown in MIC (Fig. 3e) and time-growth curve assays (Supplementary Fig. 3h). These findings suggest that unlike the depletion of ergosterol resulting from Erg11 or Ncp1 deficiency, the blockade of the Erg11-Ncp1 interaction induces additional detrimental effects beyond ergosterol depletion.

Impairing the Erg11-Ncp1 interaction increased misfolded proteins and induced ER stress

The catalytic function of Erg11 is dependent on the presence of Ncp1 to facilitate electron transfer¹⁶. The efficient exchange of electrons between these redox partners is contingent upon the formation of a specific high-affinity 1:1 complex between Erg11 and Ncp1¹⁷. In cases where the interaction between Erg11 and Ncp1 is disrupted, electrons may escape from the hydroxylation process and instead be directed towards O₂, leading to the production of ROS. We conducted RNA-seq analysis to compare the transcriptome differences between the erg11^m/erg11Δ mutant and the ERG11/erg11Δ mutant following 16 h of treatment with 4 μg/mL FLC. Pathway enrichment analysis identified notable regulatory distinctions in response to ROS, apoptotic processes, oxidative stress, and chaperone-mediated protein complex assembly between the ERG11/erg11Δ and erg11^m/erg11Δ mutants (Fig. 4a). We further employed the flow cytometer to detect ROS production with the fluorescent dye 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) and observed that ROS levels elevated 35 ± 0.5% (p < 0.0001) in the erg11^m/erg11Δ mutant compared with the ERG11/erg11Δ mutant (Fig. 4b). Additionally, we discovered that more ROS was produced in the erg11^m/erg11Δ mutant treated with 8 μg/mL menadione, an oxidative stress inducer²⁸, with an increase of 60 ± 7.8% (p < 0.001) relative to the ERG11/erg11Δ mutant (Fig. 4b). Similarly, MIC and time-growth curve assays demonstrated that the erg11^m/erg11Δ mutant exhibited greater susceptibility to menadione compared to the ERG11/erg11Δ mutant (Supplementary Fig. 4a, b). These findings suggested that blocking the Erg11-Ncp1 interaction enhances ROS generation.

**Fig. 4: Impairing the Erg11-Ncp1 interaction triggers ER stress.**

Increased ROS can oxidize proteins within the ER, resulting in misfolded proteins²⁹. Tetraphenylethene maleimide (TPE-MI) shows aggregation-induced emission, becoming fluorescent when molecules aggregate, which limits non-radiative decay. It binds to thiol groups in misfolded proteins, causing fluorescence³⁰. TPE-MI can identify many misfolded proteins because thiol exposure is linked to protein misfolding, with cysteine residues being exposed during unfolding³¹. Dithiothreitol (DTT) was selected as a positive control due to its ability to disrupt disulfide bonds in proteins, resulting in the aggregation of misfolded proteins³². The misfolded protein content in the erg11^m/erg11Δ mutant has a 29 ± 1.4% (p < 0.0001) increase compared to the ERG11/erg11Δ mutant. Additionally, the misfolded protein content increases 73 ± 5.3% (p < 0.0001) in the erg11^m/erg11Δ mutant after treatment with 4 μg/mL FLC, compared to the ERG11/erg11Δ mutant (Fig. 4c).

Elevated levels of misfolded proteins lead to ER stress, as evidenced by the increased susceptibility of the erg11^m/erg11Δ mutant to tunicamycin, an inducer of ER stress³³, compared to the ERG11/erg11Δ mutant (Supplementary Fig. 4c). The UPR pathway in C. albicans, activated by increased misfolded proteins, involves Ire1 and Hac1. Ire1, sensing ER stress, initiates a cascade by splicing Hac1 mRNA to upregulate genes related to protein folding, degradation, and ER-associated degradation³⁴. We further found that the mRNA expression levels of IRE1 and HAC1 increased to 1.9 ± 0.1 fold (p < 0.001) and 1.8 ± 0.1 fold (p < 0.001) in the ERG11^m/erg11Δ mutant compared with the ERG11/erg11Δ mutant. Under 4 μg/mL FLC treatment for 8 h, the mRNA expression levels of IRE1 and HAC1 elevated to 1.7 ± 0.2 fold (p < 0.01) and 2.2 ± 0.1 fold (p < 0.001) in the erg11^m/erg11Δ mutant compared to the ERG11/erg11Δ mutant. Disruption of the Erg11-Ncp1 interaction results in the activation of the UPR signaling pathway, akin to the effects observed with the ER stress inducer DTT (Fig. 4d).

ER stress leads to the release of Ca²⁺ from the ER and accumulation in the mitochondria

Under ER stress, Ca²⁺ in the ER may leak into the cytoplasm, potentially resulting in an increase in the availability of Ca²⁺ within the mitochondria^35,36. This suggests that the inhibition of the Erg11-Ncp1 interaction will result in the buildup of Ca²⁺ within mitochondria. Laser confocal assays revealed that there was no difference in the Ca²⁺ content within the vacuoles in both the ERG11/erg11Δ and erg11^m/erg11Δ mutants (Supplementary Fig. 5a, b). However, in comparison to the ERG11/erg11Δ mutant, the erg11^m/erg11Δ mutant cells exhibited an enhanced green fluorescence signal (representing Ca²⁺ by Fluo-3 AM) that did not overlap with the red fluorescence (indicative of the ER by ER-Tracker Red) (Fig. 5a and Supplementary Fig. 5c). Quantitative analysis the integrated green fluorescence intensity that remained green after merging with red fluorescence within individual cells and found that in the erg11^m/erg11Δ mutant, the integrated green fluorescence intensity (post-merge) was 3.7 ± 0.2 times higher than that in the ERG11/erg11Δ mutant (p < 0.0001) (Fig. 5b). This suggests that disrupting the Erg11-Ncp1 interaction leads to the leakage of Ca²⁺ into the cytoplasm.

**Fig. 5: Impairing the Erg11-Ncp1 interaction leads to the release of Ca²⁺ from the ER and accumulation in the mitochondria.**

We further used a Ca²⁺ fluorescent probe, Rhod-2 AM, which specifically accumulates in mitochondria and emits red fluorescence, to measure the concentration of Ca²⁺ within the mitochondria. Laser confocal assays demonstrated a significant increase in mitochondrial Ca²⁺ levels in the erg11^m/erg11Δ mutant compared to the ERG11/erg11Δ mutant (Fig. 5c and Supplementary Fig. 5d). The integrated fluorescence intensity in the erg11^m/erg11Δ mutant was found to be 6.9 ± 1.1 times (p < 0.01) higher than in the ERG11/erg11Δ mutant (Fig. 5d). Collectively, the induction of ER stress through inhibition of the Erg11-Ncp1 interaction leads to the release of Ca²⁺ from the ER, followed by its accumulation in mitochondria.

Mitochondrial Ca²⁺ accumulation results in mitochondrial dysfunction and apoptosis of C. albicans cells

Elevated intracellular Ca²⁺ levels can lead to mitochondrial overload, disrupting the mitochondrial membrane potential and impairing ATP synthesis. This disruption can trigger the opening of the mitochondrial permeability transition pore, leading to the release of pro-apoptotic factors and ultimately resulting in cell death³⁷, and thus, the buildup of Ca²⁺ within mitochondria serves as an important contributor to mitochondrial dysfunction. Depolarization of the mitochondrial membrane is an important characteristic of mitochondrial dysfunction³⁸. Rhodamine 123, a cationic dye, assesses mitochondrial function by accumulating in active mitochondria with a negative membrane potential. A drop in its fluorescence signifies mitochondrial dysfunction due to potential loss^39,40. The results indicated a significant decrease of 26 ± 2.7% (p < 0.001) in the mitochondrial membrane potential of the erg11^m/erg11Δ mutant, similar to the effects induced by the ER stressor DTT (Fig. 6a).

**Fig. 6: The impaired Erg11-Ncp1 interaction leads to mitochondrial dysfunction.**

Mitochondrial dysfunction leads to an increase in ROS production within the mitochondria. Mitochondrial ROS levels were quantified using MitoSOX Red, a dye that oxidizes in mitochondria and binds to nucleic acids, producing red fluorescence⁴¹. The results indicated a significant 20 ± 0.7% (p < 0.0001) increase in mitochondrial ROS levels in the erg11^m/erg11Δ mutant (Fig. 6b). EGTA is a chelator of Ca²⁺ that is widely utilized due to its capacity to form stable complexes with Ca²⁺⁴². As well, in the erg11^m/erg11Δ mutant, exposure to 200 μM EGTA led to a 21 ± 1.8% decrease in mitochondrial ROS levels (p < 0.0001) (Fig. 6b). These results indicated a correlation between elevated mitochondrial ROS levels and heightened mitochondrial Ca²⁺ content. Increased ROS caused mitochondrial oxidized proteins to be misfolded. We quantified misfolded protein mass in the mitochondrial matrix using MitoTracker Green, a dye that selectively targets this area and binds to exposed cysteine residues on misfolded proteins⁴³. The data indicated a significant increase in the content of mitochondrial misfolded proteins by 40 ± 5.9% (p < 0.001) in the erg11^m/erg11Δ mutant compared to the ERG11/erg11Δ mutant (Fig. 6c). Moreover, the erg11^m/erg11Δ mutant exhibited a reduction in mitochondrial misfolded protein mass by 15 ± 0.6% (p < 0.0001) following treatment with 200 μM EGTA, suggesting a relationship between increased mitochondrial misfolded proteins and elevated mitochondrial Ca²⁺ levels (Fig. 6c).

Mitochondrial dysfunction and increased ROS produced by mitochondria will lead to apoptosis of C. albicans cells. In the Annexin V/Propidium Iodide (PI) assay, Annexin V, a protein that binds to phosphatidylserine on apoptotic cells, is conjugated to a fluorescent marker (FITC). Cells positive for Annexin V indicate apoptosis⁴⁴. Our results indicate that inhibition of the Erg11-Ncp1 interaction resulted in enhanced apoptosis (Fig. 6d), with an apoptosis induction rate of 10.5 ± 0.3% (p < 0.0001) in the erg11^m/erg11Δ mutant compared to 3.4 ± 0.5% in the ERG11/erg11Δ mutant (Fig. 6e).

Ellipticine acts as an inhibitor of the Erg11-Ncp1 interaction by targeting Ncp1

Given that inhibiting the interaction between Erg11 and Ncp1 enhances the susceptibility of C. albicans to FLC, we employed the MYTH system to characterize potential inhibitors that could augment the antifungal effects of azoles. If a compound obstructs the Erg11-Ncp1 interaction, the reconstitution of the split ubiquitin is prevented, preventing activation of the two auxotrophic genes, HIS3 and ADE2. Furthermore, the candidate inhibitor of the Erg11-Ncp1 interaction must not exert any influence on the interaction between APP (amyloid A4 precursor protein) and Fe65 (amyloid beta A4 precursor protein-binding family B member 1), two other proteins giving a positive signal in the MYTH assay, thus ensuring the candidate compound has no general effect on the assay. The extent of yeast growth in the SD-Leu-Trp-His-Ade medium compared to that in the SD-Leu-Trp medium is indicative of the inhibition of the Erg11-Ncp1 interaction by the compound (Fig. 7a).

**Fig. 7: Ellipticine acts as an inhibitor of the Erg11-Ncp1 interaction.**

In theory, the inhibition of the Erg11-Ncp1 interaction may be achieved by the binding of a small molecule compound to either Erg11, Ncp1, or both simultaneously. We searched three categories of compounds through a literature review: (1) azoles targeting Erg11 (n = 5); (2) compounds potentially targeting Ncp1 (n = 12); (3) compounds preventing electron transfer between Ncp1 and Erg11 (n = 5) (Supplementary Table 2). Six compounds were identified as inhibitors of the Erg11-Ncp1 interaction: arachidonic acid (57 ± 3.1% inhibition), carbonyl cyanide m-chlorophenylhydrazone (75 ± 3.6% inhibition), ellipticine (57 ± 2.5% inhibition), menadione (45 ± 3.3% inhibition), piperine (37.9 ± 9.9% inhibition), and tamoxifen (68 ± 2.1% inhibition) (Fig. 7b).

Considering that inhibiting the Erg11-Ncp1 interaction leads to heightened susceptibility of C. albicans to FLC, we tested if compounds targeting the Erg11-Ncp1 interaction could augment the antifungal efficacy of FLC. The dose-matrix titration assays showed that only ellipticine can enhance the efficacy of FLC (Fig. 7c and Supplementary Fig. 6a–e). We further observed that ellipticine markedly decreased both the MIC and SMG values of FLC in FLC-resistant and -tolerant clinical strains of C. albicans (n = 13). However, in strains that were only FLC-tolerant (n = 4), ellipticine did not further reduce the MIC values, although it did decrease the SMG values, thereby lowering the tolerance level of C. albicans to FLC. Conversely, in non-FLC-resistant and -tolerant clinical C. albicans strains (n = 8), the addition of ellipticine did not result in further reductions in either MIC or SMG values for FLC. For FLC-resistant and -tolerant Nakaseomyces glabratus (formerly Candida glabrata) strains (n = 9), ellipticine significantly reduced both the MIC and SMG values of FLC. However, ellipticine did not enhance the efficacy of FLC against FLC-resistant and -tolerant clinical Candidozyma auris (formerly Candida auris) strains (n = 9) (Supplementary Table 3). Given the phylogenetic divergence between C. auris and C. albicans, which leads to substantial differences in their carbon utilization, lipid composition, and protein content⁴⁵, this divergence may account for the observed enhancement of the antifungal activity of FLC by ellipticine against C. albicans, but not against C. auris. In addition, considering that the disruption of the Erg11-Ncp1 interaction causes additional detrimental effects beyond ergosterol depletion, ellipticine, a potential inhibitor of Erg11-Ncp1 interaction, enhances the antifungal activity of FLC against the ERG11/erg11Δ mutant in the presence of ergosterol, as demonstrated through time-growth curve assays (Fig. 7d).

We further used the co-IP assay to evaluate the inhibitory effect of ellipticine on the Erg11-Ncp1 interaction. Cell lysates from the Erg11-13Myc/3HA-Ncp1 mutant were exposed to 25 μg/mL ellipticine for 1 h. The relative protein content of Ncp1 and Erg11 in the absence of drug treatment was established as 100%. Upon exposure to concentrations of ellipticine as above, the ratios of Ncp1 to Erg11 protein content decreased to 64 ± 8.3% (p < 0.05) (Fig. 7e). Taken together, this suggests ellipticine acts as an inhibitor of the Erg11-Ncp1 interaction.

The combination of FLC and ellipticine resulted in a significant increase in intracellular ROS levels of 2.6 ± 0.1 times (p < 0.0001) in the ERG11/erg11Δ mutant, as detected using the dye DCFH-DA (Fig. 7f). Additionally, this combination elevated misfolded protein levels by 2.3 ± 0.01 times (p < 0.0001) in the ER, detected using the dye TPE-MI (Fig. 7g) and in mitochondria by 20.3 ± 2.3% (p < 0.01), detected using the dye MitoTracker Green (Fig. 7h). Intracellular Ca²⁺ concentration increased by 29.0 ± 0.7% (p < 0.0001), as measured by Fluo-3AM (Fig. 7i). Concurrently, a significant decrease of 41.2 ± 8.8% (p < 0.01) in mitochondrial membrane potential was observed using Rhodamine 123 assays (Fig. 7j). The CYC3 gene encodes cytochrome c heme lyase, crucial for cytochrome c maturation, electron transport, and mitochondrial respiration. Its expression indicates mitochondrial health and functionality⁴⁶. As anticipated, the expression of the CYC3 gene exhibited a significant 1.9 ± 0.1-fold (p < 0.001) upregulation, specifically after 8 h of co-treatment with 16 μg/mL ellipticine and 4 μg/mL FLC (Fig. 7k). Subsequently, we utilized the Annexin V/PI double staining assay to demonstrate the induction of apoptosis. A notable 23.9 ± 1.1% (p < 0.0001) of cells underwent apoptosis when exposed to ellipticine in conjunction with 4 μg/mL FLC (Fig. 7l, m). In summary, ellipticine disrupts the interaction between Erg11 and Ncp1 and induces ER stress and mitochondrial dysfunction.

We used drug affinity responsive target stability (DARTS) assays to reveal the inhibitory mechanism of ellipticine on the Erg11-Ncp1 interaction. The theory of the method is that a protein becomes stable upon binding to a small molecule compound, contributing to the protection of the target protein from degradation by proteases⁴⁷. The mutant Erg11-13Myc/3HA-Ncp1 was employed, and its lysates were digested with different concentrations of pronases. It was discovered that Ncp1 was progressively degraded in the absence of ellipticine. When no pronase was added, the relative protein content was 100 ± 1%. Nevertheless, without ellipticine protection, it was reduced to 78.0 ± 4.6% (p < 0.01) and 62.0 ± 7.0% (p < 0.0001) in the presence of 8 μg/mL and 16 μg/mL pronases, respectively (Fig. 7n). However, we did not observe this protective function of ellipticine on Erg11 (Supplementary Fig. 6f, g). Hence, ellipticine appears to act as an inhibitor of the Erg11-Ncp1 interaction by targeting Ncp1.

Ellipticine and its analog phiKan 083 enhance the antifungal activity of FLC in vivo

The therapeutic efficacy of ellipticine against the wild-type C. albicans strain SN152, was tested in Galleria mellonella infections. The larvae were randomly assigned to one of four groups: (1) control group receiving PBS treatment, (2) group receiving 1 mg/kg FLC alone, (3) group receiving 4 mg/kg ellipticine alone, and (4) group receiving a combination of 1 mg/kg FLC and 4 mg/kg ellipticine. Treatment was administered 2 h post-infection and was administered as a single dose. The administration of 4 mg/kg ellipticine significantly enhanced the antifungal effect of FLC (1 mg/kg) over a 15-day experimental period (Fig. 8a). The survival rate of the ellipticine-FLC combination group was notably higher compared to the other three groups (p = 0.0003, compared to the control group; p = 0.0021, compared to the FLC alone group; p < 0.0001, compared to the ellipticine-treated group; assessed using the log-rank test). These in vivo data suggest that ellipticine can synergize with FLC to create potential antifungal effects.

The CMP1 gene encodes the catalytic subunit of calcineurin, crucial for FLC tolerance in C. albicans. Deletion of the C-terminal autoinhibitory domain of Cmp1 results in a constitutively active calcineurin complex^48,49,50. Consequently, the cmp1-aidΔ/cmp1-aidΔ mutant, with the C-terminal 110 residues of Cmp1 deleted, exhibits high FLC tolerance⁵¹. In addition, we validated that cmp1-aidΔ/cmp1-aidΔ mutant did not cause the ROS generation or the elevation of misfolded proteins and Ca²⁺ content compared with wild-type SN152 (Supplementary Fig. 7a–c). We further constructed Galleria mellonella infection model used the FLC-tolerant cmp1-aidΔ/cmp1-aidΔ strain to evaluate the therapeutic efficacy of ellipticine. The survival rate of the ellipticine-FLC combination group was notably higher compared to the other three groups (Fig. 8b). Regrettably, the enhancement effect of ellipticine on the antifungal activity of FLC in a murine model of systemic C. albicans infection was suboptimal (Supplementary Fig. 8a, b). This outcome may be attributed to the inadequate pharmacokinetic parameters of ellipticine⁵², which likely hinder its efficacy in enhancing the antifungal action of FLC in this context.

Analogues of ellipticine were pursued through an examination of its core structure, which is composed of two benzene rings surrounding a pyrrole ring. Three distinct series of ellipticine analogues were studied, each maintaining the fundamental structure of the parent nucleus: the a-series, which investigated substitutions on the benzene ring on the left side of the parent nucleus structure; the b-series, which concentrated on alterations to a nitrogen atom of the pyrrole ring; and the c-series, which explored modifications to the connecting substituents on the benzene ring on the right side of the parent nucleus structure (Supplementary Fig. 9). Dose-matrix titration assays and spotting assays identified three compounds, namely 7H-Dibenzo[c,g]carbazole (Supplementary Fig. 10a), 10-Bromo-7H-benzo[c]carbazole (Supplementary Fig. 10b), and phiKan 083 (Supplementary Fig. 10c), that demonstrated promising antifungal properties through the inhibition of FLC tolerance. Subsequently, we conducted further evaluation of the antifungal activity of these compounds through time-growth curves, leading to the discovery that only phiKan 083 demonstrated dose-dependent inhibition of fungal growth when combined with FLC (Supplementary Fig. 10d–f). Similar to ellipticine, phiKan 083 can also impair mitochondrial function (Supplementary Fig. 10g, h).

Several studies have demonstrated that mitochondrial dysfunction hinders the hyphal growth of C. albicans^53,54. Therefore, we hypothesize that ellipticine and phiKan 083, known for impairing mitochondrial function, may inhibit the hyphal growth of C. albicans. The C. albicans SN152 and cmp1-aidΔ/cmp1-aidΔ mutants were induced to form hyphae in RPMI 1640 medium. Interestingly, 4 μg/mL FLC did not prevent this morphological change. However, the combination of FLC with 8 μg/mL ellipticine or 32 μg/mL phiKan 083 significantly inhibited the hyphal growth of C. albicans. The average length of hyphae in the C. albicans SN152 strain and the cmp1-aidΔ/cmp1-aidΔ mutants was 44.5 ± 4.42 μm and 50.3 ± 4.49 μm, respectively, while under FLC treatment, the average lengths were 28.1 ± 4.77 μm and 30.3 ± 4.23 μm, respectively. When FLC was combined with 8 μg/mL ellipticine or 32 μg/mL phiKan 083, the average hyphal lengths were 11.4 ± 1.50 μm and 11.0 ± 2.08 μm in the C. albicans SN152 strain (Supplementary Fig. 11a, b), and 3.1 ± 0.60 μm and 10.8 ± 1.74 μm in the cmp1-aidΔ/cmp1-aidΔ mutants (Supplementary Fig. 11c, d and Supplementary Table 4). The data presented indicates that disrupting the Erg11-Ncp1 interaction results in mitochondrial dysfunction, thereby impeding the morphological transition of C. albicans from yeast to hyphae.

To assess the antifungal efficacy of phiKan 083 in vivo, C. albicans wild-type SN152 and highly FLC-tolerant cmp1-aidΔ/cmp1-aidΔ strains were utilized to establish a model of systemic candidiasis in C57BL/6 mice via tail vein injection. Following infection, all mice were randomly assigned to one of four groups: (1) control group, (2) FLC alone group at a dosage of 0.5 mg/kg, (3) phiKan 083 alone group at a dosage of 6 mg/kg, and (4) combination group receiving FLC (0.5 mg/kg) in conjunction with phiKan 083 (6 mg/kg). The initial intraperitoneal injection was administered 2 h post-infection, with subsequent administrations given at one-day intervals for a total of three consecutive days. On the seventh day post-infection, six mice from each group were euthanized, and the fungal load in their kidneys, livers, and spleens was evaluated. Given that there was no significant interaction between drug treatment and sex, we decided to pool the data from male and female mice together for further analysis. The results indicated a clear decrease in fungal load in the kidneys of mice treated with the drug combination (Fig. 8c, d). However, the impact of the combination therapy on fungal burden in the liver and spleen has no physiological decrease observed compared to FLC monotherapy (Supplementary Fig. 11e, f). Similar to the inhibitory effect observed in vitro with the combination of FLC and phiKan 083 on the formation of C. albicans hyphae, histopathological analysis utilizing Periodic Acid Schiff (PAS) staining revealed the absence of hyphae in the kidneys of the group treated with FLC in combination with phiKan 083. In contrast, a significant presence of hyphae was observed in the kidneys of both the C. albicans infected group, the FLC alone group and phiKan 083 alone group (Fig. 8e, f). These findings demonstrated that phiKan 083 enhanced the antifungal efficacy of FLC in vivo, especially in the highly FLC-tolerant C. albicans cmp1-aidΔ/cmp1-aidΔ.

To further evaluate the therapeutic efficacy of phiKan 083 against the wild-type C. albicans strain SN152 and the highly FLC-tolerant C. albicans cmp1-aidΔ/cmp1-aidΔ, systemic fungal infections were simulated in Galleria mellonella. The larvae were randomly assigned to one of four groups: (1) the control group receiving PBS treatment, (2) the group treated with 1 mg/kg FLC alone, (3) the group treated with 4 mg/kg phiKan alone, and (4) the group treated with a combination of 1 mg/kg FLC and 4 mg/kg phiKan 083. Each group consisted of ten larvae, which were treated 2 h post-infection with a single dose of the respective drug. Our observations indicate that the combination of phiKan 083 with FLC resulted in a 60% survival rate. In contrast, the remaining three groups exhibited a 100% mortality rate over the 20-day observation period (Fig. 8g, h). Taken together, phiKan083 significantly enhance the antifungal activity of azoles.

Discussion

In this study, we have identified the Erg11_200-251 region and the Ncp1_1-63 region as crucial for the interaction between Erg11 and Ncp1. Our findings also highlight the significance of amino acid residues V234, F235, and L238 in Erg11 for this interaction. Mutating these three amino acids effectively disrupts the Erg11-Ncp1 interaction, resulting in heightened susceptibility of C. albicans to azoles. This disruption of the Erg11-Ncp1 interaction results in elevated ROS levels, protein oxidation, and misfolding in the ER, culminating in ER stress. This stress triggers the release of Ca²⁺ from the ER and its accumulation in mitochondria, leading to mitochondrial dysfunction, heightened ROS production, and, ultimately, apoptosis in C. albicans cells. Additionally, our investigation revealed that ellipticine functions as an inhibitor of the Erg11-Ncp1 interaction by specifically targeting Ncp1, thereby enhancing the efficacy of FLC in the Galleria mellonella infection model caused by the FLC-tolerant C. albicans strain.

Our research has revealed that the cascade amplification of ROS production can impair mitochondrial function and augment the antifungal efficacy of azoles. By disrupting the Erg11-Ncp1 interaction, electrons may escape from the hydroxylation process and be redirected to oxygen, leading to the generation of ROS within the ER. ROS originating from the ER may act as a “trigger,” initiating ER stress. This stress induces the release of Ca²⁺ from the ER. The flow of Ca²⁺ may act as a “fuse,” causing an accumulation of Ca²⁺ in the mitochondria and subsequent mitochondrial dysfunction. The dysfunction of the mitochondria further increases ROS generation within mitochondria. This heightened mitochondrial ROS production may serve as an “explosive package,” ultimately leading to apoptosis in C. albicans cells and demonstrating potential for enhancing the antifungal activities of azoles (Fig. 9). It is important to note that other mechanisms might underlie these phenomena. For instance, the potential disruption of the Erg11-Ncp1 interaction could change the content and composition ratio of sterols in the membrane, thereby affecting mitochondrial functions. Furthermore, the direct impact of ROS on mitochondria can lead to apoptosis, independent of Ca²⁺ homeostasis. Additionally, there may be intricate crosstalk between the ER and mitochondria involving various signaling pathways that are not solely reliant on Ca²⁺, such as the UPR.

**Fig. 9: The schematic representation illustrates how the disruption of the Erg11-Ncp1 interaction increases the sensitivity of azoles.**

The elucidation of the interaction between Erg11 and Ncp1 offers a promising approach for the advancement of antifungal drugs inhibiting the activity of Erg11. The ergosterol biosynthesis pathway has been identified as a traditional therapeutic target for addressing invasive candidiasis⁵⁵. Azoles function by inhibiting the activity of Erg11 through binding to the heme region of Erg11¹⁴, making them a preferred choice for primary treatment options due to their favorable bioavailability and safety profile⁵⁶. However, the emerging issue of azole resistance and tolerance poses a substantial obstacle⁷ and underscores the necessity for alternative approaches to impede the function of Erg11. The formation of a complex between Erg11 and Ncp1 is essential for the catalytic activity of Erg11⁵⁷. Thus, disrupting the Erg11-Ncp1 interaction has the potential to inhibit Erg11 activity. Nevertheless, the precise mechanism of the Erg11-Ncp1 interaction has remained elusive⁵⁷. This study showcases the physical interaction between Erg11 and Ncp1 using the MYTH system and co-IP. Furthermore, we elucidate the crucial domain of the Erg11-Ncp1 interaction through fragment analysis, emphasizing the significance of the Erg11_200-251 region in facilitating this interaction. Additionally, we pinpoint key amino acid residues V234, F235, and L238 in Erg11 that are required for the Erg11-Ncp1 interaction through molecular dynamic simulations, the MYTH system, and co-IP. Our study further revealed that the disruption of the Erg11-Ncp1 interaction led to increased susceptibility of C. albicans to azoles as demonstrated by in vitro antifungal drug susceptibility tests.

Our research introduces a potential approach to impair mitochondrial function and trigger apoptosis in C. albicans cells by inhibiting the interaction between Erg11 and Ncp1. This results in inducing ER stress, causing Ca²⁺ leakage from the ER and its accumulation in mitochondria, ultimately leading to mitochondrial dysfunction. Our previous investigations have demonstrated that baicalin, berberine, and osthole can elevate ROS levels in C. albicans, thereby bolstering the antifungal potency of azoles^58,59,60. However, there is substantial conservation in the proteins of the mitochondrial respiratory chain between C. albicans and mammalian species. As a result, although the dysfunction of the mitochondrial respiratory chain induced by these compounds can lead to elevated ROS production in C. albicans cells, they can also cause an increase in ROS levels in mammalian cells, ultimately resulting in oxidative damage to host cells. In response to this issue, we suggest an alternative approach to inhibit the fungal-specific interaction between Erg11 and Ncp1. In this approach, elevated levels of ROS within the ER serve as a stimulus, inducing the release of Ca²⁺ to the mitochondria akin to a detonation fuse. Ultimately, the escalation of ROS levels within the mitochondria leads to the death of C. albicans cells (Fig. 9).

Ellipticine is a naturally occurring alkaloid with significant antineoplastic properties, originally isolated from Ochrosia elliptica⁶¹. An earlier Phase I clinical study conducted in cancer patients has shown that the half-life of ellipticine is ~23.7 h⁶². In this study, ellipticine has been identified as an inhibitor of the Erg11-Ncp1 interaction, specifically targeting Ncp1 to enhance the antifungal efficacy of azoles. These findings were achieved through various methods, including the MYTH system, in vitro antifungal drug susceptibility tests, co-IP, and DARTS. Ellipticine’s inhibition of the Erg11-Ncp1 interaction resulted in increased misfolded protein levels, intracellular Ca²⁺ concentration, ROS generation, mitochondrial dysfunction, and, ultimately, apoptosis, thereby augmenting the antifungal effects of FLC. After analyzing the molecular structure of ellipticine, it was discovered that its derivative, phiKan083, exhibits a notable ability to augment the efficacy of FLC in both in vivo and in vitro settings against fungal infections. The results suggest that focusing on the Erg11-Ncp1 interaction for the discovery of azole enhancers is a viable approach.

As Erg11 and Ncp1 are membrane-anchored proteins, challenges arise in expressing and purifying these proteins, as well as in obtaining their crystalline structures. Consequently, this study did not focus on structure-activity relationships when seeking analogues of ellipticine to enhance its antifungal efficacy. Future investigations should prioritize obtaining the crystal structure of the Erg11-Ncp1 complex to develop more effective antifungal inhibitors targeting the Erg11-Ncp1 interaction.

Methods

Ethics statement

All animal experiments comply with relevant ethical regulations. The study protocol was approved by the Animal Ethics Committee of Tongji University.

Strains, primers, agents, and cultural conditions

All strains, plasmids, and primers utilized in this study are presented in Supplementary Tables 5, 6, and 7. The C. albicans strains were cultured in YPD medium containing 2% dextrose (Sangon Biotech, A610219-0500), 1% yeast extract (Oxoid, LP0021B), and 2% peptone (Oxoid, LP0137) at 30°C. The SD medium containing 0.67% yeast nitrogen-based (Sangon Biotech, A610507-0100), required amino acids, and 2% dextrose was used to mutate target genes. To conduct the dual membrane functional assay, we employed SD-Leu-Trp (Takara, Cat#630316) and SD-Leu-Trp-His-Ade (Takara, Cat#630322) selective media. FLC (E129360), tamoxifen (T137974), arachidonic acid (A424359), piperine (P107402), menadione (M408896), terbinafine (T129208), amphotericin B (A408132), 5-fluorocytosine (F123460), and myriocin (M102400) are from Aladdin in Shanghai, China. Congo red (A427695-0100), dimethyl sulfoxide (DMSO) (A610163-0250) and 10% sodium dodecyl sulfate (SDS) solution (B548118-0100) were from Sangon Biotech in Shanghai, China. Carbonyl cyanide m-chlorophenylhydrazone (T7081) and ellipticine (T1166) were from TargetMol in Boston, USA; phiKan 083 (HY-108637) is from MedChemExpress in Shanghai, China. Ergosterol (Sangon Biotech, A351890-0100) is prepared into a 10 mM solution using Tween 80 and ethanol (1:1).

MYTH assays

The MYTH system was operated in accordance with the user manual for the dual membrane starter kits (P01201-P01229) user manual from Dualsystems Biotech, Schlieren, Switzerland⁶³. The yeast strains obtained were subjected to serial dilution and spotted onto selective plates lacking either two amino acids (leucine and tryptophan) or four amino acids (leucine, tryptophan, histidine, and adenine). The bait plasmid pTSU2-APP, expressing the type I integral membrane protein APP (amyloid beta A4 precursor protein), and the prey plasmid pNubG-Fe65, expressing the cytosolic protein Fe65 (amyloid beta A4 precursor protein-binding family B member 1), were used as positive controls. The co-transformation with the control plasmids pTSU2-APP and the pPR3-N prey vector served as the negative controls. 3-AT (Aladdin, A107202) acts as a competitive inhibitor of the HIS3 gene product, and increasing concentrations of 3-AT enhance the stringency of HIS3 selection. The selective plates were then sealed and incubated at 30 °C for 4–6 days.

To identification of inhibitors of the Erg11-Ncp1 interaction using the MYTH assays. The yeasts were subjected in the 96-well plates using fluid selective medium. 4 mM 3-AT was added in the SD-Leu-Trp-His-Ade selective medium to eliminate the self-activation of Erg11. The pTSU2-APP and pNubG-Fe65 were chosen to determine whether the compounds can specifically inhibit Erg11-Ncp1 interaction. The yeasts were sealed and cultured for 16 h at 30 °C and adjusted to 1 × 10³ cells/mL. The compounds were initiated at a concentration of 64 μg/mL and subsequently diluted in duplicate using SD-Leu-Trp and SD-Leu-Trp-His-Ade (containing 4 mM 3-AT) selective media. After incubating at 30 °C for 48 h, the 96-well plates were tested for OD₆₀₀ at the full-wavelength microplate instrument (Thermo Fisher Scientific, Massachusetts, USA). The degree to which a compound disrupts the interaction between Erg11 and Ncp1 can be quantified by evaluating yeast growth in SD-Leu-Trp-His-Ade (containing 4 mM 3-AT) medium compared to its growth in SD-Leu-Trp medium. The inhibition rate of compounds is determined by subtracting the result of the subtraction in yeast growth rates in these two media. Compounds exhibiting ≥30% inhibition are considered potential inhibitors of the Erg11-Ncp1 interaction.

Spotting assay

The spotting assay was performed as follows: Overnight cultured strains were diluted ten-fold with sterilized phosphate-buffered saline (PBS) (Sangon Biotech, B040100-0005) in five gradients. Subsequently, 5 μL of each gradient tube was aliquoted onto spotting plates, with various compounds added to the solid plates as required by the experimental design. The plates were then incubated at 30 °C for 48 h before being photographed using an automatic chemiluminescence image analyzer (Tanon, Shanghai, China).

Co-IP assay

The co-IP assay was conducted as follows: Overnight C. albicans cultures of 10 mL were incubated at 30 °C. The collected C. albicans cells were washed twice with ice-cold sterilized water and then suspended in detergent-free lysis buffer [(PBS containing: 5 mM EDTA (Sangon Biotech, B540625-0500), 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sangon Biotech, A610425-0025), and protease inhibitor cocktail (TargetMol, C0001)]. The cells were lysed by adding 3 volumes of ice-cold lysis buffer and 3 volumes of glass beads per volume of pelleted yeast cells. Then, the C. albicans cell suspension was vigorously vortexed with 425–600 μm glass beads (Sigma-Aldrich, G9268) for four 1-min intervals, with the cells being kept on ice for 1 min between each period. Cell lysates totaling 800 μL were incubated with 8 μL of either anti-Myc (Santa Cruz, sc-47694) or anti-HA (Santa Cruz, sc-7392) antibody for 20 min at room temperature. The sample, pre-incubated with the antibody, was then applied to Protein A columns as per a Co-IP Kit’s (Clontech, 635721) instructions, which are designed to capture antibody-bound target protein complexes. In our investigation of the potential inhibitory effects of ellipticine on the Erg11-Ncp1 interaction, we encountered difficulties with compound binding to Protein A columns. To address this issue, we utilized an anti-Myc (20587ES03) or anti-HA agarose affinity gel (20586ES03) from Yeasen Biotechnology in Shanghai, China. The resulting 1000 μL cell lysates were then individually exposed to 10 μL of either anti-Myc or anti-HA affinity gel for 2 h at room temperature. Finally, the agarose beads containing the protein complexes were retrieved, subjected to three washes with TBS buffer (Sangon Biotech, B040126-0005), and subsequently analyzed through SDS-PAGE and western blot analysis.

Western blot

In the western blot procedure, proteins were initially separated by molecular weight using SDS-PAGE with an 8–20% precast PAGE gel (Beyotime, P0471M). Subsequently, the separated proteins were electroblotted onto a 0.45 μm polyvinylidene fluoride (PVDF) membrane (Millipore, IPVH00010). Following blocking, the primary antibodies were used: c-Myc (1:1000, Santa Cruz, sc-47694), HA (1:1000, Santa Cruz, sc-7392), β-Tubulin (1:1000, Abbkine, ABL1030). After incubating with an anti-mouse IgG secondary antibody m-IgGκ BP-HRP (Santa Cruz, sc-516102) at a dilution of 1:10,000, the Omni-ECL™Femto Light Chemiluminescence Kit (Epizyme Biotech, SQ201) was utilized to reveal the chemiluminescence signal, which was captured by an automatic chemiluminescence image analyzer (Tanon, Shanghai, China)²⁸.

Molecular dynamics simulations

The crystal structure of Erg11 was obtained from the Protein Data Bank (PDB) (ID: 5V5Z) at a resolution of 2.9 Å. Due to the unavailability of the crystal structure of Ncp1 in the existing protein database, the predicted protein structure of Ncp1 generated by AlphaFold was used for molecular docking. The docking procedure was carried out using ZDOCK 3.0.2 software (University of California, San Francisco, USA)⁶⁴, with default Hdock parameters. The analysis of the 10 docking results yielded the selection of the highest-scoring outcomes for subsequent molecular dynamics simulations. The docked complex structure was enclosed within a 20 Å cubic water box utilizing the TIP3P explicit solvent model. To maintain electrical neutrality, 24 Na⁺ ions were incorporated based on the volume of the box and the electronegativity of the protein. This process completed the construction of the molecular dynamics model of the complex, facilitating the commencement of traditional molecular dynamics simulation. A conventional molecular dynamics simulation was conducted for a total duration of 50 ns using the AMBER 18 software (University of California, San Francisco, USA), employing the assisted model building with energy refinement method.

Disk diffusion assays

The C. albicans cultures were incubated at 30 °C for 16 h and subsequently diluted 100-fold with sterilized PBS. The diluted cultures were then evenly spread onto drug-free or drug-containing YPD plates using a cotton swab. Following the drying of the plates, a 25 μg FLC (Liofilchem, 9166) paper disk was positioned at the center of each plate. The plates were then placed in an incubator at 30 °C and observed and photographed after 48 h.

Antifungal susceptibility testing

Antifungal susceptibility testing was performed using the following methods: Compounds were diluted in a 96-well plate through a two-fold dilution from column 2 to 11, with 100 μL discarded from column 11. Subsequently, overnight cultures of C. albicans were diluted to a concentration of 1 × 10³ cells/mL in the YPD medium. Following this, 100 μL of the diluted C. albicans cell suspension was added to each well of the 96-well plate. Incubate the microdilution plates at 30 °C for 24 h and observe for the presence or absence of visible growth. Use a reading mirror to score the microdilution wells. Compare the turbidity in each well with that of the drug-free growth control well. Assign a numerical score to each well based on the following scale: 0, optically clear; 1, slightly hazy; 2, prominent decrease in turbidity (approximately 50% as determined visibly); 3, slight reduction in turbidity; and 4, no reduction of turbidity. In this study, the MIC is defined as the lowest concentration at which a score of 2 (prominent decrease in turbidity) is observed. MIC values of >64 μg/mL was defined as 128 μg/mL⁶⁵.

The dose-matrix titration assay was utilized to assess the efficacy of a drug combination with C. albicans culture dilution methods consistent with those employed in MIC assays. The two drugs were diluted at a 2-fold ratio in separate 96-well plates, with one drug diluted horizontally and the other vertically. Subsequently, the contents of one plate were transferred to the other, followed by the addition of 100 μL of 1 × 10³ cells/mL cell suspension to each well. The 96-well plate was incubated for 24 h to visually read the MIC for the calculation of the FICI and subsequently incubation was extended to 48 h for OD₆₀₀ measurement using a full-wavelength microplate instrument (Thermo Fisher Scientific, Waltham, USA). FICI is calculated as follows: FICI = MIC_AB/MIC_A + MIC_BA/MIC_B. Here, MIC_AB refers to the MIC of drug A in the presence of drug B; MIC_A stands for the MIC of drug A alone. Similarly, MIC_BA refers to the MIC of drug B in the presence of drug A; MIC_B stands for the MIC of drug B alone. The interaction was categorized as synergic if the FICI was ≤0.5, additive if the FICI was >0.5–1.0, indifferent if the FICI was >1.0–4.0 and antagonistic if the FICI was >4.0⁶⁶. The tolerance, characterized by incomplete growth inhibition over an extended range of antifungal concentrations, was recorded after 48 h of incubation⁶⁷. We quantified SMG at 48 h (average growth per well above the MIC, normalized to growth in the absence of the drug)³.

Growth inhibition curve assay

C. albicans colonies were introduced into the YPD medium, and the concentration of fungal liquid was standardized to an optical density of approximately 0.2 at a wavelength of 600 nm. Subsequently, compounds were diluted in a 96-well plate as required for the experimental procedure. A volume of 75 μL was then introduced into the 96-well plate. The completed plate was subsequently placed into the Infinite 200 PRO Multifunctional Microplate Reade (Tecan, California, USA) for incubation and analysis. The temperature was set at 30 °C, with OD₆₀₀ measurements taken and recorded at 2-h intervals for 48 h.

RNA sequencing

The cultures of ERG11/erg11Δ and erg11^m/erg11Δ mutants were incubated for 16 h with 4 μg/mL FLC in YPD at 30 °C. RNA was isolated and a DNA probe was utilized for rRNA hybridization. Subsequently, DNase I (Takara, 2270A) digestion of the DNA probe and selective digestion of the DNA/RNA hybridization chain by RNase H (Takara, 2641Q) facilitated the isolation of the desired RNA. The isolated RNA was then reverse-transcribed into the cDNA, which was further synthesized into double-stranded DNA with the use of primers to amplify the ligation products. The purified double-stranded DNA was mixed with an end-repair reaction mix containing DNA polymerase, ATP, and dNTPs to repair the ends of the DNA fragments. Then, the 3’ ends of the DNA fragments were added to an adenine (A) nucleotide via incubation with a dATP and terminal transferase enzyme mix. The DNA fragments were thermally denatured into a single strand, and then a bridge primer was used to cyclize the single-strand DNA to obtain a single-strand circular DNA library and then sequenced (BGI Genomics, Shenzhen, China).

Determination of misfolded protein levels

To determination of intracellular and mitochondrial misfolded protein levels, overnight cultures of C. albicans were inoculated at a 1:100 dilution and grown in the presence of respective compounds (or DMSO equivalent) for 4 h at 30 °C. Exposure to 5 mM DTT (Sangon Biotech, A620058-0025) for 2 h as the positive control. Cell suspensions of 1 × 10⁷ cells/mL were incubated with 50 μM TPE-MI (MedChemExpress, HY-143218) at 30 °C or 0.1 μM MitoTracker Green (MedChemExpress, HY-135056) at 37 °C for 30 min. The excitation and emission wavelength of TPE-MI were 350 nm and 470 nm, respectively, while the MitoTracker Green fluorescence intensities were measured with excitation at 490 nm and emission at 523 nm. Fluorescence intensity measurements were obtained using a BD FACSverse flow cytometer (BD Biosciences, New York, USA). Forward Scatter (FSC) and Side Scatter (SSC) were adjusted at 419 V and 150 V, respectively to encircle the C. albicans cells to exclude cell debris. The gating process was concluded upon reaching a stopping criterion of 10,000 events.

Measurement of ROS generation

The levels of intracellular and intra-mitochondrial ROS were quantified using a BD FACSVerse flow cytometer (BD Biosciences, New York, USA). The overnight cultures of C. albicans were inoculated at a 1:100 dilution and grown in the presence of respective compounds (or DMSO equivalent) for 4 h at 30 °C. Exposure to 5 mM DTT for 2 h as the positive control. Subsequently, Cell suspensions of 1 × 10⁷ cells/mL were incubated with either 10 μM DCFH-DA (Sigma-Aldrich, D6883) at 37 °C or 5 μM MitoSOX Red (MedChemExpress, HY-D1055) at 30 °C for 30 min. The DCFH-DA fluorescence intensities were detected with an excitation wavelength of 492 nm and an emission wavelength of 517 nm, while the MitoSOX Red fluorescence intensities were detected with an excitation wavelength of 510 nm and an emission wavelength of 580 nm. FSC and SSC were adjusted at 415 V and 150 V, respectively to encircle the C. albicans cells to exclude cell debris. The gating process was concluded upon reaching a stopping criterion of 10,000 events.

Determination of the mitochondrial membrane potential

The mitochondrial membrane potential of C. albicans cells was assessed using Rhodamine 123 (Aladdin, R299309) staining. Cells cultured overnight were diluted 1:100 and treated with drugs for 8 h. A volume of 10 mL of cell suspensions was stained with 10 μM Rhodamine 123 at 30 °C for 30 min in the absence of light. Fluorescence intensity was measured with excitation at 507 nm and emission at 529 nm using a BD FACSVerse flow cytometer (BD Biosciences, New York, USA).

Apoptosis assay

The C. albicans cells required protoplast preparation prior to Annexin V and PI staining. Overnight cultured C. albicans were diluted 1:100 and further incubated in the presence or absence of compounds (or DMSO equivalent) for 8 h at 30 °C. The purified C. albicans cells were then exposed to a solution containing 80 μL of 2.4 M sorbitol (comprising 0.1 M PBS, 5 mM EDTA, 50 mM DTT, 50 mM Na₂HPO₄, 50 mM NaH₂PO₄, and 40 mM 2-mercaptoethanol) and 5 μL Zymolyase (ZYMO RESEARCH, R1002). After incubating at 37 °C for 30 min, 160 μL of 0.1 M sodium citrate containing 0.1% Triton X-100 was added to the processed protoplasts. Subsequent procedures were carried out in accordance with the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, 556547). FITC fluorescence density was measured using 488 nm excitation and 525 nm emission settings, while PI fluorescence density was measured with an excitation wavelength of 535 nm and an emission wavelength of 615 nm. FSC and SSC were adjusted at 380 V and 200 V, respectively to locate the C. albicans cells using the unstained tube. Then proceeding with the samples stained singly with FITC and singly with PI and adjusting the compensation so that the cell population is positioned in the lower left corner of 10⁴. Control the flow rate of the cells at 1000 cells per second.

Measurement of the content of Ca²⁺

To assess the levels of the cytosolic Ca²⁺, the overnight cultures of C. albicans were diluted 1:100 and exposed to 4 μg/mL FLC and 16 μg/mL ellipticine (or DMSO equivalent) for 4 h. Subsequently, the cells were stained with Fluo-3 AM (5 μM) (HY-D0716) from MedChemExpress, Shanghai, China, and incubated in darkness for 30 min. The fluorescence intensities of Fluo-3 AM were measured using an excitation wavelength of 488 nm and an emission wavelength of 526 nm. Fluorescence intensity measurements were obtained using a BD FACSverse flow cytometer (BD Biosciences, New York, USA). FSC and SSC were adjusted at 419 V and 150 V, respectively to encircle the C. albicans cells to exclude cell debris. The gating process was concluded upon reaching a stopping criterion of 10,000 events.

Confocal microscope analysis

The co-localization of ER or vacuole with Ca²⁺ and localization of mitochondrial Ca²⁺ were observed using the Nikon Laser Scanning Confocal Microscope System (Nikon, Tokyo, Japan). Prior to imaging, C. albicans cells were pre-stained with ER-Tracker Red (2 μM, 30 min) (MedChemExpress, HY-D1431), or Cell Tracker Blue (25 uM, 30 min) (MedChemExpress, HY-D1462), and co-stained with Fluo-3 AM (5 μM, 30 min) at 37 °C. Bright red fluorescence indicated ER, blue fluorescence indicated vacuole, while green fluorescence indicated Ca²⁺. To detect the level of Ca²⁺ in mitochondria, C. albicans cells were stained with Rhod-2 AM (5 μM, 30 min) (MedChemExpress, HY-D0989) at 37 °C. The images were acquired utilizing a confocal microscope (Nikon, Tokyo, Japan). The excitation and emission wavelengths for the fluorescent probes used in this study are as follows: ER-Tracker Red (587 nm/615 nm), Rhod-2AM (549 nm/578 nm), Fluo-3 AM (488 nm/526 nm), and Cell Tracker Blue (372 nm/470 nm).

DARTS assay

The DARTS assay was operated according to the reported protocols⁴⁷. The 10 mL overnight C. albicans cultures were harvested. Following two washes with ice-cold sterilized water, C. albicans cells were resuspended in detergent-free lysis buffer. The resuspended cells were transferred a tube containing 0.5-mm glass beads and vortexed at maximum speed for 1 min, followed by a 1-min rest on ice. Perform four cycles of 1-min vertexing and 1-min resting. The samples underwent four cycles of 1-min vertexing and 1-min resting. Subsequently, the tube containing lysates were centrifuged at 20,000 × g for 10 min at 4 °C. Following centrifugation, 1000 μL of cell lysates with either 5 μL of DMSO or 5 μL of ellipticine (5 mg/mL) separately for 1 h at room temperature. The protein samples were then divided into six aliquots, each containing 50 μL, and subject to a 30-min digestion with an appropriate concentration of pronase. The digestion process was halted by adding SDS loading buffer to each sample, which were then heated at 100 °C for 10 min. Finally, the samples were analyzed using SDS-PAGE and Western blot analysis.

Systemic candidiasis model

Mice were maintained in a controlled environment with a 12-h light/dark cycle, temperature set between 18 and 25 °C, and relative humidity of 30–70%. Mice had unrestricted access to food and water throughout the experiment. C57BL/6 mice (specific-pathogen-free, 6–8 weeks) (Slaccas, Shanghai, China) weighing 17–19 g were chosen for the establishment of a systemic candidiasis model. For each group, a total of 12 mice (half male and half female) were systemically infected through the tail vein injection of 2 × 10⁵ C. albicans cells. In the treatment group, the initial intraperitoneal administration occurred 2 h post-infection and was administered for three consecutive days with one-day intervals between doses. Following completion of dosing, all mice were euthanized at 3-day intervals. Subsequently, the liver, kidney, and spleen of each mouse were extracted and processed in PBS, ground, and plated onto SDA (4% dextrose, 1% peptone, and 2% agar) solid plates at varying dilutions. The resulting colonies on each SDA plate were enumerated, and the colony-forming units were calculated⁶⁸.

Histopathology

Following the systemic candidiasis model, the left kidney of each mouse was excised and placed in 4% paraformaldehyde for 3 days for histological examination. Subsequently, the finished fixed kidney tissue was sectioned thinly, stained with PAS, and subjected to photographic documentation and analysis.

Statistics and reproducibility

All experiments (except those described otherwise in the legend) were performed independently at least twice with similar results. The number of independent experiments is reported in the figure legend. Statistical analysis was conducted using two-tailed unpaired t-tests. The Log-rank (Mantel–Cox) test was used to manifest differences between survival curves. For all statistical analyses, p < 0.05 was considered significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The RNA-seq raw data can be acquired from the sequence read archive (SRA) section of the National Center for Biotechnology Information (NCBI) repository under the accession code PRJNA1083027 [http://submit.ncbi.nlm.nih.gov]. Source data are provided with this paper.

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Acknowledgements

This study received financial support from the National Natural Science Foundation of China (No. 82020108032 to Y.J.), the Innovation Program of Shanghai Municipal Education Commission (202101070007-E00094 to Y.J.) and the National Key Research and Development Program of China (No.2022YFC2303004 to Y.J.).

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Department of Pharmacy, Shanghai Tenth People’s Hospital, School of Medicine, Tongji University, Shanghai, China
Wanqian Li, Sijin Hang, Jinhua Yu, Hui Lu & Yuanying Jiang
Department of Biology, Concordia University, Montreal, QC, Canada
Malcolm Whiteway

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Wanqian Li
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Jinhua Yu
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Hui Lu
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Contributions

W.L. conducted most of the experiments and performed data analysis. W.L., H. L., M.W., and Y.J. wrote the manuscript draft and revised the manuscript. W.L., S.H., and J.Y. constructed mutant strains. W.L., S.H., and J.Y. evaluated the antifungal activities and data collection. W.L., H. L., M. W., and Y. J. discussed and analyzed the data. H.L. and Y.J. conceived the idea. H.L. and Y. J. directed the experiments.

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Correspondence to Hui Lu or Yuanying Jiang.

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Li, W., Whiteway, M., Hang, S. et al. Impairing the interaction between Erg11 and cytochrome P450 reductase Ncp1 enhances azoles’ antifungal activities. Nat Commun 16, 6821 (2025). https://doi.org/10.1038/s41467-025-62131-z

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Received: 01 July 2024
Accepted: 11 July 2025
Published: 24 July 2025
DOI: https://doi.org/10.1038/s41467-025-62131-z