High throughput screen reveals four compounds with novel antifungal activity, including a metal-responsive candidate with in vivo efficacy against Candida albicans.

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Learn more: PMC Disclaimer | PMC Copyright Notice Sci Rep . 2026 Apr 12;16:17019. doi: 10.1038/s41598-026-48741-7 High throughput screen reveals four compounds with novel antifungal activity, including a metal-responsive candidate with in vivo efficacy against Candida albicans Jana Nysten 1 , Lara Van Campenhout 1 , Eliane Vanhoffelen 2 , Greetje Vande Velde 2 , Patrick Van Dijck 1, ✉ Author information Article notes Copyright and License information 1 Department of Biology, Laboratory of Molecular Cell Biology, KU Leuven, 3001 Heverlee, Belgium 2 Department of Imaging and Pathology, Biomedical MRI, KU Leuven, 3000 Leuven, Belgium ✉ Corresponding author. Received 2026 Jan 5; Accepted 2026 Apr 9; Collection date 2026. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13230819  PMID: 41968200 Abstract Fungal infections represent a growing global health concern, aggravated by the paucity of effective antifungal therapies and the rapid rise of drug resistance. The development of new treatments is challenging due to the shared eukaryotic biology of fungi and human hosts, which restricts selective molecular targets and contributes to issues such as host toxicity and fungistatic activity. These challenges underscore the pressing need for new agents with distinct modes of action. In this study, we identified four promising compounds with novel antifungal activity from a high-throughput screen of 20,000 compounds against the opportunistic fungal pathogen, Candida albicans , namely Z1199266541, Z1024453766, Z56842335, and Z126932704. Subsequently, the compounds were characterized in vitro to assess their spectrum of activity, efficacy against a fluconazole-resistant isolate (CaCi-17), and effectiveness in combination treatments, followed by in vivo evaluation in a Galleria mellonella infection model. Among the candidates, Z56842335 emerged as a lead compound, effectively clearing infection caused by the Candida albicans in this invertebrate model. Mechanistic analyses revealed that the antifungal activity of Z56842335 is mitigated by supplementation with iron, copper, and zinc, indicating a metal-dependent mode of action. Notably, despite its apparent metal-binding capacity, Z56842335 displays a narrow spectrum of activity, targeting only C. albicans and, to a lesser extent C. dubliniensis . This selectivity suggests that, in addition to metal availability, species-specific factors such as differences in metal acquisition, intracellular metal buffering, or compound uptake or accumulation may contribute to susceptibility, highlighting a promising yet mechanistically intriguing avenue for further investigation. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-026-48741-7. Subject terms: Biochemistry, Drug discovery, Microbiology Introduction Fungal infections have become more prevalent over recent decades and now represent a serious global health threat 1 , 2 . An estimated 1.7 billion people are affected every year. Although the majority of these infections are superficial (e.g., on nails and skin), fungi can also cause severe, life-threatening systemic infections with high mortality rates 3 , 4 . Recent estimates suggest that mycoses result in approximately 3.8 million attributable deaths annually, with Candida species accounting for nearly one-third of these fatalities 5 . Candida albicans is a leading cause of nosocomial infections among Candida species. This opportunistic pathogen commonly colonizes the gastrointestinal tract, skin, mouth, and genitourinary tract of over 70% of the global population 6 . While C. albicans typically exists as a harmless commensal in its human host, it can transition to a pathogenic state under certain conditions, leading to diseases ranging from mucosal infections to life-threatening invasive candidiasis 7 . These invasive infections have a mortality rate of approximately 63.6%, prompting the World Health Organization to recently classify C. albicans as a pathogen of critical concern to public health that requires immediate attention 5 , 8 , 9 . Importantly, in addition to C. albicans , non- albicans Candida species, such as Candida glabrata (recently renamed as Nakaseomyces glabratus) and Candida auris (recently renamed as Candidozyma auris ) , are becoming increasingly prevalent. These species often display intrinsic or acquired resistance to commonly used antifungal drugs, thereby further complicating treatment 10 – 12 . Despite their impact, Fungal pathogens have long been understudied, underestimated, and neglected because they were considered relatively rare, and most attention was given to bacterial and viral pathogens 13 , 14 . Consequently, only three major antifungal drug classes are available to treat invasive mycoses. Drug development is hindered by the close evolutionary relationship between fungi and humans, which limits the number of selective molecular targets and contributes to challenges such as host toxicity, fungistatic activity, and escalating antifungal resistance 15 – 17 . These challenges underscore the pressing need for new antifungal agents with distinct modes of action 18 . To address this gap, a library of 20,000 compounds was screened to identify new compounds with anti- Candida albicans activity. Here, we report the discovery and characterization of four structurally distinct novel compounds with potent antifungal activity, namely Z1199266541 (Z41), Z1024453766 (Z66), Z56842335 (Z35), and Z126932704 (Z04). Hereafter, these compounds are referred to as Z41, Z66, Z35, and Z04 throughout the text and figures. Compound Z41 displayed activity against C. albicans , Candida dubliniensis , and C andida tropicalis . In contrast, Z66 and Z04 exhibited broader antifungal spectra, with Z66 additionally demonstrating activity against Cryptococcus neoformans , Aspergillus fumigatus , and S. aureus . Furthermore, all four compounds show promise in combination therapies. Among them, Z35 showed the most favorable in vivo potential in a Galleria mellonella infection model. Its antifungal activity decreases progressively with increasing concentrations of iron, copper, and zinc, indicating that the compound disrupts fungal metal homeostasis. These metals play essential roles in cellular metabolism, the oxidative stress response, and virulence, and are tightly regulated 19 – 22 . Consistent with a metal-dependent mode of action, the addition of metal chelators enhanced the compound’s activity, and the chemical structure of the compound contains two strong chelating moieties. Interestingly, Z35 selectively inhibits C. albicans , and to a lesser extent, C. dubliniensis , which is unexpected for a metal-chelating compound that would typically be predicted to exert broad-spectrum antifungal activity. This suggests that species-specific aspects of metal acquisition, regulation, or compound uptake may contribute to its mechanism of action. Altogether, our results highlight a promising and mechanistically intriguing avenue for further investigation. Results Fungal pathogens pose a growing threat to human health, yet antifungal drug discovery remains limited, resulting in an urgent need for new antifungal agents 18 . To address this need, a structurally diverse library of 20,000 compounds was screened against a leading opportunistic fungal pathogen of humans, Candida albicans, at 50 µM, as previously described 23 (Fig. 1 A). Here, we investigate four promising and structurally distinct compounds identified in this screen: (Z41), (Z66), (Z35), and (Z04). These compounds had not previously been reported to exhibit antimicrobial bioactivity. The structures of the four hits are depicted in Fig. 1 B. Fig. 1. Open in a new tab Compounds with antimicrobial activity. ( A ) Four hit compounds were identified from a high-throughput screen of 20,000 compounds at 50 µM as previously described 23 : Z1199266541 (Z41), Z1024453766 (Z66), Z56842335 (Z35), and Z126932704 (Z04). Absorbance was measured after 24 h, and the percent growth was calculated by normalizing the absorbance of the wells with compound to that of the untreated controls. A rib1Δ/Δ strain was used as a positive control because it mimics the effect of a compound that inhibits an essential pathway, specifically riboflavin biosynthesis, which represents an attractive target for the development of novel antifungal drugs 25 . ( B ) Structures of prioritized compounds. ( C ) A two-fold dose–response assay was conducted in various media, and growth was assessed after 48 h. The relative growth of two averaged technical repeats is shown. ( D ) Prioritized compounds were tested against a panel of prevalent Candida species, C. neoformans , A. fumigatus , Escherichia coli and Staphylococcus aureus . Growth was assessed after 48 h except for C. neoformans , which was incubated for 72 h. Two technical repeats were averaged, and the relative growth was shown. Afterward, cidality was assessed by stamping medium from dose–response assay plates onto compound-free YPD agar (fungi) or MH agar (bacteria). These plates were grown for 48 h at 30 °C or 24 h for A. fumigatus and the bacterial species, which were grown at 35 °C and 37 °C, respectively. ( E ) The bioactivity and cidality of compound Z04 were evaluated against an azole-tolerant (CaCi-2) and an azole-resistant strain (CaCi-17), as described in panel D. ( F ) Checkerboard assays of compounds with caspofungin against C. albicans SC5314 are shown. Absorbance at 600 nm was measured after 48 h. The relative growth of two averaged technical repeats is shown. The compounds were tested in two-fold dose–response assays against C. albicans in RPMI, YPD, and synthetic complete (SC) media with or without 10% fetal bovine serum as indicated. Growth was quantified by measuring the absorbance at 600 nm after 48 h of incubation at 30 °C. Three out of four compounds exhibited different bioactivity depending on the media used. Compounds Z66 and Z04 demonstrated activity in all media tested (Fig. 1 C). Given the activity of all compounds in SC medium, subsequent experiments were continued under these conditions. To evaluate the antimicrobial spectrum of activity, the compounds were tested against a panel of clinically relevant fungal and bacterial strains (Fig. 1 D). All strains used in this study are listed in Supplementary Table 1 . Z41 displayed activity against C. albicans , Candida dubliniensis , and C andida tropicalis . In contrast, Z66 and Z04 exhibited broader antifungal spectra, with Z66 additionally demonstrating activity against Cryptococcus neoformans , Aspergillus fumigatus , and S. aureus . Z35 exhibited activity against C. albicans and C. dubliniensis. However, inhibition of C. dubliniensis was only visible at 24 h and had disappeared by 48 h, as this compound’s activity decreases over time. (Fig. 1 D (48 h) and S1B (24 h)). Only Z66 and Z04, the compounds that exhibited the broadest spectrum of activity, showed fungicidal activity against certain species (Fig. 1 D and S1A). The compounds were also tested against fluconazole-tolerant and -resistant C. albicans strains, CaCi-2 and CaCi-17, respectively 24 (Figs. 1 E and S1 C). Our findings showed that only Z04 retained fungicidal activity against the fluconazole-resistant C. albicans strain. Finally, checkerboard assays were performed to assess if the prioritized compounds showed enhanced activity in the presence of common antifungal drugs, such as fluconazole and caspofungin. The four compounds showed an additive effect in combination with caspofungin with FIC 80 values ≥ 0.5 and < 1.0 (Fig. 1 F), and indifferent effects in the presence of fluconazole with FIC 80 values ranging from 1 to 1.5 (Fig. S1 D). Next, we assessed the toxicity and in vivo efficacy of the compounds using a Galleria mellonella model. G. mellonella larvae were bred in-house, and ten healthy 6th-instar larvae of 300 ± 50 mg were selected for each condition of the experiment. During the experiment, the larvae were housed individually in 12-well plates at 37 °C, without food and in the dark. To evaluate toxicity, larvae were injected daily with 10 µL of each compound or vehicle control directly through the last proleg of the larvae into the hemocoel using a Hamilton syringe (model 701SN, 31 gauge). Larvae were treated daily with 50 mg/kg of the compound, a relatively high dose selected to assess host toxicity at elevated exposure levels and to maximize the likelihood of detecting antifungal efficacy in subsequent experiments 26 – 28 . At this dose of 50 mg/kg, none of the tested compounds caused a significant reduction in survival, in contrast to the amphotericin B control, which resulted in substantial mortality (Fig. 2 A). This suggests that the compounds do not induce severe toxicity in this model. However, larval health (scored based on movement, melanization, and survival as previously described 26 ) declined with daily administration of Z66, Z35, and Z04 (Fig. 2 B). For amphotericin B, health deterioration was evident early in the experiment, whereas Z35 and Z66 only caused a significant reduction in health scores on the final day. Fig. 2. Open in a new tab Toxicity and treatment efficacy studies in Galleria mellonella . ( A ) Survival curves of 6 th -instar G. mellonella larvae (300 ± 50 mg) injected daily with 50 mg/kg of compound or vehicle control. Data are represented as the mean ± standard error of the mean (SEM) (n = 10). Statistical significance was determined using the Log-rank (Mantel-Cox test). ( B ) Health scores of the same larvae as in ( A ). The mean ± SEM is shown. Statistical analysis was performed using two-way ANOVA with Dunnett correction, and longitudinal effects between vehicle control and treatment groups were determined using two-way ANOVA. ( C ) Experimental set-up for assessing treatment efficacy in G. mellonella . ( D ) Survival curves of G. mellonella larvae infected with 6 × 10 4 bioluminescent C. albicans cells/larvae with daily compound treatment of 50 mg/kg or vehicle. Data are shown as mean ± SEM. Statistical significance was assessed using the Log-rank (Mantel-Cox) test. ( E ) Daily health scores of the larvae described in ( D ). The mean ± SEM is shown. Statistical analysis was performed using two-way ANOVA with Dunnett correction, and longitudinal effects between vehicle control and treatment groups were determined using two-way ANOVA. ( F ) Fungal burden over time, measured by BLI. For deceased larvae, the last BLI measurement prior to death was included in the plot, but these values were excluded from statistical analysis. Data are shown as mean ± SEM. Statistical analysis was performed using mixed-effects analysis with multiple comparisons and Dunnett correction, and longitudinal effects between DMSO and treated conditions were determined using mixed-effects analysis with multiple comparisons and Šidák correction. */#, p < 0.05, **/## P < 0.01, ***/### P < 0.001; and ****/#### P < 0.0001, with significant longitudinal effects marked using #. To evaluate the in vivo antifungal efficacy, larvae were injected with 6 × 10 4 cells of a bioluminescent C. albicans reporter strain 27 and were treated daily with 50 mg/kg of compound or vehicle control (Fig. 2 C). The survival, health, and fungal load were monitored daily. Ten minutes before every bioluminescence imaging (BLI) session, the larvae were injected with 10 µL of 40 µg/g D-Luciferin into the hemocoel and were transferred to a black 12-well plate with a transparent bottom (IBL Baustoff + Labor GmbH, Austria). Subsequently, they were incubated at 37 °C for 10 min 23 , 26 , 28 . Bioluminescence emission was measured using an IVIS Spectrum imaging system (Revvity), as previously described 23 , 28 . Among the tested compounds, Z35 demonstrated the most promising activity. It improved survival (Fig. 2 D), maintained significantly higher health scores throughout the experiment (Fig. 2 E), and reduced fungal burden longitudinally, as measured by BLI, with a clear treatment effect observed as early as 24 h post-infection (p.i.) (Fig. 2 F). Furthermore, larvae treated with Z35 showed the highest survival rate (90%), compared to 60% for the vehicle control, 10% for Z41, 30% for Z66, and 0% for Z04. Notably, compounds Z41, Z66, and Z04 caused increased mortality in infected larvae compared to the vehicle control. This likely reflects the combined physiological stress of C. albicans infection and compound exposure, as these compounds showed less adverse effects in uninfected larvae. Compound Z66 also induced a significant longitudinal reduction in fungal burden, with a significant decrease observed at day 4 p.i. However, this compound was associated with reduced survival and overall health, indicating potential toxicity. The remaining compounds did not significantly reduce fungal burden. In conclusion, Z35 demonstrated the most favorable in vivo profile, combining antifungal efficacy with low toxicity in the G. mellonella model. To elucidate the mode of action of Z35, the compound with the strongest in vivo potential, we explored the impact of metal ions on its antifungal activity. Given that the compound lost efficacy in YPD and serum, we hypothesized that metal ions could interfere with its activity. To test this, we assessed growth inhibition in the presence of biologically relevant metals at concentrations near their maximal tolerated levels 29 . Compounds were added at concentrations that inhibited fungal growth by ~ 85% under metal-free conditions. The addition of FeCl 3 , CuSO₄, and ZnSO₄ significantly restored growth (Fig. 3 A), suggesting that these ions mitigate the compound’s antifungal effect. Moreover, Z35 was inactive in YPD, however, supplementation with chelator bathophenanthrolinedisulfonate (BPS; Bathophenanthrolinedisulfonic acid disodium salt hydrate) restored antifungal activity (Fig. 3 B), supporting a role for metal availability in modulating compound efficacy. Dose–response assays further showed that increasing concentrations of FeCl 3 , CuSO₄, and ZnSO₄ progressively reduced the compound’s activity (Fig. 3 C), suggesting that Z35 may interact with or sequester these metals. Furthermore, the addition of BPS and ethylenediaminetetraacetic acid (EDTA) enhanced compound activity. Notably, both EDTA and BPS can also influence C. albicans growth by limiting the availability of essential metals. The absolute growth values of Fig. 3 B and C are shown in supplementary Fig. 2 . Fig. 3. Open in a new tab Z35 loses antifungal activity in the presence of iron, copper, and zinc. ( A ) Relative growth of C. albicans cultured in SC medium under various treatment conditions: untreated, treated with 100 µM Z35, 200 µM BPS (positive control), 200 µM EDTA, or 5 µg/mL fluconazole (negative control). Treatment concentrations were selected to inhibit approximately 85% of growth. To assess whether serum or specific metals could mitigate compound activity, cultures were grown in SC medium alone or supplemented with 10% serum, 1 mM FeCl 3 , 250 µM FeSO 4 , 1 mM MgSO 4 , 250 µM CuSO 4 , or 125 µM ZnSO 4 . Growth is measured by absorbance at 600 nm after 48 h at 30 °C. Data represent the mean ± standard deviation of two technical replicates. Statistical analysis was performed using two-way ANOVA with Dunnett’s multiple comparisons test. *p < 0.05, **p < 0.01, ****p ≤ 0.0001. ( B ) Two-fold compound dilutions of Z35 in YPD medium supplemented with increasing concentrations of BPS. Growth was assessed by measuring absorbance at 600 nm after 48 h at 30 °C, and the relative growth was plotted. ( C ) Two-fold compound dilutions with Z35 in SC medium supplemented with increasing concentrations of FeCl 3 , CuSO 4 or ZnSO 4 , in the presence or absence of 50 µM EDTA or 50 µM BPS. Growth was assessed by measuring absorbance at 600 nm after 48 h at 30 °C, and the relative growth was plotted. Consistent with our observations, Z35 contains two strong chelating moieties, and we hypothesize that its nitrogen atoms serve as key coordination sites for metal binding, enabling the compound to chelate Fe 3 , Cu 2 ⁺, and Zn 2 ⁺. Discussion and conclusion This work characterized four compounds with novel antifungal activity identified through a high-throughput screen: Z41, Z66, Z35 and Z04. Compound Z41 displayed activity against C. albicans , Candida dubliniensis , and C andida tropicalis . In contrast, Z66 and Z04 exhibited broader antifungal spectra, with Z66 additionally demonstrating activity against Cryptococcus neoformans , Aspergillus fumigatus , and S. aureus . Z35 exhibited activity against C. albicans as well as against C. dubliniensis , albeit to a lesser extent. All compounds showed additive effects when combined with caspofungin, suggesting potential for combination therapy. Among these compounds, Z35 showed the most favorable in vivo potential in a G. mellonella model, reducing fungal burden and improving overall larval health. Our results show that Z35’s antifungal activity is modulated by the presence of iron, copper, and zinc. These essential micronutrients are involved in a wide range of biological functions, including cellular metabolism, the oxidative stress response, and virulence 19 – 22 . Supplementation with these metals progressively reduced the antifungal activity of Z35, indicating that metal availability modulates compound efficacy. Conversely, the addition of the metal chelators BPS or EDTA enhanced Z35 activity in both YPD and SC media. Because EDTA and BPS can themselves affect C. albicans growth by limiting the availability of essential metals, the enhanced activity observed in their presence may reflect multiple contributing factors. These include reduced metal availability, potential synergistic effects between metal limitation and compound exposure, or additive stress imposed by the chelators 30 . Structurally, Z35 contains two nitrogen-containing moieties that could serve as coordination sites for metal binding, providing a possible explanation for the observed sensitivity of compound activity to metal availability. While our data are consistent with a model in which Z35 interacts with biologically relevant metal ions, further biochemical studies will be required to determine whether the compound directly chelates metals or instead perturbs metal homeostasis through a more indirect mechanism. Moreover, while interference with metal availability appears to be an important component of Z35 activity, additional metal-independent mechanisms cannot be excluded. Further studies will therefore be required to determine whether the antifungal effect primarily results from extracellular metal chelation or from a more direct intracellular interaction with fungal targets. Interestingly, Z35 exhibited a narrow spectrum of activity, which is unusual for compounds whose activity depends on metal chelation, as such mechanisms might be expected to affect multiple microbial species. The selective inhibition of C. albicans , and to a lesser extent C. dubliniensis suggests that additional species-specific factors contribute to its mode of action. These may include differences in metal acquisition pathways, regulation of metal homeostasis, intracellular metal buffering capacity, or compound uptake and accumulation 31 . Furthermore, the narrow spectrum of activity of Z35 may underlie the compound’s minimal effect on host cells, as reflected by the reduced fungal burden and improved host health in the infection model, mitigating concerns about systemic metal chelation. However, this should be further investigated in additional infection models. Interference with metal homeostasis represents an attractive antifungal strategy, as pathogens must carefully balance the acquisition, storage, and detoxification of metal ions during infection 31 , 32 . At the same time, host-imposed nutritional immunity limits metal availability by actively sequestering essential micronutrients to restrict microbial growth 32 , 33 . Consequently, compounds that exploit vulnerabilities in fungal metal acquisition or utilization may be especially effective in host environments with limited metal availability, such as the skin or mucosal surfaces 34 . Metal chelators are often considered unsuitable for systemic antifungal therapy as they can disrupt host metal homeostasis, and they are therefore mostly used to treat superficial fungal infections. However, there is a precedent for the therapeutic use of metal chelators in systemic settings, particularly by enhancing the efficacy of other antimicrobial drugs 35 , 36 . For example, EDTA can enhance the efficacy of amphotericin B in an invasive pulmonary aspergillosis rat model, without causing observable toxicity 35 , 36 . Consistent with this concept, exploring the combination treatment of Z35 and caspofungin in a systemic setting would be interesting. This is especially true given the encouraging results of Z35’s in vivo efficacy and absence of detectable host toxicity. While several antifungal compounds exploit metal chelation, Z35 seems to be mechanistically distinct from other known classes such as pyridinones 37 , 38 , thiosemicarbazone derivatives 39 , siderophore-mimetic compounds (e.g., VL-2397) 40 , novel chelating compound (e.g. APC6) 41 , and adjuvant chelators such as DTPA 35 . Overall, while interference with metal availability appears to represent an important component of Z35 activity, further work will be required to fully elucidate its mechanism of action. The unusual specificity of Z35 toward C. albicans , together with its in vivo efficacy, highlights a promising yet mechanistically intriguing avenue for further investigation. Methods Strains and growth conditions All strains used in this study are listed in Table 1 . Strains were grown in SC medium consisting of 0.19% yeast nitrogen base without amino acids (Formedium), 0.079% complete supplement mixture (MP Biomedicals), and 2% glucose with an adjusted pH of 5.5. RPMI (1.04% RPMI 164 (Thermo Fisher Scientific), 3.453% MOPS, 2% glucose at pH 7) and YPD (1% yeast extract, 2% peptone, 2% glucose) media were used when indicated. All fungal strains were grown at 30 °C except for A. fumigatus . This fungus was first grown at 26 °C on potato dextrose agar (0.4% potato extract, 1.5% agar and 2% glucose) for six days, and then spores were collected according to the method described by Debonne et al. 42 . In short, the spores were scraped from the petri dish by adding 5 mL of cold 0.1% Tween80-H 2 O, and subsequently the spore solution was filtered twice using a sterile cotton filter to remove agar. The solution was centrifuged for 15 min at 3750 rpm at 4 °C. The supernatants was removed and resuspended in 25 mL cold 0.1% Tween80-PBS. The solution was centrifuged again for 15 min at 3750 rpm at 4 °C and the pellet was resuspended in 25 mL cold PBS. The spores were counted in a bürker chamber. The spores were stored at 4 °C for no more than one week. E. coli and S. aureus were grown on Mueller–Hinton broth at 37 °C. Table 1. Strains used in this study. Strain name Species Strain ID Genotype Reference SC5314 C. albicans Cat#MYA-2876 Wild type strain, clinical Candida albicans isolate ATCC 43 rib1Δ/Δ C. albicans JN19 SC5314 with rib1∆/∆ 25 bioluminescent C. albicans reporter strain C. albicans 4222 CIP10 NoHA + optimized firefly luciferase 27 CaCi-2 C. albicans CaLC79 Clinical isolate 44 CaCi-17 C. albicans CaLC91 Clinical isolate 44 CD36 C. dubliniensis 36 CdLC743 Clinical isolate 45 C. tropicalis 1685 C. tropicalis 1685 CtLC575 Clinical isolate Heitman Lab ATCC® 22019™ C. parapsilosis CpLC6144 Clinical isolate ATCC B8441 C. auris 0387 CauLC5294 Clinical isolate Clade I 46 , 47 C. krusei 1680 C. krusei 1680 CkLC571 Clinical isolate Heitman Lab BG2 C. glabrata CgLC1002 Clinical isolate 48 H99 C. neoformans CnLC3142 Clinical isolate 49 A. fumigatus A. fumigatus FY 554 Clinical isolate Katrien Lagrou lab MG1655 Escherichia coli Escherichia coli BS 82 WT 50 ATCC® 25923™; subsp. aureus Rosenbach S. aureus BS 103 Reference strain for antimicrobial tests ATCC Open in a new tab To assess the impact of metal ions on antifungal activity, FeCl₃, CuSO₄, and ZnSO₄ were added to the medium where indicated, either alone or in combination with the metal chelators EDTA (Sigma) and BPS (Sigma). High-throughput screen A library of 20,000 drug-like compounds, acquired from Enamine and made available through the VIB screening core Ghent was screened at 50 µM against C. albicans SC5314 in LoFlo medium at 30 °C, as previously described 23 . SC5314 and rib1Δ/Δ (positive control) were grown overnight and subsequently washed three times in LoFlo medium. The rib1Δ/Δ strain was used as a positive control because it mimics the effect of a compound that inhibits an essential pathway, specifically riboflavin biosynthesis, which represents an attractive target for the development of novel antifungal drugs 25 . The cultures were diluted to OD 600 0.1 in 384 well plates. An echo acoustic dispenser was used to add compound or DMSO to the plates, which were subsequently incubated for 24 h at 30 °C with shaking before measuring the OD 600 . Susceptibility testing Two-fold dose–response assays were performed in either 96-well or 384-well plates and incubated at 30 °C in dark static conditions. Overnight yeast cultures were diluted to a final density of 2 × 10 3 cells per well, and serial two-fold dilutions of compounds were added. Optical density at 600 nm was measured at designated time points using a spectrophotometer (Molecular Devices). Growth inhibition was reported after 48 h to capture sustained antifungal or antibacterial effects and to account for potential regrowth. For compound Z35, a visible decrease in activity was observed over time; therefore, results at both 24 h and 48 h are reported. For C. neoformans , growth was assessed after 72 h to accommodate its slower growth kinetics. For Aspergillus fumigatus , dose–response assays were performed in SC medium at 35 °C using an inoculum of 1.5 × 10 5 spores/mL. Bacterial dose–response assays were performed with 5 × 10 5 CFU/mL in Mueller–Hinton broth, and the plates were incubated at 37 °C. Data analysis included background subtraction using medium-only controls, averaging of technical replicates, and normalization to the no-drug control. Relative growth values were visualized as heat maps. To evaluate compound cidality, treated cultures were stamped onto drug-free YPD agar plates or Mueller–Hinton agar for bacterial growth. Growth was assessed after 48 h for fungal growth and 24 h for bacterial growth. Checkerboard assays were performed similarly to the susceptibility assays, but two compound gradients were added to the plate. Toxicity and efficacy tests in Galleria mellonella G. mellonella larvae were bred in-house, and ten healthy 6th-instar larvae of 300 ± 50 mg were selected for each condition of the experiment. During the experiment, the larvae were housed individually in 12-well plates at 37 °C, without food and in the dark. To evaluate toxicity, 10 µL of compound or vehicle was injected with a Hamilton syringe (model 701SN, 31 gauge; Hamilton Company, Switzerland) through the last proleg into the hemocoel of the larvae. The larvae were injected daily and were monitored for five to six days. The health score was determined by assessing the survival, movement, and melanization of the larvae as previously described 26 . To evaluate the efficacy of eradicating C. albicans in vivo, a bioluminescent C. albicans reporter strain 27 was used to monitor fungal burden over the course of the infection. The larvae were infected with 6 × 10 4 bioluminescent C. albicans reporter cells and were treated every day of the experiment with 50 mg/kg compound or vehicle control. This dosage was selected based on previously reported antifungal studies in the Galleria mellonella model 51 – 53 and does not directly correspond to in vitro data, as direct extrapolation is difficult, as pharmacokinetic parameters in this model are not sufficiently characterized. The survival and health, and fungal load were monitored daily. Ten minutes before every BLI imaging session, the larvae were injected 10 µL of 40 µg/g D-Luciferin into the hemocoel and they were transferred to a black 12-well plate with a transparent bottom (IBL Baustoff + Labor GmbH, Austria) and were incubated at 37 °C for 10 min 23 , 26 , 28 , 54 . Bioluminescence emission was measured with an IVIS Spectrum imaging system (Revvity) and five consecutive images were taken with open filter settings, F/stop 1, subject height 0.5 cm, medium binning, and a 30 s exposure time per image. Living Image Software (version 4.5.4) was used to quantify the total photon flux (p/s) per larva by defining a circular region of interest (ROI) with a 2.5 cm diameter. Supplementary Information Supplementary Information. (632.3KB, docx) Acknowledgements We are grateful to Dr. Nicole Robbins and Prof. Leah E. Cowen for their support and for providing valuable feedback on the manuscript. Furthermore, we acknowledge the excellent support with animal and imaging facilities by Kasia Błażejczyk. J.N. was supported by a PhD fellowship strategic basic research, a travel grant for a long research stay abroad by the Fund for Scientific Research Flanders (1S18121N and V410324N) and by a personal grant from KU Leuven VTI-24-00176. E.V. was supported by a PhD fellowship strategic basic research by FWO (1SF2224N). This work was supported by the KU Leuven Research Council (C14/22/075) to P.V.D. and C3/23/005 to G.V.V. Author contributions Conceptualization: J.N.; Formal analysis: J.N.; Funding Acquisition: J.N., G.V.V. and P.V.D.; Investigation: J.N., and L.V.C. Methodology: J.N., E.V., and G.V.V. Resources: E.V., G.V.V., and P.V.D.; Supervision: J.N., G.V.V., and P.V.D.; Writing – original draft: J.N.; All authors edited and/or approved the manuscript. Data availability All data supporting the findings of this study are available in the paper and its Supplementary material. Declarations Competing interests The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References 1. Guarner, J. & Brandt, M. E. Histopathologic diagnosis of fungal infections in the 21st century. Clin. Microbiol. Rev. 24 , 247–280 (2011). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 2. Casadevall, A. Fungal diseases in the 21st century: The near and far horizons. Pathog. Immun. 3 , 183–196 (2018). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 3. Brown, G. D. et al. Hidden killers: Human fungal infections. Sci. Transl. Med. 4 , 165rv13 (2012). [ DOI ] [ PubMed ] [ Google Scholar ] 4. Pathakumari, B., Liang, G. & Liu, W. Immune defence to invasive fungal infections: A comprehensive review. Biomed. Pharmacother. 130 , 110550 (2020). [ DOI ] [ PubMed ] [ Google Scholar ] 5. Denning, D. W. Global incidence and mortality of severe fungal disease. Lancet. Infect. Dis. 10.1016/S1473-3099(23)00692-8 (2024). [ DOI ] [ PubMed ] [ Google Scholar ] 6. Noble, S. M., Gianetti, B. A. & Witchley, J. N. Candida albicans cell-type switching and functional plasticity in the mammalian host. Nat. Rev. Microbiol. 15 , 96–108 (2017). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 7. Nobile, C. J. & Johnson, A. D. Candida albicans biofilms and human disease. Annu. Rev. Microbiol. 69 , 71–92 (2015). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 8. Pfaller, M. A. & Diekema, D. J. Epidemiology of invasive candidiasis: A persistent public health problem. Clin. Microbiol. Rev. 20 , 133–163 (2007). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 9. Organization GWH. 2022. WHO fungal priority pathogens list to guide research, development and public health action. Licence: CC BY-NC-SA 30 IGO. 10. Deorukhkar, S. C., Saini, S. & Mathew, S. Non- albicans Candida infection: An emerging threat. Interdiscip. Perspect. Infect. Dis. 2014 , 615958 (2014). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 11. Carolus, H. et al. Genome-wide analysis of experimentally evolved Candida auris reveals multiple novel mechanisms of multidrug resistance. MBio 10.1128/mBio.03333-20 (2021). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 12. Vale-Silva, L. A. & Sanglard, D. Tipping the balance both ways: Drug resistance and virulence in Candida glabrata . FEMS Yeast Res. 15 , fov025 (2015). [ DOI ] [ PubMed ] [ Google Scholar ] 13. Richardson, M. D. Changing patterns and trends in systemic fungal infections. J. Antimicrob. Chemother. 56 (Suppl 1), i5–i11 (2005). [ DOI ] [ PubMed ] [ Google Scholar ] 14. Rodrigues, M. L. & Nosanchuk, J. D. Fungal diseases as neglected pathogens: A wake-up call to public health officials. PLoS. Negl. Trop. Dis. 14 , e0007964 (2020). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 15. Robbins, N., Wright, G. D. & Cowen, L. E. Antifungal drugs: The current armamentarium and development of new agents. Microbiol. Spectr. 10.1128/microbiolspec.FUNK-0002-2016 (2016). [ DOI ] [ PubMed ] [ Google Scholar ] 16. Pierce, C. G., Srinivasan, A., Ramasubramanian, A. K. & Lopez-Ribot, J. L. From biology to drug development: New approaches to combat the threat of fungal biofilms. Microbiol. Spectr. 10.1128/microbiolspec.MB-0007-2014 (2015). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 17. Fisher, M. C., Hawkins, N. J., Sanglard, D. & Gurr, S. J. Worldwide emergence of resistance to antifungal drugs challenges human health and food security. Science 360 , 739–742 (2018). [ DOI ] [ PubMed ] [ Google Scholar ] 18. Pierce, C. G. & Lopez-Ribot, J. L. Candidiasis drug discovery and development: New approaches targeting virulence for discovering and identifying new drugs. Expert Opin. Drug Discov. 8 , 1117–1126 (2013). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 19. Garcia-Santamarina, S. & Thiele, D. J. Copper at the fungal pathogen-host axis. J. Biol. Chem. 290 , 18945–18953 (2015). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 20. Festa, R. A. & Thiele, D. J. Copper: An essential metal in biology. Curr. Biol. 21 , R877–R883 (2011). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 21. Chen, L., Min, J. & Wang, F. Copper homeostasis and cuproptosis in health and disease. Signal Transduct Target Ther 7 , 378 (2022). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 22. Wilson, D. The role of zinc in the pathogenicity of human fungal pathogens. Adv. Appl. Microbiol. 117 , 35–61 (2021). [ DOI ] [ PubMed ] [ Google Scholar ] 23. Nysten, J. et al. Novel antifungal compound Z247611722 exhibits antifungal activity by inhibiting serine palmitoyltransferase. Res. Sq. 10.21203/rs.3.rs-6810020/v1 (2025). 24. White, T. C. Increased mRNA levels of ERG16 , CDR , and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob. Agents Chemother. 41 , 1482–1487 (1997). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 25. Nysten, J. et al. The riboflavin biosynthetic pathway as a novel target for antifungal drugs against Candida species. MBio 15 , e0250224 (2024). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 26. Vanhoffelen, E. et al. Powerful and real-time quantification of antifungal efficacy against triazole-resistant and -susceptible Aspergillus fumigatus infections in Galleria mellonella by longitudinal bioluminescence imaging. Microbiol. Spectr. 11 , e0082523 (2023). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 27. Jacobsen, I. D., Luttich, A., Kurzai, O., Hube, B. & Brock, M. In vivo imaging of disseminated murine Candida albicans infection reveals unexpected host sites of fungal persistence during antifungal therapy. J. Antimicrob. Chemother. 69 , 2785–2796 (2014). [ DOI ] [ PubMed ] [ Google Scholar ] 28. Vanhoffelen, E. et al. Sensitive bioluminescence imaging of cryptococcosis in Galleria mellonella improves antifungal screening under in vivo conditions. Virulence 15 , 2327883 (2024). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 29. Fallah, S. et al. A chemical screen identifies structurally diverse metal chelators with activity against the fungal pathogen Candida albicans . Microbiol. Spectr. 12 , e0409523 (2024). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 30. Gomez, M. et al. Malfunctioning of the iron-sulfur cluster assembly machinery in Saccharomyces cerevisiae produces oxidative stress via an iron-dependent mechanism, causing dysfunction in respiratory complexes. PLoS ONE 9 , e111585 (2014). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 31. Simm, C. et al. Disruption of iron homeostasis and mitochondrial metabolism are promising targets to inhibit Candida auris . Microbiol. Spectr. 10 , e0010022 (2022). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 32. Fourie, R., Kuloyo, O. O., Mochochoko, B. M., Albertyn, J. & Pohl, C. H. Iron at the centre of Candida albicans interactions. Front. Cell Infect. Microbiol 8 , 185 (2018). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 33. Potrykus, J., Ballou, E. R., Childers, D. S. & Brown, A. J. Conflicting interests in the pathogen-host tug of war: Fungal micronutrient scavenging versus mammalian nutritional immunity. PLoS Pathog 10 , e1003910 (2014). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 34. Wright, J. A., Richards, T. & Srai, S. K. The role of iron in the skin and cutaneous wound healing. Front. Pharmacol. 5 , 156 (2014). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 35. Polvi, E. J. et al. Metal chelation as a powerful strategy to probe cellular circuitry governing fungal drug resistance and morphogenesis. PLoS Genet. 12 , e1006350 (2016). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 36. Hachem, R., Bahna, P., Hanna, H., Stephens, L. C. & Raad, I. EDTA as an adjunct antifungal agent for invasive pulmonary aspergillosis in a rodent model. Antimicrob. Agents Chemother. 50 , 1823–1827 (2006). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 37. Mena, L. et al. Two new compounds containing pyridinone or triazine heterocycles have antifungal properties against Candida albicans . Antibiotics 10.3390/antibiotics11010072 (2022). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 38. Kamauchi, H. et al. Synthesis and antifungal activity of polycyclic pyridone derivatives with anti-hyphal and biofilm formation activity against Candida albicans . Bioorg. Med. Chem. Lett. 37 , 127845 (2021). [ DOI ] [ PubMed ] [ Google Scholar ] 39. Al-Amiery, A. A., Kadhum, A. A. & Mohamad, A. B. Antifungal and antioxidant activities of pyrrolidone thiosemicarbazone complexes. Bioinorg. Chem. Appl. 2012 , 795812 (2012). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 40. Mammen, M. P. et al. First-in-human phase 1 study to assess safety, tolerability, and pharmacokinetics of a novel antifungal drug, VL-2397, in healthy adults. Antimicrob. Agents Chemother. 63 (2019). [ DOI ] [ PMC free article ] [ PubMed ] 41. Shinohara, T. et al. A zinc-chelating cyclic alkyl polyamine compound is efficient and safe in a murine model of multidrug-resistant Candida auris infection. Antimicrob. Agents Chemother. 69 , e0085625 (2025). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 42. Debonne EVB, F., De Leyn, I., Devlieghere, F. & Eeckhout, M. Validation of in-vitro antifungal activity of thyme essential oil on Aspergillus niger and Penicillium paneum through application in par-baked wheat and sourdough bread. LWT 10.1016/j.lwt.2017.09.007 (2018). [ Google Scholar ] 43. Gillum, A. M., Tsay, E. Y. & Kirsch, D. R. Isolation of the Candida albicans gene for orotidine-5’-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations. Mol. Gen. Genet. 198 , 179–182 (1984). [ DOI ] [ PubMed ] [ Google Scholar ] 44. White, T. C. The presence of an R467K amino acid substitution and loss of allelic variation correlate with an azole-resistant lanosterol 14alpha demethylase in Candida albicans . Antimicrob. Agents Chemother. 41 , 1488–1494 (1997). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 45. Sullivan, D. J., Westerneng, T. J., Haynes, K. A., Bennett, D. E. & Coleman, D. C. Candida dubliniensis sp. nov.: Phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV-infected individuals. Microbiology 141 (Pt 7), 1507–1521 (1995). [ DOI ] [ PubMed ] [ Google Scholar ] 46. Lockhart, S. R. et al. Simultaneous emergence of multidrug-resistant Candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses. Clin. Infect. Dis. 64 , 134–140 (2017). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 47. Lutgring, J. D. FDA-CDC antimicrobial resistance isolate bank: A publicly available resource to support research, development, and regulatory requirements. J. Clin. Microbiol. 56 (2018). [ DOI ] [ PMC free article ] [ PubMed ] 48. Cormack, B. P. & Falkow, S. Efficient homologous and illegitimate recombination in the opportunistic yeast pathogen Candida glabrata . Genetics 151 , 979–987 (1999). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 49. Granger, D. L., Perfect, J. R. & Durack, D. T. Virulence of Cryptococcus neoformans . Regulation of capsule synthesis by carbon dioxide. J. Clin. Invest. 76 , 508–516 (1985). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 50. Blattner, F. R. et al. The complete genome sequence of Escherichia coli K-12. Science 277 , 1453–1462 (1997). [ DOI ] [ PubMed ] [ Google Scholar ] 51. Piatek, M., Sheehan, G. & Kavanagh, K. Galleria mellonella : The versatile host for drug discovery, in vivo toxicity testing and characterising host-pathogen interactions. Antibiotics 10.3390/antibiotics10121545 (2021). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 52. Ignasiak, K. & Maxwell, A. Galleria mellonella (greater wax moth) larvae as a model for antibiotic susceptibility testing and acute toxicity trials. BMC Res. Notes 10 , 428 (2017). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 53. Mishra, B. et al. A substituted diphenyl amide based novel scaffold inhibits Staphylococcus aureus virulence in a Galleria mellonella infection model. Front. Microbiol. 12 , 723133 (2021). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 54. Vanhoffelen, E. et al. Combinations of posaconazole and tacrolimus are effective against infections with azole-resistant Aspergillus fumigatus . Front. Cell. Infect. Microbiol. 15 , 1550457 (2025). [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Supplementary Materials Supplementary Information. 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