https://orcid.org/0009-0008-0632-3747
Figures
Abstract
Cryptococcosis is a major health threat among immunocompromised individuals, resulting in life-threatening consequences secondary to brain invasion. The limited treatment options and the increased incidence of treatment failure and relapses urge the need for more effective therapies. In this study, we investigated the therapeutic potential of nitroxoline (NTX) in treating cryptococcal infection. NTX exhibited potent fungicidal activity against Cryptococcus neoformans/gattii clinical isolates, with an MIC90 of 1 µg/mL. In addition, NTX substantially inhibits the main virulence traits of cryptococcal cells, including the polysaccharide capsule, melanin production, and the urease enzyme, which are crucial for the infection progression. In exploring the potential mechanism of action, our results revealed the relationship between the metal-chelating properties of the NTX scaffold and its antifungal activity. Interfering metal chelation, either through structural modification or exogenous supplementation with varied divalent cations, significantly reduced the antifungal and anti-virulence activities of NTX. Given its potential therapeutic role, we evaluated the interaction between NTX and the clinically used antifungals amphotericin B, flucytosine and fluconazole. Additionally, we investigated the tendency of cryptococcal cells to develop resistance following repeated exposure to NTX, as well as its efficacy in the Caenorhabditis elegans nematode model of cryptococcal infection. Notably, cryptococcal cells exhibited consistent susceptibility to NTX upon repeated exposure to a subinhibitory concentration. In the C. elegans model, NTX significantly reduced the fungal burden of the infected worms, achieving a 2-log10 reduction, and consistently enhanced their survival compared to untreated group. These findings highlight the potential role of the NTX scaffold in combating cryptococcal infection that warrants further investigation.
Author summary
Nitroxoline (NTX) antibiotic has been prescribed for decades to treat acute and recurrent bacterial urinary tract infections. Its extensive urinary excretion has limited its therapeutic utility for invasive bacterial infections. However, its favorable safety profile, excellent oral bioavailability, and the low potential of resistance, have encouraged its repurposing against a diverse range of pathogens. In this study, we investigated NTX activity against Cryptococcus fungal pathogen that can disseminate to the central nervous system, leading to fatal meningoencephalitis in immunocompromised patients. The mortality rate of this opportunistic infection can exceed 60% especially in resource-limited regions. NTX exhibited potent fungicidal activity against proliferated cryptococcal cells, with an inhibitory activity on the main virulence factors involved in the initial colonization, including capsule formation, melanin pigmentation and urease enzyme. This anti-virulence activity is directly related to the metal chelation nature of the NTX scaffold, enabling the depletion of the intracellular content of essential cations. Furthermore, cryptococcal cells exhibited a negligible resistance tendency during continuous exposure to NTX. Overall, these findings demonstrate that NTX exerts both direct antifungal and anti-virulence activities on Cryptococcus through a metal chelation-dependent mechanism and highlight its potential role as a repurposed therapeutic for the treatment of cryptococcosis.
Citation: Alkashef NM, Khan AA, Seleem MN (2026) Investigating the therapeutic potential of the antibacterial drug nitroxoline in combating cryptococcal infection. PLoS Negl Trop Dis 20(8): e0014627. https://doi.org/10.1371/journal.pntd.0014627
Editor: Francelise Bridi Cavassin, Faculdades Pequeno Príncipe: Faculdades Pequeno Principe, BRAZIL
Received: March 18, 2026; Accepted: July 29, 2026; Published: August 17, 2026
Copyright: © 2026 Alkashef et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting information files.
Funding: This work was supported by the National Institutes of Health (R01AI141439 to MNS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Cryptococcosis is an opportunistic fungal infection that primarily affects individuals with compromised immune system. This invasive infection begins by inhaling the fungal spores of the C. neoformans/gattii species, which colonize the alveolar space, establishing an active infection or remaining dormant depending on the competence of the immune system [1,2]. The invading cryptococcal cells can then disseminate towards the central nervous system, resulting in life-threatening meningitis [3,4]. Annually, cryptococcal infections affect approximately 194,000 immunocompromised individuals worldwide, primarily HIV/AIDS patients, with a mortality rate exceeding 75.8%, especially in low to middle-income regions [5,6].
The intrinsic resistance of cryptococcal cells to echinocandins limits the current therapeutic arsenal to three systemic antifungals: amphotericin B (AmB), fluconazole (FLC), and flucytosine (5-FC) [7,8]. Current clinical guidelines predominantly rely on the combination of AmB and 5-FC to elicit early fungal clearance during the induction stage of treatment [9,10]. On the other hand, FLC has been proven to be effective in preventing relapses during both consolidation and maintenance stages of treatment [9]. However, the scarcity of the first-line antifungals, AmB and 5-FC, in resource-limited regions and their related toxicities, requiring continuous treatment monitoring, make it challenging to overcome the high fatality and escalating relapses of this infection with alternative FLC monotherapy, even with higher doses up to 1200 mg/daily [11–13].
Recent efforts to reposition approved drugs for cryptococcal infections have efficiently replenished the therapeutic pipeline with a variety of candidate molecules [14,15]. Previously, we identified the antifungal activity of the 5-nitro-8-hydroxyquinoline scaffold, nitroxoline (NTX), against the Cryptococcus pathogen through screening of 3,700 therapeutic molecules, including FDA-approved drugs and investigational molecules [16]. Over the past 50 years, NTX has been used primarily in combating acute and recurrent infections caused by a broad spectrum of urinary tract pathogens [17]. This antibacterial activity is considered to be related to its ability to chelate essential metals needed for the activity of bacterial RNA polymerase enzymes [18]. Its favorable safety profile and superior oral bioavailability have encouraged its repurpose against a diverse range of bacterial, viral, and parasitic pathogens [19–23]. However, its short plasma half-life and extensive urinary excretion have limited its clinical usage to urinary infections [24].
In this study, we investigated the therapeutic potential of NTX against the Cryptococcus yeast. NTX demonstrated potent antifungal activity against the tested cryptococcal isolates, with a fungicidal activity on proliferating cryptococcal cells. NTX also demonstrated potent inhibitory activity against the key virulence factors of cryptococcal cells involved in the alveolar colonization and dissemination. Additionally, we investigated the relationship between the metal chelation activity of the NTX scaffold and its antifungal properties. Most importantly, the tendency of cryptococcal cells to develop resistance to NTX upon repeated exposure was investigated to highlight its therapeutic potential. The efficacy of NTX was also evaluated in the Caenorhabditis elegans nematode model of cryptococcal infection.
Materials and methods
Strains, drugs, and chemicals
Clinical isolates of the C. neoformans/gattii complex were obtained from the American Type Culture Collection, ATCC (Manassas, VA, USA) and BEI Resources (Manassas, VA, USA). Prior to any experiment, fungal isolates were recovered from 20% glycerol stocks stored at −80°C on YPD agar (Becton, Dickinson and Company, MD, USA). Tested drugs including NTX (Cayman chemicals, MI, USA), NTX analogs (Mcule, CA, USA) and standard antifungals, including AmB (Chem-Impex International, IL, USA), FLC (Thermo Fisher, NC, USA), and 5-FC (Thermo Scientific, MA, USA), were solubilized in DMSO (Thermo Fisher, NC, USA) and stored at -20 °C. RPMI 1640 medium (Gibco, NY, USA) was prepared according to the manufacturer’s guidelines and was buffered with 3-(N-morpholino)propanesulfonic acid (Fisher Bioreagents, NJ, USA). Metal cations: CuSO4, ZnCl2, MgCl2, CaCl2, FeCl2, and MnCl2 (Sigma-Aldrich, MO, USA) were dissolved in sterilized double-distilled H2O.
In vitro evaluation of the antifungal activity
The minimum inhibitory concentration (MIC) for tested compounds and standard antifungals was determined using the standard microdilution technique following the Clinical and Laboratory Standards Institute (CLSI) guidelines M27-A3 for yeast [25]. Cryptococcal cells from overnight cultures in YPD broth were diluted in RPMI-MOPS medium to achieve a final density of 1 × 103 CFU/mL. The MIC was assessed after 72 hours of incubation at 35 °C. Complete growth inhibition, compared to the untreated culture, was used as the endpoint for both NTX and AmB, while a 50% reduction in fungal growth was considered the endpoint for fluconazole and 5-FC.
Microdilution checkerboard assay
The impact of combining NTX with standard antifungals, including AmB and FLC, was evaluated using the microdilution checkerboard method as previously described [16,26]. The efficacy of varied combinations was evaluated after incubation at 35 °C for 72 hrs. The optical density was measured at 540 nm and expressed relative to the growth of the untreated inoculum. The fractional inhibitory concentration index (ΣFICI) was calculated to interpret the interaction between combined drugs. Combined drugs were considered to interact synergistically if FICI values were ≤ 0.5. On the other hand, FICI values > 0.5 and 1 indicate additive interaction, whereas values between 1 and 2 or > 4 indicate indifferent and antagonistic interaction, respectively [27].
Time-kill assay
The impact of NTX on the growth kinetics of Cryptococcal cells was evaluated as previously described [28]. Logarithmically growing cells from an overnight culture of C. neoformans H99 in YPD broth were collected and adjusted to ~ 5 × 103 CFU/mL in RPMI-MOPS medium. Equal aliquots were treated with different concentrations of NTX and AmB, as standard antifungal, and were incubated at 35 °C for 72 hours. The number of viable cells was monitored at various points (0, 2, 4, 6, 12, 24, 48, and 72 hrs) by 1:10 diluting with PBS and platting on YPD agar.
Anti-virulence characterization
Urease activity.
Cells from an overnight culture of C. neoformans H99 strains were adjusted to a final density of ~1 × 104 CFU/mL in urease broth and incubated in the presence of sublethal concentrations of NTX (up to 0.5x MIC), alongside acetohydroxamic acid (3.33 mM) as a positive control, at 30 °C for 72 hours. Following the incubation period, cells were removed by centrifugation at 10,000 rpm for 5 minutes, and the absorbance of the cell-free aliquots was measured at 560 nm [29,30].
Induction of Melanin pigmentation.
The induction of melanin production was evaluated using liquid minimal medium (15 mM glucose, 10 mM MgSO4, 29.4 mM KH2PO4, 13 mM glycine, 3 µM thiamine) supplemented with 1 mM L-DOPA as previously described [29,31]. Minimal medium was inoculated with C. neoformans H99 strain (~1 × 104 CFU/mL) and equal aliquots were dispensed into 24-well microtiter plates in the presence of subinhibitory concentrations of NTX (up to 0.5x MIC), alongside 5-FC as a positive control. Plates were incubated in the dark at 30 °C and were monitored for 10 days. The mean gray value of the treated cultures was quantified in grayscale using ImageJ software as previously described [32,33].
Capsule size analysis.
Capsule formation was induced in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2 for 3 days in the presence of a sublethal concentration of NTX (0.5x MIC). AmB (0.0156 µg/mL) was included as a positive control. Cells were collected by centrifugation at 10,000 rpm for 5 minutes and were subjected to microscopic analysis using India ink (Thermo Scientific, MA, USA). Measurement of the whole cell diameter and cell body diameter was conducted using ImageJ software. The impact of varied treatments on capsule thickness was assessed for 50 cells from each treatment by subtracting the cell body diameter from the entire cell diameter [34–36].
PGFL assay
The effect of NTX treatment on the intracellular content of metal cations was evaluated using the fluorescent probe PhenGreen FL diacetate (PGFL), as previously described [37]. Logarithmically grown C. neoformans H99 were adjusted to ~1 × 106 CFU/mL in RPMI-MOPS medium and were treated with varying concentrations of NTX (up to 0.5 µg/mL) for 6 hours at 30°C. Cells were rinsed and incubated with 2.5 µM PGFL (Invitrogen, CA, USA) for 30 minutes. Following the incubation time, cells were rinsed again for three times with distilled water and transferred to an opaque 96-well microtiter plate. The fluorescence intensity was measured at excitation and emission wavelengths of 490 nm and 520 nm, respectively.
Drug resistance analysis
The tendency of cryptococcal cells to adapt to the activity of NTX was evaluated using frequent passaging as previously described [16,38,39]. C. neoformans H99 cells (~1x105 CFU/mL) in RPMI-MOPS medium were treated with a 0.5x MIC of the tested compounds, including 5-FC (2 μg/mL) as a positive control, for 48 hrs at 35°C. Treated cells were collected by centrifugation at 10,000 rpm for 5 minutes and were adjusted to the same density for the following passage. Frequent passaging of cryptococcal cells was continued for 20 passages, and the susceptibility of treated cells was evaluated after each passage.
The nematode model of Caenorhabditis elegans
The efficacy of NTX was evaluated in the C. elegans model as previously described [28,40]. Worms of C. elegans AU37 (Δglp-4; Δsek-1) were synchronized and allowed to reach L4 larval stage on a lawn of Escherichia coli OP50 strain on standard nematode growth medium [41]. Synchronized larva were then infected with C. neoformans H99 (~1x106 CFU/mL) in M9 buffer, supplemented with 50 μg/ml of gentamicin to inhibit the growth of E. coli OP50, for 6 hrs at 30 °C. Infected worms (300 worm/mL) were rinsed 5 times with an equal volume of M9 buffer and treated with different concentrations of NTX for 24 hrs at 30 °C. Treated worms were then rinsed, resuspended in M9 buffer, and were homogenized with 0.5 mm silicon/zirconia beads (BioSpec Products, OK, USA) using Next advance homogenizer (Next Advance, NY, USA) at 4800 cycle/minute for 6 minutes at room temperature. The fungal burden of differently treated worms was evaluated by diluting and plating their homogenate on gentamicin supplemented YPD agar (100 μg/mL). The survival of treated worms was also monitored for 10 days and data was presented using a Kaplan-Meier survival curve.
Statistical analysis
The statistical significance of the collected data was assessed by one-way analysis of variance (ANOVA) test for multiple comparisons and log-rank test for Kaplan-Meier survival curve using GraphPad Prism 8.0 Software (La Jolla, CA, USA). Data sets with p values of ≤ 0.05 were considered statistically significant.
Results
Validating the antifungal activity of NTX against Cryptococcus yeast
The activity of NTX was tested on a panel of 16 clinical isolates representing the C. neoformans/gattii complex. NTX exhibited potent inhibitory activity against the tested isolates, with an MIC90 of 1 µg/mL (Table 1). Additionally, the impact of NTX on the growth kinetics of cryptococcal cells was evaluated for 72 hours. NTX exerted concentration-dependent fungicidal activity (Fig 1A), depleting the entire population of the C. neoformans H99 strain within 6 hrs at 4x MIC.
(A) Dose-dependent effect of NTX on the growth kinetics of treated cryptococcal cells. Aliquots of H99 cultures (~5 × 103 CFU/mL) in RPMI-MOPS medium were treated with NTX at different concentrations (up to 4× MIC), along with AmB (1 µg/mL) as a control antifungal and DMSO (0.2%). Cell proliferation was monitored at the designated time points over 72 hours. The Lower limit of detection (LOD) was 1 CFU/mL, determined by platting 1 ml of treated aliquots on YPD agar. (B) Chemical structure of 5-nitro-8-hydroxyquinoline (NTX).
NTX exhibits potent anti-virulence activity on cryptococcal cells
We examined the impact of NTX on the key virulence traits of cryptococcal cells, including urease activity, melanin pigmentation, and capsule formation. Cryptococcal cells exhibited reduced urease activity when grown in urea-containing broth in the presence of sublethal levels of NTX (Fig 2A and 2C). Similarly, NTX significantly impaired the oxidation of L-DOPA to the dark melanin pigment, resulting in non-melanized cultures, even at low concentrations (Fig 2B and 2D).
(A) Urease activity of C. neoformans H99 cultures treated with different subinhibitory concentrations of NTX (up to 0.5x MIC) and DMSO (0.25%). Acetohydroxamic acid (AHA) was also included as a positive control at a concentration of 3.33 mM. The color intensity was measured at 560 nm and expressed relative to untreated cultures. Data represent the average of two independent experiments (5 replicates each), relative to the untreated cells. (B) Mean gray values of C. neoformans H99 cultures induced for melanin production in the presence of varied sublethal concentrations of NTX (up to 0.5x MIC), alongside 5-FC (0.0312 μg/mL) as a positive control and DMSO (0.25%). Data represent the average of two independent experiments. (C) Colored photograph for cell-free aliquots of C. neoformans H99 cultures in urea broth, in the presence of varied concentrations of NTX and AHA as a positive urease inhibitor. (D) Colored photograph for C. neoformans H99 in liquid minimal medium supplemented with L-dopa for 10 days at 30 °C, in the presence of different concentrations of NTX and 5-FC as a positive inhibitor for melanin pigmentation. Data was analyzed using one-way analysis of variance (ANOVA) for multiple comparison, and p values of ≤ 0.05 indicate statistical significance of compared data sets.
Additionally, we investigated the impact of NTX on the formation of capsular polysaccharide in cryptococcal cells grown under induction conditions. Cryptococcal cells treated with a sublethal concentration of NTX (0.5x MIC) produced significantly smaller capsules, with an average thickness of 2.13 ± 0.72 µm. On the other hand, untreated cryptococcal cells grown under the same conditions produced capsules with an average thickness of 4.59 ± 0.89 µm (Fig 3A and 3B).
(A) Capsule size measurement of cryptococcal cells grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (FBS) in the presence of subinhibitory concentration of NTX (0.5x MIC), AmB (0.0156 µg/mL) as a positive control, or DMSO (0.2%). Data represent the average measurement of 50 cells from each individually treated aliquots of C. neoformans H99 strain. (B) Corresponding light microscopy images for individually treated aliquots stained with India ink (1:1). Data was analyzed using one-way analysis of variance (ANOVA) for multiple comparison, and p values of ≤ 0.05 indicate statistical significance of compared data sets.
Metal chelation is a major contributor controlling NTX activity
Initially, we tested whether exogenous supplementation with various divalent cations would affect the antifungal activity of NTX. Supplementing RPMI-MOPS medium with increasing concentrations of copper ions significantly impaired NTX activity, resulting in a 32-fold increase in the MIC at 25 µM. Supplementation with Zn2+ and Fe2+ had a similar effect on NTX activity, resulting in 8- and 2-fold increases in its MIC, respectively. In contrast, the susceptibility of cryptococcal cells to NTX remained unchanged in the presence of other divalent cations, including Ca2+, Mn2+, and Mg2+, highlighting the metal preference of the NTX molecule (Fig 4A).
(A) The impact of divalent cation supplementation on the antifungal activity of NTX. The change in the MIC was evaluated in RPMI-MOPS medium supplemented with increasing concentrations of various divalent cations (up to 50 μM). (B) PGFL assay for the intracellular metal content of cryptococcal cells (~1 × 106 CFU/mL) treated with varied concentrations of NTX or DMSO (0.5%). (C) Effect of CuSO4 supplementation (12.5 µM) on the intracellular metal content of cryptococcal cells treated with NTX (0.25 µg/mL) and DMSO (0.25%). Data represent the average of two independent experiment relative to the untreated cells. (D) Effect of CuSO4 supplementation (12.5 µM) on the growth of C. neoformans H99 (~ 5 × 103 CFU/mL) in RPMI-MOPS medium after 72 hrs in the presence of 4x MIC of NTX (2 µg/mL) or DMSO (0.2%). Data was analyzed using one-way analysis of variance (ANOVA) for multiple comparison, and p values of ≤ 0.05 indicate statistical significance of compared data sets.
Secondly, we utilized the PhenGreen FL diacetate (PGFL) fluorescent probe to gain direct insight into the intracellular metal content of cryptococcal cells in response to NTX treatment. Cryptococcal cells demonstrated a significant dose-dependent increase in the PGFL fluorescence following exposure to different concentrations of NTX, indicating the stripping of essential metal cations (Fig 4B). This fluorescence upshift was significantly reversed upon supplementation with CuSO4 (12.5 µM), resulting in the quenching of PGFL in the presence of excess metal (Fig 4C). Consistently, supplementing RPMI-MOPS medium with CuSO4 (12.5 µM) rescued cryptococcal cells treated with NTX at 4x MIC (Fig 4D).
Furthermore, we tested whether the loss of the metal-chelating center would affect the antifungal activity of the NTX scaffold. All 4 tested analogs with disabled chelating activity due to the absence of the electron-donating 8-hydroxyl group within the chelation center of the NTX scaffold (Fig 5A), including 5-nitro-1,4-dihydroquinolin-4-one, 5-nitroquinoline, 8-methoxy-2-methyl-5-nitroquinoline, and 8-methyl-5-nitroquinoline, did not show antifungal activity against cryptococcal cells up to 64 µg/mL (Fig 5B). Similarly, these tested analogs did not affect the activity of urease enzyme or melanin pigmentation of treated cryptococcal cells (Fig 5C), highlighting the relationship between metal chelation and the antifungal attributes of NTX scaffold.
(A) Chemical structures of NTX analogs with modified chelation center. (B) Antifungal activity of the tested NTX analogs on C. neoformans H99 strain. OD was measured at 540 nm, and the growth intensity was presented relative to the control growth as a heat map. (C) Anti-virulence activity of tested NTX analogs. The upper and lower panels show the effects of the tested analogs on melanin production and urease enzyme activity of cryptococcal cells treated with 0.5x MIC of each analog.
Drug interaction with standard antifungals
The interaction between NTX and clinically used antifungals, including parenteral AmB and oral 5-FC and FLC, was evaluated using a microdilution checkerboard assay. Combining NTX with AmB showed indifferent interaction (FICI = 2) with no change in the individual MICs of both combined drugs (Fig 6A). On the other hand, NTX exhibited enhanced activity when combined with either 5-FC or FLC, resulting in an additive interaction with a FICI of 1 and 0.75, respectively (Fig 6B and 6C), Which highlights the beneficial outcome of these combinations.
The efficacy of the combination between NTX (0.125-4 μg/mL) and either AmB (0.0078-4 μg/mL), 5-FC (0.25-128 μg/mL), or FLC (0.0625-32 μg/mL) was evaluated on C. neoformans H99 strain using checkerboard microdilution. Following incubation for 72 hrs at 30°C, the optical density (OD) was measured at 540 nm, and growth intensity was presented relative to the control growth as heat maps. The fractional inhibitory concentration index (ΣFICI) was used to interpret the outcome combined drugs where FICI ≤ 0.5 or between > 0.5 and 1 indicate synergistic and additive interaction, respectively. FICI values between 1 and 2, or > 4 indicate both indifferent and antagonistic interactions, respectively.
Evaluation of resistance development to NTX treatment during serial passaging
The tendency of cryptococcal cells to develop resistance to NTX was evaluated by repeated exposure to a sublethal concentration (0.5 × MIC) of NTX, along with 5-FC as a positive control. Notably, cryptococcal cells showed consistent susceptibility to NTX with no change in initial MIC (0.5 μg/mL) over 20 consecutive passages (Fig 7A). In contrast, cells treated with a sublethal concentration of 5-FC showed a 16-fold increase in the initial MIC (4 μg/mL) during the first four passages.
A) Resistance-tendency analysis of cryptococcal cells to NTX. Aliquots of C. neoformans H99 strain in RPMI-MOPS (~1 × 105 CFU/mL) were continuously exposed to a subinhibitory concentration (0.5 MIC) of NTX, along with 5-FC (2 μg/mL) as a positive control. Cells from individually treated cultures were evaluated for their susceptibility following each passage, and the fold change in MIC was plotted versus the corresponding passage number. (B) Efficacy of NTX in the C. elegans model. Infected L4 larvae (300 worms per mL) were individually treated with NTX (up to 4x MIC), along with AmB and FLC as positive controls and DMSO (0.4%), for 24 hrs at 30°C. Treated worms were homogenized and plated onto gentamicin-supplemented YPD agar to assess the change in fungal burden. Data represent the average of two independent experiments (4 replicates each). Data was analyzed using one-way analysis of variance (ANOVA) for multiple comparison, and p values of ≤ 0.05 indicate statistical significance of compared data sets. (C) A Kaplan-Meier survival curve of C. elegans worm groups treated with NTX (up to 4x MIC), FLC (2x MIC) as a positive control or DMSO (0.4%). Statistical significance was evaluated using log-rank test, and **** indicates statistical significance at p value of ≤ 0.0001.
Efficacy of NTX in the Caenorhabditis elegans model of cryptococcal infection
The efficacy of NTX was further assessed in the C. elegans model, alongside the standard antifungals AmB and FLC. NTX treatment significantly reduced the fungal burden of the treated worms. Following treatment for 24 hours, NTX treatment at different concentrations (up to 4x MIC) achieved ~2 log10 reduction in the colony counting of homogenized worms compared with the untreated worms (Fig 7B). Similarly, AmB treatment depleted the fungal population within the treated worms, achieving ~ 2.35 log10 reduction compared with untreated worms. On the other hand, FLC (2x MIC) didn’t achieve a significant reduction in the fungal burden of treated worms (-0.71 log10). Next, we assessed whether NTX treatment would affect the survival of C. elegans worms infected with C. neoformans H99 (Fig 7C). At 10 days postinfection, the untreated larval group showed a survival rate of ~35.6% while groups treated with NTX at 2x MIC and 4x MIC exhibited a significantly enhanced survival rate of ~ 73.5% and 86.6% (p < 0.0001), respectively.
Additionally, we evaluated the impact of combination between FLC and NTX on the fungal burden and the survival of infected worms. Compared to untreated worm group, combining NTX at a sublethal level (0.5x MIC) with FLC (2x MIC) resulted in ~ 0.9 log10 reduction (S1A Fig). On the other hand, FLC/NTX combination didn’t affect the survival of treated worm groups (S1B Fig).
Discussion
Cryptococcus pathogen is the leading cause of fungal meningoencephalitis among immunocompromised individuals, accounting for 19% of HIV-related mortalities [5,6]. The treatment outcome of this invasive infection mainly relies on achieving early fungal clearance, particularly within the brain parenchyma and CSF [42]. The pharmacodynamic properties of the used antifungals and their dynamics within targeted body compartments are the main factors affecting treatment outcomes [43]. Combining intravenous AmB with oral 5-FC is the optimal treatment for CM infection due to its fungicidal activity and efficient penetration to the brain compartments [42,44]. However, limited access to these antifungals and the need for continuous treatment monitoring, especially with hepatic and renal comorbidities, urge the alternative use of FLC which results in treatment failure and relapses due to its fungistatic nature [7,45]. Hence, current clinical outcomes for cryptococcal infections, especially in resource-limited regions, underscore the need for more effective antifungals with improved tolerability and wider availability.
Repurposing drugs with established pharmacokinetic and safety profiles provides a fast track toward potentially effective antifungals, avoiding substantial costs related to preclinical development [46]. Structural optimization can further expedite the development of candidate entities by enhancing their efficacy, selectivity, and distribution within the targeted body compartments [47]. In this study, we identified the antifungal activity of NTX against Cryptococcus pathogen, with a MIC90 of 1 µg/mL on the tested panel of clinical isolates. In the time-kill assay, NTX exhibited potent fungicidal activity on proliferating cryptococcal cells, which is a notable advantage for candidate molecules targeting cryptococcal infections. Consistently, NTX has been reported to be active against other superficial and invasive fungal pathogens, including azole-resistant Aspergillus and multidrug-resistant Candida spp. [48–50], highlighting the spectrum of its antifungal activity.
Targeting the key virulence traits of cryptococcal cells is another advantage for NTX, further supporting its use during the consolidation and maintenance stages of treatment, in combination with oral FLC [51]. Among the existing antifungal arsenal, only 5-FC exhibits anti-virulence activity on cryptococcal cells by inhibiting melanin production, which may contribute to its efficacy when combined with AmB at the early treatment stage [31]. The survival of the cryptococcal cells within the alveolar space mainly depends on the protective function of the capsular polysaccharide and melanin [52,53]. On the other hand, the activity of the urease enzyme is crucial for breaching the BBB endothelium, thereby allowing brain invasion [54,55]. Hence, disrupting the function of these traits could significantly impact the outcome of cryptococcal infection.
The ability of NTX to inhibit these two metallo-centric enzymes, laccase and urease, is primarily related to the metal-chelating activity of the nitrogen atom of the quinoline ring and the 8-hydroxyl moiety of this scaffold [56,57]. This anti-virulence trait aligns with the reported activity of NTX on metallo-β-lactamase-producing pathogens through the chelation of zinc [57–59]. Cryptococcal laccase enzyme utilizes copper ions as a cofactor to oxidize norepinephrine and other abundant catecholamines within the brain parenchyma into dark melanin pigmentation [60,61]. The limited level of copper metal within the brain and its essentiality for the cerebral progression of cryptococcal infection highlights the potential role of NTX in halting cryptococcal growth within the brain parenchyma by depriving the invading cells of copper [62]. Similarly, inhibiting the activity of the urease enzyme by targeting nickel ions within the enzyme complex could significantly impede the transmigration of cryptococcal cells toward the brain [55].
While there is debate over whether NTX could affect other molecular targets, our results indicate that the antifungal activity of the NTX scaffold is directly related to metal chelation, which significantly affects various cellular activities necessary for pathogen survival [63,64]. Similar to other cell-permeable metal chelators, NTX treatment resulted in a dose-dependent increase in PGFL fluorescence, indicating depletion of essential metals within cryptococcal cells [65]. Consistently, NTX was reported to affect metal homeostasis in Acanthamoeba castellanii amoebae and A. fumigatus, leading to the depletion of iron and copper metals, respectively [66,67]. The impact of exogenous supplementation with various divalent cations, particularly Cu2+ and Zn2+, on NTX activity also supports metal homeostasis as the primary target of NTX in cryptococcal cells. Notably, NTX metal preference varies among other pathogens. While Mn2+ and Mg2+ significantly affect the antibacterial activity of NTX against E. coli, supplementation with these two cations doesn’t exert any change on the MIC of NTX against cryptococcal cells [56]. Additionally, the loss of both lethality and anti-virulence activity with tested NTX analogs, secondary to the substitution of 8-hydroxyl moiety at the chelation center, underscores the crucial link between metal chelation and the activity of the NTX scaffold against cryptococcal cells.
The beneficial interaction between NTX and standard antifungals, especially oral FLC, highlights the potential role of NTX in addressing frequent relapses and treatment failures related to FLC monotherapy [68]. FLC is often the only treatment option for cryptococcal infection even during the induction stage treatment due to the restricted access to AmB and 5-FC in resource-limited regions [69,70]. Even at higher doses (800–1200 mg/day), FLC monotherapy showed frequent treatment failure and relapses with mortality rate exceeding 70% within the first year comparing to AmB based regimens [13,71–73]. Hence, combining FLC and NTX could leverage the efficiency of FLC monotherapy considering the fungicidal activity and the anti-virulence advantage of NTX scaffold. Additionally, the low propensity of cryptococcal cells to develop resistance to NTX during continuous exposure, under the used in vitro conditions, also supports its potential role during treatment stages, which needs further validation considering the extended length of the cryptococcal infection treatment and other host related factors.
The pharmacokinetic prosperities of the NTX scaffold pose a significant challenge to its clinical utility against invasive fungal infection [24]. While the oral dosage of NTX achieves adequate plasma level (~ 5.5 µg/mL), its extensive hepatic degradation and excretion via the urinary tract (> 60% of administered dose) limit its systemic exposure [24,74,75]. Despite the scarce data regarding its level within cerebral and pulmonary compartments, reported NTX efficiency in slowing glioma proliferation in mice and resolving granulomatous amebic encephalitis in human, in combination with the standard treatment regimen, highlights its ability to reach brain parenchyma and its potential role in combating the cerebral progression of cryptococcal infection [76,77]. Additionally, recent derivatives of the NTX scaffold, including ASN-1733 and ASN-1213, could exhibit superior therapeutic efficacy against cryptococcal infection. Their metabolic stability could allow better systemic exposure for the targeted organs, brain and lung, considering their plasma level of ~ 52.36 and 20 µg/mL and their extended half-life of ~ 22 hrs. and 14.3 hrs., respectively [78]. Moreover, direct drug delivery via inhalation can provide an alternative approach to overcome the pharmacokinetic limitation of NTX, allowing reaching the alveolar spaces with high concentration comparing to the oral route [79,80]. While there is no approved inhaled antifungal for treating pulmonary fungal infections like invasive aspergillosis, results from preclinical and clinical studies for inhaled antifungals including azoles (voriconazole and itraconazole) supports the benefit of this approach in overcoming the PK limitations and reducing potential toxicities and drug-drug interactions [80–83].
In conclusion, this study elucidates various aspects of NTX activity against the Cryptococcus pathogen, including its modulation for the primary virulence traits of cryptococcal cells and its potential mechanism of action. Additionally, our study highlights the beneficial interaction between NTX and standard antifungals, especially FLC, and its efficacy in the nematode model of C. elegans, which necessitates further investigation in mammalian model to confirm its in vivo efficacy.
Supporting information
S1 Raw Data. In vitro evaluation of NTX efficacy.
https://doi.org/10.1371/journal.pntd.0014627.s001
(XLSX)
S1 Fig. Evaluation the efficacy of NTX/FLC combination in the Caenorhabditis elegans nematode model.
(A) Impact of NTX on the fungal burden of infected C. elegans in combination with FLC. Infected L4 larvae (300 worms per mL) were individually treated with NTX at a sub-inhibitory concentration (0.5x MIC), FLC (2x MIC), or their combination for 24 hrs at 30°C. Treated worms were homogenized and plated onto gentamicin-supplemented YPD agar to assess the change in the fungal burden. Data was analyzed using one-way analysis of variance (ANOVA) for multiple comparison, and p values of ≤ 0.05 indicate statistical significance of compared data sets. (B) A Kaplan-Meier survival curve of C. elegans worm groups individually treated with NTX (0.5x MIC), FLC (2x MIC), or their combination. Statistical significance was evaluated using log-rank test.
https://doi.org/10.1371/journal.pntd.0014627.s002
(TIFF)
S2 Fig. Growth of cryptococcal cells in the presence of a sublethal concentration of NTX or positive controls used in the virulence study.
Inoculum of the C. neoformans H99 strain was adjusted to the exact density used for each virulence experiment and treated with NTX (0.5x MIC) and other positive controls including amphotericin B (AmB), acetohydroxamic acid (AHA), and flucytosine (5-FC). Following the end of each incubation time, aliquots of treated cryptococcal cells were serially diluted with phosphate-buffered saline and platted on YPD agar.
https://doi.org/10.1371/journal.pntd.0014627.s003
(TIFF)
Acknowledgments
We sincerely appreciate BEI Resources for providing the fungal isolates utilized in this study.
References
- 1. Maziarz EK, Perfect JR. Cryptococcosis. Infect Dis Clin North Am. 2016;30(1):179–206.
- 2. Firacative C, Trilles L, Meyer W. Recent Advances in Cryptococcus and Cryptococcosis. Microorganisms. 2021;10(1).
- 3. Rodrigues ML, Alviano CS, Travassos LR. Pathogenicity of Cryptococcus neoformans: virulence factors and immunological mechanisms. Microbes Infect. 1999;1(4):293–301. pmid:10602663
- 4. Esher SK, Zaragoza O, Alspaugh JA. Cryptococcal pathogenic mechanisms: a dangerous trip from the environment to the brain. Mem Inst Oswaldo Cruz. 2018;113(7):e180057. pmid:29668825
- 5. Rajasingham R, Govender NP, Jordan A, Loyse A, Shroufi A, Denning DW, et al. The global burden of HIV-associated cryptococcal infection in adults in 2020: a modelling analysis. Lancet Infect Dis. 2022;22(12):1748–55. pmid:36049486
- 6. Denning DW. Global incidence and mortality of severe fungal disease. Lancet Infect Dis. 2024;24(7):e428–38. pmid:38224705
- 7. Iyer KR, Revie NM, Fu C, Robbins N, Cowen LE. Treatment strategies for cryptococcal infection: challenges, advances and future outlook. Nat Rev Microbiol. 2021;19(7):454–66. pmid:33558691
- 8. Sionov E, Chang YC, Garraffo HM, Kwon-Chung KJ. Heteroresistance to fluconazole in Cryptococcus neoformans is intrinsic and associated with virulence. Antimicrob Agents Chemother. 2009;53(7):2804–15. pmid:19414582
- 9. Chang CC, Harrison TS, Bicanic TA, Chayakulkeeree M, Sorrell TC, Warris A, et al. Global guideline for the diagnosis and management of cryptococcosis: an initiative of the ECMM and ISHAM in cooperation with the ASM. Lancet Infect Dis. 2024;24(8):e495–512. pmid:38346436
- 10. Perfect JR, Dismukes WE, Dromer F, Goldman DL, Graybill JR, Hamill RJ, et al. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of america. Clin Infect Dis. 2010;50(3):291–322. pmid:20047480
- 11. Loyse A, Burry J, Cohn J, Ford N, Chiller T, Ribeiro I, et al. Leave no one behind: response to new evidence and guidelines for the management of cryptococcal meningitis in low-income and middle-income countries. Lancet Infect Dis. 2019;19(4):e143–7. pmid:30344084
- 12. Rothe C, Sloan DJ, Goodson P, Chikafa J, Mukaka M, Denis B, et al. A prospective longitudinal study of the clinical outcomes from cryptococcal meningitis following treatment induction with 800 mg oral fluconazole in Blantyre, Malawi. PLoS One. 2013;8(6):e67311. pmid:23840659
- 13. Longley N, Muzoora C, Taseera K, Mwesigye J, Rwebembera J, Chakera A, et al. Dose response effect of high-dose fluconazole for HIV-associated cryptococcal meningitis in southwestern Uganda. Clin Infect Dis. 2008;47(12):1556–61. pmid:18990067
- 14. Truong M, Monahan LG, Carter DA, Charles IG. Repurposing drugs to fast-track therapeutic agents for the treatment of cryptococcosis. PeerJ. 2018;6:e4761. pmid:29740519
- 15. Butts A, DiDone L, Koselny K, Baxter BK, Chabrier-Rosello Y, Wellington M, et al. A repurposing approach identifies off-patent drugs with fungicidal cryptococcal activity, a common structural chemotype, and pharmacological properties relevant to the treatment of cryptococcosis. Eukaryot Cell. 2013;12(2):278–87. pmid:23243064
- 16. Alkashef NM, Salama EA, Anandakrishnan R, Hazbun TR, Hoernig MV, Brown AM, et al. In vitro and in vivo efficacy of the antimycobacterial molecule SQ109 against the human pathogenic fungus, Cryptococcus neoformans. PLoS Negl Trop Dis. 2025;19(12):e0013837. pmid:41401231
- 17. Kresken M, Körber-Irrgang B. In vitro activity of nitroxoline against Escherichia coli urine isolates from outpatient departments in Germany. Antimicrob Agents Chemother. 2014;58(11):7019–20. pmid:25182654
- 18. Krajewski W, Łaszkiewicz J, Tomczak W, Nowak Ł, Chorbińska J, Sójka A, et al. Nitroxoline: treatment and prevention of urinary tract infections from the urologist’s perspective. Cent European J Urol. 2024;77(2):339–43. pmid:39345309
- 19. Zhang Z-R, Zhang H-Q, Li X-D, Deng C-L, Wang Z, Li J-Q, et al. Generation and characterization of Japanese encephalitis virus expressing GFP reporter gene for high throughput drug screening. Antiviral Res. 2020;182:104884. pmid:32750466
- 20. Laurie MT, et al. Functional assessment of 2,177 U.S. and international drugs identifies the quinoline nitroxoline as a potent amoebicidal agent against the pathogen Balamuthia mandrillaris. mBio. 2018;9(5).
- 21. Shah S, Dalecki AG, Malalasekera AP, Crawford CL, Michalek SM, Kutsch O, et al. 8-hydroxyquinolines are boosting agents of copper-related toxicity in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2016;60(10):5765–76. pmid:27431227
- 22. Cacace E, Tietgen M, Steinhauer M, Mateus A, Schultze TG, Eckermann M, et al. Uncovering nitroxoline activity spectrum, mode of action and resistance across Gram-negative bacteria. Nat Commun. 2025;16(1):3783. pmid:40263263
- 23. Hoffmann AM, Wolke M, Rybniker J, Plum G, Fuchs F. In vitro activity of repurposed nitroxoline against clinically isolated mycobacteria including multidrug-resistant Mycobacterium tuberculosis. Front Pharmacol. 2022;13:906097. pmid:35721137
- 24. Wijma RA, Huttner A, Koch BCP, Mouton JW, Muller AE. Review of the pharmacokinetic properties of nitrofurantoin and nitroxoline. J Antimicrob Chemother. 2018;73(11):2916–26. pmid:30184207
- 25.
Institute CALS. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard. 3rd ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.
- 26. Spadari CDC, Vila T, Rozental S, Ishida K. Miltefosine has a postantifungal effect and induces apoptosis in Cryptococcus yeasts. Antimicrob Agents Chemother. 2018;62(8):e00312–18. pmid:29844051
- 27. Zhai B, Wu C, Wang L, Sachs MS, Lin X. The antidepressant sertraline provides a promising therapeutic option for neurotropic cryptococcal infections. Antimicrob Agents Chemother. 2012;56(7):3758–66. pmid:22508310
- 28. Alkashef NM, Seleem MN. Novel combinatorial approach: Harnessing HIV protease inhibitors to enhance amphotericin B’s antifungal efficacy in cryptococcosis. PLoS One. 2024;19(8):e0308216. pmid:39088434
- 29. Baker RP, Casadevall A. Reciprocal modulation of ammonia and melanin production has implications for cryptococcal virulence. Nat Commun. 2023;14(1):849. pmid:36792633
- 30. Fu MS, Coelho C, De Leon-Rodriguez CM, Rossi DCP, Camacho E, Jung EH, et al. Cryptococcus neoformans urease affects the outcome of intracellular pathogenesis by modulating phagolysosomal pH. PLoS Pathog. 2018;14(6):e1007144. pmid:29906292
- 31. Zimbres ACG, Reuwsaat JCV, Barcellos VA, Joffe LS, Fonseca FL, Staats CC, et al. Pharmacological inhibition of pigmentation in Cryptococcus. FEMS Yeast Res. 2019;19(1). pmid:30418573
- 32.
Albuquerque P, et al. Measuring laccase activity and melanin production in Cryptococcus neoformans. In: McClelland EE, editor. Cryptococcus neoformans: Methods and Protocols. New York, NY: Springer US; 2024. pp. 257–68.
- 33. de Sousa HR, de Oliveira GP Jr, Frazão SDO, Gorgonha KCDM, Rosa CP, Garcez EM, et al. Faster Cryptococcus melanization increases virulence in experimental and human Cryptococcosis. J Fungi (Basel). 2022;8(4):393. pmid:35448624
- 34. Trevijano-Contador N, et al. Capsule enlargement in Cryptococcus neoformans is dependent on mitochondrial activity. Front Microbiol. 2017;8.
- 35. Diehl C, Pinzan CF, de Castro PA, Delbaje E, García Carnero LC, Sánchez-León E, et al. Brilacidin, a novel antifungal agent against Cryptococcus neoformans. mBio. 2024;15(7):e0103124. pmid:38916308
- 36. Brandão FA, Derengowski LS, Albuquerque P, Nicola AM, Silva-Pereira I, Poças-Fonseca MJ. Histone deacetylases inhibitors effects on Cryptococcus neoformans major virulence phenotypes. Virulence. 2015;6(6):618–30. pmid:26103530
- 37. Haeili M, Moore C, Davis CJC, Cochran JB, Shah S, Shrestha TB, et al. Copper complexation screen reveals compounds with potent antibiotic properties against methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2014;58(7):3727–36. pmid:24752262
- 38. Chang W, Liu J, Zhang M, Shi H, Zheng S, Jin X, et al. Efflux pump-mediated resistance to antifungal compounds can be prevented by conjugation with triphenylphosphonium cation. Nat Commun. 2018;9(1):5102. pmid:30504815
- 39. Zhou M, Liu L, Cong Z, Jiang W, Xiao X, Xie J, et al. A dual-targeting antifungal is effective against multidrug-resistant human fungal pathogens. Nat Microbiol. 2024;9(5):1325–39. pmid:38589468
- 40. Eldesouky HE, Salama EA, Lanman NA, Hazbun TR, Seleem MN. Potent synergistic interactions between lopinavir and azole antifungal drugs against emerging multidrug-resistant Candida auris. Antimicrob Agents Chemother. 2020;65(1):e00684–20. pmid:33046487
- 41. Mylonakis E, Ausubel FM, Perfect JR, Heitman J, Calderwood SB. Killing of Caenorhabditis elegans by Cryptococcus neoformans as a model of yeast pathogenesis. Proc Natl Acad Sci U S A. 2002;99(24):15675–80. pmid:12438649
- 42. O’Connor L, Livermore J, Sharp AD, Goodwin J, Gregson L, Howard SJ, et al. Pharmacodynamics of liposomal amphotericin B and flucytosine for cryptococcal meningoencephalitis: safe and effective regimens for immunocompromised patients. J Infect Dis. 2013;208(2):351–61. pmid:23599314
- 43. Bellmann R, Smuszkiewicz P. Pharmacokinetics of antifungal drugs: practical implications for optimized treatment of patients. Infection. 2017;45(6):737–79. pmid:28702763
- 44. Perfect JR, Bicanic T. Cryptococcosis diagnosis and treatment: What do we know now. Fungal Genet Biol. 2015;78:49–54. pmid:25312862
- 45. Hope W, Stone NRH, Johnson A, McEntee L, Farrington N, Santoro-Castelazo A, et al. Fluconazole monotherapy is a suboptimal option for initial treatment of Cryptococcal meningitis because of emergence of resistance. mBio. 2019;10(6):e02575–19. pmid:31796539
- 46. Oprea TI, Mestres J. Drug repurposing: far beyond new targets for old drugs. AAPS J. 2012;14(4):759–63. pmid:22826034
- 47. Ashley ED. Antifungal drugs: special problems treating central nervous system infections. J Fungi (Basel). 2019;5(4):97. pmid:31614505
- 48. Hoffmann A, et al. In vitro activity of nitroxoline (5-nitro-8-hydroxyquinoline) against Aspergillus species. Mycoses. 2025;68(11):e70131.
- 49. Fuchs F, Aldejohann AM, Hoffmann AM, Walther G, Kurzai O, Hamprecht AG. In vitro activity of nitroxoline in antifungal-resistant Candida species isolated from the urinary tract. Antimicrob Agents Chemother. 2022;66(6):e0226521. pmid:35543103
- 50. Fuchs F, Hof H, Hofmann S, Kurzai O, Meis JF, Hamprecht A. Antifungal activity of nitroxoline against Candida auris isolates. Clin Microbiol Infect. 2021;27(11):1697.e7–1697.e10. pmid:34245904
- 51. Vu K, Garcia JA, Gelli A. Cryptococcal meningitis and anti-virulence therapeutic strategies. Front Microbiol. 2019;10.
- 52. Noverr MC, Williamson PR, Fajardo RS, Huffnagle GB. CNLAC1 is required for extrapulmonary dissemination of Cryptococcus neoformans but not pulmonary persistence. Infect Immun. 2004;72(3):1693–9. pmid:14977977
- 53. Bednarek JM, Brown JCS. Elements of dissemination in cryptococcosis. mBio. 2024;15(12):e0215523. pmid:39470312
- 54. Olszewski MA, Noverr MC, Chen G-H, Toews GB, Cox GM, Perfect JR, et al. Urease expression by Cryptococcus neoformans promotes microvascular sequestration, thereby enhancing central nervous system invasion. Am J Pathol. 2004;164(5):1761–71. pmid:15111322
- 55. Singh A, Panting RJ, Varma A, Saijo T, Waldron KJ, Jong A, et al. Factors required for activation of urease as a virulence determinant in Cryptococcus neoformans. mBio. 2013;4(3):e00220–13. pmid:23653445
- 56. Pelletier C, Prognon P, Bourlioux P. Roles of divalent cations and pH in mechanism of action of nitroxoline against Escherichia coli strains. Antimicrob Agents Chemother. 1995;39(3):707–13. pmid:7793877
- 57. Wykowski R, Fuentefria AM, de Andrade SF. Antimicrobial activity of clioquinol and nitroxoline: a scoping review. Arch Microbiol. 2022;204(8):535. pmid:35907036
- 58. He P, Huang S, Wang R, Yang Y, Yang S, Wang Y, et al. Novel nitroxoline derivative combating resistant bacterial infections through outer membrane disruption and competitive NDM-1 inhibition. Emerg Microbes Infect. 2024;13(1):2294854. pmid:38085067
- 59. Principe L, Vecchio G, Sheehan G, Kavanagh K, Morroni G, Viaggi V, et al. Zinc chelators as carbapenem adjuvants for metallo-β-lactamase-producing bacteria: in vitro and in vivo evaluation. Microb Drug Resist. 2020;26(10):1133–43. pmid:32364820
- 60. Jiang N, Sun N, Xiao D, Pan J, Wang Y, Zhu X. A copper-responsive factor gene CUF1 is required for copper induction of laccase in Cryptococcus neoformans. FEMS Microbiol Lett. 2009;296(1):84–90. pmid:19459959
- 61. Baker RP, Chrissian C, Stark RE, Casadevall A. Cryptococcus neoformans melanization incorporates multiple catecholamines to produce polytypic melanin. J Biol Chem. 2022;298(1):101519. pmid:34942148
- 62. Waterman SR, Hacham M, Hu G, Zhu X, Park Y-D, Shin S, et al. Role of a CUF1/CTR4 copper regulatory axis in the virulence of Cryptococcus neoformans. J Clin Invest. 2007;117(3):794–802. pmid:17290306
- 63. Robinson JR, Isikhuemhen OS, Anike FN. Fungal-metal interactions: a review of toxicity and homeostasis. J Fungi (Basel). 2021;7(3).
- 64. Jung WH, Sham A, White R, Kronstad JW. Iron regulation of the major virulence factors in the AIDS-associated pathogen Cryptococcus neoformans. PLoS Biol. 2006;4(12):e410. pmid:17121456
- 65. Petrat F, Rauen U, de Groot H. Determination of the chelatable iron pool of isolated rat hepatocytes by digital fluorescence microscopy using the fluorescent probe, phen green SK. Hepatology. 1999;29(4):1171–9. pmid:10094962
- 66. Chen L, Han W, Jing W, Feng M, Zhou Q, Cheng X. Nitroxoline evidence amoebicidal activity against Acanthamoeba castellanii through DNA damage and the stress response pathways. Int J Parasitol Drugs Drug Resist. 2025;27:100578. pmid:39764873
- 67. Zhang Y, Han Y, Cao D, Yuan X, Xu P, Xiang Y, et al. Nitroxoline exerts potent anti-Aspergillus fumigatus activity by disrupting copper homeostasis and inducing oxidative stress. Antimicrob Agents Chemother. 2026;70(5):e0183125. pmid:41914973
- 68. Sionov E, Lee H, Chang YC, Kwon-Chung KJ. Cryptococcus neoformans overcomes stress of azole drugs by formation of disomy in specific multiple chromosomes. PLoS Pathog. 2010;6(4):e1000848. pmid:20368972
- 69. Bongomin F, Ekeng BE, Kwizera R, Salmanton-García J, Kibone W, van Rhijn N, et al. Fungal diseases in Africa: Closing the gaps in diagnosis and treatment through implementation research and advocacy. J Mycol Med. 2023;33(4):101438. pmid:38358796
- 70. Bongomin F, Ebbs DS, Henao-Martínez AF, Denning DW. Health disparities and the burden of fungal infections in Africa. Lancet Region Health - Afr. 2026;4:100014.
- 71. Gaskell KM, Rothe C, Gnanadurai R, Goodson P, Jassi C, Heyderman RS, et al. A prospective study of mortality from cryptococcal meningitis following treatment induction with 1200 mg oral fluconazole in Blantyre, Malawi. PLoS One. 2014;9(11):e110285. pmid:25375145
- 72. Nussbaum JC, Jackson A, Namarika D, Phulusa J, Kenala J, Kanyemba C, et al. Combination flucytosine and high-dose fluconazole compared with fluconazole monotherapy for the treatment of cryptococcal meningitis: a randomized trial in Malawi. Clin Infect Dis. 2010;50(3):338–44. pmid:20038244
- 73. Sloan DJ, Dedicoat MJ, Lalloo DG. Treatment of cryptococcal meningitis in resource limited settings. Curr Opin Infect Dis. 2009;22(5):455–63. pmid:19587589
- 74. Bergogne-Berezin E, Berthelot G, Muller-Serieys C. Present status of nitroxoline. Pathol Biol (Paris). 1987;35(5 Pt 2):873–8.
- 75. Ghoneim MM, El-Desoky HS, Abdel-Galeil MM. Electrochemistry of the antibacterial and antifungal drug nitroxoline and its determination in bulk form, pharmaceutical formulation and human blood. Bioelectrochemistry. 2011;80(2):162–8. pmid:20833114
- 76. Spottiswoode N, Pet D, Kim A, Gruenberg K, Shah M, Ramachandran A, et al. Successful treatment of Balamuthia mandrillaris granulomatous amebic encephalitis with nitroxoline. Emerg Infect Dis. 2023;29(1):197–201. pmid:36573629
- 77. Lazovic J, Guo L, Nakashima J, Mirsadraei L, Yong W, Kim HJ, et al. Nitroxoline induces apoptosis and slows glioma growth in vivo. Neuro Oncol. 2015;17(1):53–62. pmid:25074541
- 78. He P, Huang S, Wang R, Yang Y, Yang S, Wang Y, et al. Novel nitroxoline derivative combating resistant bacterial infections through outer membrane disruption and competitive NDM-1 inhibition. Emerg Microbes Infect. 2024;13(1):2294854. pmid:38085067
- 79. Ito K. Inhaled antifungal therapy: benefits, challenges, and clinical applications. Expert Opin Drug Deliv. 2022;19(7):755–69. pmid:35634895
- 80. Hoenigl M, et al. The antifungal pipeline: Fosmanogepix, Ibrexafungerp, Olorofim, Opelconazole, and Rezafungin. Drugs. 2021;81(15):1703–29.
- 81. Murray A, Cass L, Ito K, Pagani N, Armstrong-James D, Dalal P, et al. PC945, a novel inhaled antifungal agent, for the treatment of respiratory fungal infections. J Fungi (Basel). 2020;6(4):373. pmid:33348852
- 82. Holle J, Leichsenring M, Meissner PE. Nebulized voriconazole in infections with Scedosporium apiospermum--case report and review of the literature. J Cyst Fibros. 2014;13(4):400–2. pmid:24263169
- 83. Hava DL, Tan L, Johnson P, Curran AK, Perry J, Kramer S, et al. A phase 1/1b study of PUR1900, an inhaled formulation of itraconazole, in healthy volunteers and asthmatics to study safety, tolerability and pharmacokinetics. Br J Clin Pharmacol. 2020;86(4):723–33. pmid:31696544