Development Of Antifungal Drugs Research Articles

P-1086. COT-832, a Novel Polyene Macrolide Antifungal (I): In Vitro Antifungal Activity against Candida, Aspergillus, and Mucor, etc., Hemolytic Activity, and Cytotoxicity

Abstract Background A novel polyene macrolide antifungal, COT-832 was created by SHIONOGI through structurally modifying Amphotericin B(AmB). Invasive fungal infections are commonly treated with antifungal drugs such as azoles, polyenes, and echinocandins; however, their efficacy is limited and mortality rates remain high. Therefore, the development of more effective and safer antifungal drugs is needed. AmB is available in formulations such as Fungizone (FGZ), which utilizes a deoxycholic acid formulation, and AmBisome (L-AMB), which employs a liposome formulation. While these polyene antifungals exhibit activity against a wide range of fungal species, their usage is often discontinued due to the nephrotoxicity associated with AmB itself. This study aimed to evaluate the antifungal activity, hemolytic activity, and cytotoxicity of COT-832 in vitro. Methods Antifungal activity was assessed using the CLSI method on clinical isolates from Japan (2012-2016). Hemolysis activity was measured after applying compounds to 4% sheep red blood cells for 24 hours. Cytotoxicity was evaluated by applying compounds to HepG2 cells for 2 hours and measuring lactate dehydrogenase (LDH) levels in the supernatant with Cytotox96 assay for calculating cell survival rate. Results Antifungal activity: COT-832 exhibited MIC90 of 0.5-2 µg/mL against Aspergillus and Candida, matching AmB's MIC. It also showed efficacy against azole-resistant A. fumigatus and echinocandin-resistant C. glabrata. COT-832's MIC90 for Cryptococcus was 0.5 µg/mL, surpassing AmB. Similar MICs were observed for rare molds. Hemolytic activity: AmB caused hemolysis from 0.625 µg/mL, reaching 80% at 2.5 µg/mL. COT-832 displayed an 8% hemolysis rate at 2.5 µg/mL. Cytotoxicity: AmB reduced cell count from 2 µg/mL, while no noticeable decrease occurred with COT-832 up to 72 µg/mL. Conclusion COT-832 exhibits comparable antifungal activity to AmB while demonstrating reduced toxicity against various cells, up to a tenfold decrease compared to AmB. These findings suggest that COT-832 has potential for development as a safer polyene macrolide antifungal drug, offering potent antifungal activity across a wide range of fungal species. Disclosures Hideki Maki, PhD, Shionogi & Co., Ltd.: employee|Shionogi & Co., Ltd.: employee Yuri Nishiyama, MS, Shionogi & Co., Ltd.: employee Takafumi Ohara, n/a, Shionogi & Co., Ltd.: employee Hideki Sugimoto, n/a, Shionogi & Co., Ltd.: employee

Trichosporon asahii: emerging challenges in pathogenesis and drug resistance.

Trichosporon asahii (T. asahii) is an opportunistic pathogenic fungus that often causes severe infections in immunosuppressed patients. Among Trichosporon species, T. asahii is the most pathogenic and lethal species. Current research faces challenges related to unknown pathogenic mechanisms, complex resistance mechanisms, insufficiently rapid and accurate diagnostic methods, and insufficient research on susceptibility to infection. These issues need to be explored in depth. This review summarizes research progress on the origin and classification of T. asahii, its virulence factors and pathogenic mechanisms, epidemiological characteristics, infection modes, diagnostic methods, drug treatment options, and drug resistance mechanisms. Traditional culture combined with molecular biology techniques, such as polymerase chain reaction and gene sequencing, has improved the accuracy and speed of detection. Treatment relies mainly on azole antifungal drugs and amphotericin B; however, patients are facing the problem of drug resistance. New techniques, such as gene knockout and gene sequencing, have identified resistance mechanisms, thus supporting the development of novel antifungal drugs. In summary, an in-depth study of T. asahii will aid in developing more effective diagnostic and therapeutic methods and improve patient prognosis.

Dendritic Antifungal Peptides as Potent Agents against Drug-Resistant Candida albicans and Biofilm.

Candida albicans infection is a major public health problem, exacerbated by the emergence of drug-resistant fungi with the widespread use of antifungal drugs. Therefore, the development of novel antifungal drugs for drug-resistant C. albicans infections is crucial. We constructed a series of dendritic antifungal peptides (AFPs) with different chain lengths of fatty acids as hydrophobic ends and 2 or 3 protease-stable repeats (Arg-Pro) as dendritic peptide branches. Among them, C4-3RP exhibited excellent antidrug-resistant fungal and biofilm activity (GMall = 5.04 μM) and was nontoxic. Furthermore, C4-3RP demonstrated high protease stability and salt ion tolerance, making it highly effective in murine skin infection mediated by C. albicans. In addition, C4-3RP uses multiple mechanisms of action to achieve excellent antifungal effects. In conclusion, the construction of dendritic peptides holds substantial potential in the treatment of fungal infections and provides a broader perspective on the design of peptide-based antifungal drugs.

In vitro and in vivo activity of 1,2,3,4,6-O-pentagalloyl-glucose against Candida albicans.

Invasive fungal infections have become an increasingly serious threat to global human health, underscoring the urgent need for the development of new antifungal drugs. In this study, we found a natural polyphenolic compound 1,2,3,4,6-O-pentagalloyl-glucose (PGG), which is present in various plants and herbs. PGG showed broad-spectrum antifungal activities, enhancing the efficacy of fluconazole. Furthermore, PGG could protect mice against gastrointestinal and systemic infection with Candida albicans. Our mechanistic studies revealed that PGG exerts its antifungal effects partially by binding to the CaEno1 protein to inhibit its activity. As a crucial therapeutic target, Eno1 has been reported to be closely associated with cancer, hypertension, and infectious diseases. Our findings indicated that PGG, a new Eno1 inhibitor, is a potential candidate for further antifungal development.

Antifungal activity of 2-adamantylamine hydrochloride on Candida albicans and Candida parapsilosis.

Introduction. Increased virulence and drug resistance in species of Candida resulted in reduced disease control and further demand the development of potent antifungal drugs.Hypothesis. The repurposing of non-antifungal drugs and combination therapy has become an attractive alternative to counter the emerging drug resistance and toxicity of existing antifungal drugs against Candida albicans and non-albicans species.Aim. This study aimed to accelerate antifungal drug development process by drug repurposing approach.Methodology. In this study, the antifungal effects of the antiviral drug, 2-adamantylamine hydrochloride (2-AM), were explored against C. albicans and C. parapsilosis. Broth microdilution measured in vitro efficacy of 2-AM, whereas reactive oxygen species (ROS) accumulation and ergosterol quantification, cell cycle and phosphatidylserine externalization studies were detailed to investigate the antifungal mode of 2-AM action.Results. Results showed that 2-AM had fungicidal action against both the strains where, 2-AM further inhibited morphogenic transitions as well. Antibiofilm action of 2-AM on C. albicans was evidenced on urinary catheters. G2/M phase arrest and apoptosis indicated ROS induced antifungal effect of 2-AM on both strains.Conclusions. Results of in vitro studies offers insight into the antifungal activity of 2-AM and may serve as an effective antifungal repurposed candidate against C. albicans and C. parapsilosis.

The proteomic response of Aspergillus fumigatus to amphotericin B (AmB) reveals the involvement of the RTA-like protein RtaA in AmB resistance.

The polyene antimycotic amphotericin B (AmB) and its liposomal formulation AmBisome belong to the treatment options of invasive aspergillosis caused by Aspergillus fumigatus. Increasing resistance to AmB in clinical isolates of Aspergillus species is a growing concern, but mechanisms of AmB resistance remain unclear. In this study, we conducted a proteomic analysis of A. fumigatus exposed to sublethal concentrations of AmB and AmBisome. Both antifungals induced significantly increased levels of proteins involved in aromatic acid metabolism, transmembrane transport, and secondary metabolite biosynthesis. One of the most upregulated proteins was RtaA, a member of the RTA-like protein family, which includes conserved fungal membrane proteins with putative functions as transporters or translocases. Accordingly, we found that RtaA is mainly located in the cytoplasmic membrane and to a minor extent in vacuolar-like structures. Deletion of rtaA led to increased polyene sensitivity and its overexpression resulted in modest resistance. Interestingly, rtaA expression was only induced by exposure to the polyenes AmB and nystatin, but not by itraconazole and caspofungin. Orthologues of rtaA were also induced by AmB exposure in A. lentulus and A. terreus. Deletion of rtaA did not significantly change the ergosterol content of A. fumigatus, but decreased fluorescence intensity of the sterol-binding stain filipin. This suggests that RtaA is involved in sterol and lipid trafficking, possibly by transporting the target ergosterol to or from lipid droplets. These findings reveal the contribution of RtaA to polyene resistance in A. fumigatus, and thus provide a new putative target for antifungal drug development.

Exploring potential natural plant-based antifungal agents in Sri Lanka: A comprehensive review

Recent advances in antifungal drug discovery have prompted a closer inspection of the biology of fungi and the ways in which they adapt to their growing environment. Understanding these findings presents a challenge: developing antifungal agents that can withstand resistance from fungi and effectively combat it. The use of plant-based antifungal agents is gaining momentum within the antifungal drug industry. This review aims to examine the potential to propel current findings on the antifungal activity of selected medicinal plant extracts found in Sri Lanka towards their potential candidacy as novel antifungals. The benefits and adverse effects of fungi are discussed, along with an overview of synthetic and natural antifungal agents. It delves into the modes of action of antifungal agents and the ways in which they encounter resistance from fungi. The review also highlights the classes of phytochemicals in these plant extracts and the mechanisms through which they exhibit antifungal abilities. Furthermore, it summarizes the capability of natural antifungal agents to enhance the antifungal activity of synthetic antifungal drugs through possible synergistic actions. Finally, the barriers to bringing these plant-based antifungal agents to the forefront of the antifungal drug development industry, along with a few suggestions to overcome them, are considered.

Antifungal and anti-biofilm effects of hydrazone derivatives on Candida spp

Worldwide, invasive candidiasis are a burden for the health system due to difficulties to manage patients, to the increasing of the resistance of the current therapeutics and the emergence of naturally resistant species of Candida. In this context, the development of innovative antifungal drugs is urgently needed. During invasive candidiasis, yeast is submitted to many stresses (oxidative, thermic, osmotic) in the human host. In order to resist in this context, yeast develops different strategy, especially the biosynthesis of trehalose. Starting from the 3D structural data of TPS2, an enzyme implicated in trehalose biosynthesis, we identified hydrazone as an interesting scaffold to design new antifungal drugs. Interestingly, our hydrazone derivatives which demonstrate antifungal and anti-biofilm effects on Candida spp., are non-toxic in in vitro and in vivo models (Galleria mellonella).

Roles of Different Signaling Pathways in Cryptococcus neoformans Virulence.

Cryptococcus neoformans is a widespread fungal pathogen that can infect the human central nervous system (CNS) and cause fungal meningitis, leading to hundreds of thousands of deaths worldwide each year. Previous studies have demonstrated that many signal transduction pathways are crucial for the morphological development and virulence of C. neoformans. In this review, data from over 116 research articles have been compiled to show that many signaling pathways control various characteristics of C. neoformans, individually or in association with other pathways, and to establish strong links among them to better understand C. neoformans pathogenesis. Every characteristic of C. neoformans is closely linked to these signaling pathways, making this a rich area for further research. It is essential to thoroughly explore these pathways to address questions that remain and apply a molecular mechanistic approach to link them. Targeting these pathways is crucial for understanding the exact mechanism of infection pathogenesis and will facilitate the development of antifungal drugs as well as the diagnosis and prevention of cryptococcosis.

Inhibition of Virulence Associated Traits by β-Sitosterol Isolated from Hibiscus rosa-sinensis Flowers Against Candida albicans: Mechanistic Insight and Molecular Docking Studies.

The emerging drug resistance and lack of safer and more potent antifungal agents make Candida infections another hot topic in the healthcare system. At the same time, the potential of plant products in developing novel antifungal drugs is also in the limelight. Considering these facts, we have investigated the different extracts of the flowers of Hibiscus rosa-sinensis of the Malvaceae family for their antifungal efficacy against five different pathogenic Candida strains. Among the various extracts, the chloroform extract showed the maximum zone of inhibition (26.6 ± 0.5mm) against the Candida albicans strain. Furthermore, the chloroform fraction was isolated, and a sterol compound was identified as β-sitosterol. Mechanistic studies were conducted to understand the mechanism of action, and the results showed that β-sitosterol has significant antifungal activity and is capable of interrupting biofilm formation and acts by inhibiting ergosterol biosynthesis in Candida albicans cells. Microscopic and molecular docking studies confirmed these findings. Overall, the study validates the antifungal efficacy of Candida albicans due to the presence of β-sitosterol which can act as an effective constituent for antifungal drug development individually or in combination.

Impact of Fab1/Vac14 inhibition on β-1,3-glucanase localization at the tip in Saccharomyces cerevisiae

Deep mycosis is a severe fungal disease that could result in fatal outcomes. However, there is still a demand for highly effective and safe antifungal drugs, given the side effects of the existing treatments and the increase in the resistance to them. In this study, we evaluated the involvement of the lipid kinase Fab1 and its activator Vac14 (Fab1/Vac14) in tip growth in Saccharomyces cerevisiae INVSc1, along with their impact on cell proliferation, using a genetic approach to inhibit them. The results revealed that Fab1/Vac14 inhibition suppressed growth and caused an increase in the rate of β-1,3-glucanase (BGL2) fused with emerald green fluorescent protein (EmGFP) (BGL2-EmGFP) localization at the tip. The inhibition of the endocytic pathway using a lysosome inhibitor also resulted in an increased localization of BGL2-EmGFP at the tip. The overexpression of wild-type BGL2-EmGFP, but not that of the inactive mutant BGL2, led to a complete loss of the cell proliferation ability. These findings suggested that the Fab1/Vac14 complex could be a novel target for the development of antifungal drugs based on tip growth regulation, possibly via excessive cell wall degradation.

Total Synthesis Analysis of Pseuboyenes D

In this work, we propose a total synthesis route for the antifungal agent Pseuboyenes D, a compound derived from Pseudallescheria boydii CS-793. Pseuboyenes D exhibits antifungal activity comparable to Amphotericin B while offering the advantage of a simpler structure, making it a promising candidate for clinical applications. The system became the focus of this analysis due to its regioselectivity, and after exploration, a [2+2] photocycloaddition and ring expansion strategy was adopted to construct the key bridged ring system. The route employs readily available substrates and mild reaction conditions, making it scalable and suitable for large-scale production. Computational analysis of the regioselectivity of the photocycloaddition revealed that the bridged adduct was thermodynamically favored over the fused system. The synthetic route was further diversified by incorporating functional group modifications using TMS derivatives, enhancing the potential for pharmacological applications. Overall, this work establishes an efficient and scalable synthetic pathway for Pseuboyenes D, paving the way for future applications in antifungal drug development.

Comparative analysis of polysaccharide and cell wall structure in Aspergillus nidulans and Aspergillus fumigatus by solid-state NMR

Invasive aspergillosis poses a significant threat to immunocompromised patients, leading to high mortality rates associated with these infections. Targeting the biosynthesis of cell wall carbohydrates is a promising strategy for antifungal drug development and will be advanced by a molecular-level understanding of the native structures of polysaccharides within their cellular context. Solid-state NMR spectroscopy has recently provided detailed insights into the cell wall organization of Aspergillus fumigatus, but genetic and biochemical evidence highlights species-specific differences among Aspergillus species. In this study, we employed a combination of 13C, 15N, and 1H-detection solid-state NMR, supplemented by Dynamic Nuclear Polarization (DNP), to compare the structural organization of cell wall polymers and their assembly in the cell walls of A. fumigatus and A. nidulans, both of which are key model organisms and human pathogens. The two species exhibited a similar rigid core architecture, consisting of chitin, α-glucan, and β-glucan, which contributed to comparable cell wall properties, including polymer dynamics, water retention, and supramolecular organization. However, differences were observed in the chitin, galactosaminogalactan, protein, and lipid content, as well as in the dynamics of galactomannan and the structure of the glucan matrix.

Structural insights into the inhibition mechanism of fungal GWT1 by manogepix

Glycosylphosphatidylinositol (GPI) acyltransferase is crucial for the synthesis of GPI-anchored proteins. Targeting the fungal glycosylphosphatidylinositol acyltransferase GWT1 by manogepix is a promising antifungal strategy. However, the inhibitory mechanism of manogepix remains unclear. Here, we present cryo-EM structures of yeast GWT1 bound to the substrate (palmitoyl-CoA) and inhibitor (manogepix) at 3.3 Å and 3.5 Å, respectively. GWT1 adopts a unique fold with 13 transmembrane (TM) helixes. The palmitoyl-CoA inserts into the chamber among TM4, 5, 6, 7, and 12. The crucial residues (D145 and K155) located on the loop between TM4 and TM5 potentially bind to the GPI precursor, contributing to substrate recognition and catalysis, respectively. The antifungal drug, manogepix, occupies the hydrophobic cavity of the palmitoyl-CoA binding site, suggesting a competitive inhibitory mechanism. Structural analysis of resistance mutations elucidates the drug specificity and selectivity. These findings pave the way for the development of potent and selective antifungal drugs targeting GWT1.

Current Updates on Pathogenesis, Systemic Therapy, and Treatment of Invasive Fungal Infections.

Numerous health hazards are associated with fungal infections, ranging from asymptomatic cases to potentially fatal invasive diseases that are especially dangerous for those with impaired immune systems. The main causes behind these diseases are opportunistic fungi, namely Aspergillus, Candida, and Cryptococcus. Invasive fungal infections (IFIs) require a global response that includes the development of vaccines, standardized protocols for diagnosis, potent antifungal medications, and strategies to stop drug-resistant strains. Improving high-risk group diagnosis and treatment is essential to lowering death rates. This review highlights the substantial health concerns associated with fungal infections, especially in immunocompromised individuals, and identifies Aspergillus, Candida, and Cryptococcus as the main pathogens. It highlights the necessity of international efforts, such as the development of novel diagnostic instruments, imaging methods, and antifungal drugs, to combat these invasive infections. The review also addresses the increasing need for novel treatment approaches in light of the developing resistance to widely used antifungal medications. Furthermore, the significance of secretory proteins in fungal pathogenicity and the potential of combination therapy are investigated. It is also suggested that a multimodal strategy be used to fight these illnesses, given the promise of multivalent vaccinations. Overall, this study emphasizes how critical it is to develop better diagnostic and treatment strategies in order to successfully control and lessen the impact of invasive fungal diseases on the health of the world.

Critical roles of Dpb3-Dpb4 sub-complex of DNA polymerase epsilon in DNA replication, genome stability, and pathogenesis of Candida albicans.

This study explored the role of DNA polymerase epsilon, especially its non-essential structural subunits in Candida albicans biology. Apart from their role in DNA replication and genome stability, the Dpb3-Dpb4 subcomplex regulates morphological switching and virulence. Since the defective strain is locked in filamentous form and is avirulent, the complex may be targeted for anti-fungal drug development.

Cycle-ESM: Generation-assisted classification of antifungal peptides using ESM protein language model

The rising prevalence of invasive fungal infections and the emergence of antifungal resistance highlight the urgent need for new antifungal medications. Antifungal peptides have emerged as promising alternatives to traditional antimicrobial agents. The identification of natural or synthetic antifungal peptides is crucial for advancing antifungal drug development. Typically, the availability of antifungal samples is limited, and significant sequence diversity exists among antifungal peptides, posing challenges for high-throughput screening. To address the identification challenge of antifungal peptides with limited sample availability, this study introduces the Cycle ESM method. Initially, the method utilises the ESM protein language model to generate additional data on antifungal peptides, serving as a data augmentation technique to enhance model training effectiveness. Subsequently, the ESM is employed in conjunction with a textCNN model to construct a classifier for peptide prediction, with a comprehensive exploration of peptide characteristics to improve prediction accuracy. Experimental results demonstrate that the performance of the Cycle ESM method surpasses that of existing methods across three distinct antifungal peptide datasets. This study presents a novel approach to antifungal peptide prediction and offers innovative insights for addressing classification problems with limited sample availability.

Mutations across Diverse Domains of CjXDR1 Lead to Multidrug Resistance in Clarireedia jacksonii.

Recently, Clarireedia jacksonii has emerged as a significant pathogen threatening turfgrass, and its escalating resistance to multiple drugs often undermines field interventions. This study highlighted the critical role of the fungus-specific transcription factor CjXDR1 (formerly ShXDR1) in regulating multidrug resistance (MDR) in C. jacksonii. This was demonstrated through experiments involving CjXDR1-knockout and CjXDR1-complemented strains. Our sequence analysis revealed five mutations in CjXDR1: G445D, K453E, S607F, D676H, and V690A. All five gain-of-function (GOF) mutations were confirmed to directly contribute to MDR against three different classes of fungicides (propiconazole: demethylation inhibitor, boscalid: succinate dehydrogenase inhibitor, and iprodione: dicarboximide) using the genetic transformation system and in vitro fungicide-sensitivity assay. Comparative transcriptome analysis revealed that CjXDR1 and its GOF mutations led to the overexpression of downstream genes encoding a Phase I metabolizing enzyme (CYP68) and two Phase III transporters (CjPDR1 and CjAtrD) previously reported. Knockout mutants of CYP68, CjPDR1, CjAtrD, and double-knockout mutants of CjPDR1 and CjAtrD exhibited increased sensitivity to all three fungicides tested. Among these, the CYP68-knockout mutants displayed the highest sensitivity to propiconazole, while the CjPDR1 knockout mutant exhibited significantly increased sensitivity to all three fungicides. Double-knockout mutants of CjPDR1 and CjAtrD displayed greater sensitivity than the single knockouts. In conclusion, multiple GOF mutants in CjXDR1 contribute to MDR by upregulating the expression of CjPDR1, CjAtrD, and CYP68. This study enhances our understanding of the molecular mechanisms underlying MDR in plant pathogenic fungi, providing valuable insights into GOF mutation structures and advancing the development of antifungal drugs.

Disruption to de novo uridine biosynthesis alters β-1,3-glucan masking in Candida albicans.

The uridine derivatives UDP-glucose and UDP-N-acetylglucosamine are important for cell wall construction as they are the precursors for the synthesis of β-1,3-glucan and chitin, respectively. Previous studies have demonstrated attenuated virulence of uridine auxotrophs in mice, which has been attributed to insufficient uridine levels for growth in the host. We have discovered that uridine deprivation in the uridine auxotroph ura3ΔΔ disrupts cell wall architecture by increasing surface mannans, exposing β-1,3-glucan and chitin, and decreasing UDP-sugar levels. Cell wall architecture and UDP-sugars can be rescued with uridine supplementation. The cell wall architectural disruptions in the ura3ΔΔ mutant also impact immune activation since the mutant elicited greater TNFα secretion from RAW264.7 macrophages than wild type. To determine if cell wall defects contributed to decreased virulence in the ura3ΔΔ mutant, we used a murine model of systemic infection. Mice infected with the ura3ΔΔ mutant exhibited increased survival and reduced kidney fungal burden compared with mice infected with wild type. However, suppression of the immune response with cyclophosphamide did not rescue virulence in mice infected with the ura3ΔΔ mutant, indicating the attenuation in virulence of uridine auxotrophs can be attributed to decreased growth in the host but not increased exposure of β-1,3-glucan. Moreover, the ura3ΔΔ mutant is unable to grow on ex vivo kidney agar, which demonstrates its inability to colonize the kidneys due to poor growth. Thus, although uridine auxotrophy elicits changes to cell wall architecture that increase the exposure of immunogenic polymers, metabolic fitness costs more strongly drive the observed virulence attenuation.IMPORTANCECandida albicans is a common cause of bloodstream infections (candidemia). Treatment of these bloodstream infections is made difficult because of increasing antifungal resistance and drug toxicity. Thus, new tactics are needed for antifungal drug development, with immunotherapy being of particular interest. The cell wall of C. albicans is composed of highly immunogenic polymers, particularly β-1,3-glucan. However, β-1,3-glucan is naturally masked by an outer layer of mannoproteins, which hampers the detection of the fungus by the host immune system. Alteration in cell wall components has been shown to increase β-1,3-glucan exposure; however, it is unknown how the inability to synthesize precursors to cell wall components affects unmasking. Here, we demonstrate how cell wall architecture is altered in response to a deficit in precursors for cell wall synthesis and how uridine is a crucial component of these precursors.

Cryo-EM structures of Candida albicans Cdr1 reveal azole-substrate recognition and inhibitor blocking mechanisms

In Candida albicans, Cdr1 pumps azole drugs out of the cells to reduce intracellular accumulation at detrimental concentrations, leading to azole-drug resistance. Milbemycin oxime, a veterinary anti-parasitic drug, strongly and specifically inhibits Cdr1. However, how Cdr1 recognizes and exports azole drugs, and how milbemycin oxime inhibits Cdr1 remain unclear. Here, we report three cryo-EM structures of Cdr1 in distinct states: the apo state (Cdr1Apo), fluconazole-bound state (Cdr1Flu), and milbemycin oxime-inhibited state (Cdr1Mil). Both the fluconazole substrate and the milbemycin oxime inhibitor are primarily recognized within the central cavity of Cdr1 through hydrophobic interactions. The fluconazole is suggested to be exported from the binding site into the environment through a lateral pathway driven by TM2, TM5, TM8 and TM11. Our findings uncover the inhibitory mechanism of milbemycin oxime, which inhibits Cdr1 through competition, hindering export, and obstructing substrate entry. These discoveries advance our understanding of Cdr1-mediated azole resistance in C. albicans and provide the foundation for the development of innovative antifungal drugs targeting Cdr1 to combat azole-drug resistance.