A method for screening drugs that target the V-ATPase VOc' subunit of Candida albicans to inhibit its virulence and its application.

Abstract

The present application relates to the technical field of drug screening and application, and discloses a screening method of a drug for targeting Candida albicans V-ATPase V0c' subunit to inhibit its virulence and application thereof, comprising the following steps: first, querying the protein sequence of V0c' through pubchem, performing structure prediction on the protein by Alphafold2, and virtually screening small molecule compounds capable of binding to the protein on software QuickVina 2.1; then, performing fine docking of the screened compounds with a series of key amino acids on the V0c' subunit on the software QuickVina 2.1, obtaining ideal screening drugs according to the fine docking results, combining mode analysis and molecular screening; finally, performing a series of type verification and molecular mechanism exploration on the ideal screening drugs. The obtained drug N026 can effectively down-regulate the hyphae, biofilm formation and adhesion capacity of Candida albicans, thereby inhibiting the virulence traits of Candida albicans, and effectively widening the drug range for treating Candida albicans infection in clinic.

Description

Technical Field

[0001] This invention relates to the field of drug screening and application technology, specifically to a method for screening drugs that target the V-ATPaseVOc' subunit of Candida albicans to inhibit its virulence and its application. Background Technology

[0002] Candida albicans is the most common pathogenic fungal infection causing oral candidiasis in humans, and this type of infection is highly prevalent in the elderly and those with weakened immune systems. When the oral microenvironment is imbalanced and the body's immune defenses are weakened, Candida albicans can proliferate abnormally, leading to a series of pathological damages such as oral mucosal congestion, leukoplakia, erosion, and pain, severely disrupting the normal physiological structure and eating function of the oral cavity. If oral Candida albicans infection is not treated promptly and effectively, the condition can persist and further induce deep invasive fungal infections, causing continuous damage to the body and seriously threatening the health and quality of life of particularly susceptible populations. Therefore, efficient, precise, and safe prevention and control of oral Candida albicans infection is a key research direction in the field of clinical anti-infective treatment of oral diseases.

[0003] For oral infections caused by Candida albicans, current technologies primarily rely on traditional antifungal drug development and screening systems to obtain therapeutic drugs. These systems utilize conventional methods such as in vitro antifungal activity verification, strain-killing efficacy assessment, and pharmacological activity screening to screen for broad-spectrum antifungal drugs with inhibitory and bactericidal activity against Candida albicans. Existing screening technologies mainly focus on the overall antifungal and bactericidal effects, using the drug's ability to inhibit Candida albicans proliferation and its strain-clearing effect as core evaluation indicators. The screened drugs often exert their anti-infective effects through common mechanisms such as interfering with fungal cell membrane synthesis, blocking fungal basal metabolic pathways, and inhibiting fungal cell proliferation, thereby achieving clinical treatment and symptom control of oral candidiasis.

[0004] However, existing drug screening technologies and corresponding clinical treatment plans for Candida albicans infection have many technical defects that are difficult to overcome, which greatly limit the clinical treatment effect of oral candidiasis. As follows: (1) The long-term abuse of existing broad-spectrum antifungal drugs has led to a continuous increase in Candida albicans drug resistance, a significant decrease in the antibacterial and bactericidal effects of traditional drugs, and a significant reduction in the clinical treatment effectiveness. (2) Antifungal drugs obtained by existing screening systems generally have obvious limitations in clinical use. They not only have systemic toxic side effects, but also are prone to cross-reaction with other clinical drugs, resulting in poor drug safety. Furthermore, the types of antifungal drugs currently available in clinical practice are scarce, and the available treatment drug system is limited, making it difficult to meet the individualized treatment needs of complex and drug-resistant oral candidiasis. (3) Existing drug screening is mostly based on the core idea of ​​directly killing and inhibiting fungal proliferation. It lacks a new screening strategy that targets virulence regulation and does not conduct precise screening for specific pathogenic targets of Candida albicans, thus failing to avoid the problems of fungal resistance and drug toxic side effects from the root. The V-ATPase V0c' subunit of *Candida albicans* is a fungal-specific target with no homologous protein in the human body, making it a highly promising novel antifungal target. However, there is currently no drug screening system based on this specific target that inhibits fungal virulence to achieve anti-infection. In summary, existing technologies suffer from single-target approaches, traditional screening methods, a limited range of drugs, and high treatment risks. There is an urgent need to develop a novel drug screening method targeting the specific virulence of *Candida albicans* to enrich the therapeutic targets and drug types for oral candidiasis and overcome current clinical treatment bottlenecks. Summary of the Invention

[0005] The present invention aims to provide a method for screening drugs that target the V-ATPase V0c' subunit of Candida albicans to inhibit its virulence and its application, in order to solve the technical problem that the drugs obtained by existing screening systems have low clinical efficacy against drug-resistant Candida albicans.

[0006] To achieve the above objectives, the present invention employs the following technical solution: a method for screening drugs that target the V-ATPase V0c' subunit of Candida albicans to inhibit its virulence, comprising the following steps: Step S1, Deconstruction and Initial Screening: The protein sequence of V0c' was queried through PubChem, its structure was predicted using Alphafold2, and small molecule compounds that could bind to the protein were virtually screened on the software QuickVina 2.1. Step S2, Amino Acid Fine Docking: The compounds after initial screening are finely docked with the V0c' subunit using QuickVina 2.1 software; based on the fine docking results, combined with pattern analysis and molecular screening, ideal screening drugs are obtained; Step S3, Verification: Verify the inhibitory effect of the obtained drug on the in vitro and in vivo toxicity of Candida albicans through experiments.

[0007] Beneficial Effects: This screening method, focusing on the key virulence target of Candida albicans, the V-ATPase V0c' subunit, constructs a hierarchical precision drug screening system encompassing "structure prediction - virtual primary screening - precise docking of key amino acids - phenotypic and molecular mechanism verification." It overcomes the shortcomings of traditional broad-spectrum antifungal screening, such as high blindness, large off-target rates, and easy induction of drug resistance. It possesses significant advantages including clear targets, high screening efficiency, strong binding specificity, and reliable results. It can efficiently obtain candidate drugs that target and inhibit the virulence of Candida albicans, providing a precise and standardized screening technology system for the development of novel antifungal virulence inhibitors. Specifically, it offers the following advantages: 1. Precise and well-defined target, with highly targeted screening. This method targets the V-ATPase V0c' subunit, the core regulatory element of Candida albicans virulence, as its specific target. Unlike traditional antifungal drug screening methods that use bactericidal or bacteriostatic effects as a single evaluation criterion, this method focuses on inhibiting fungal virulence. This effectively avoids the drawbacks of traditional antifungal drugs, such as inducing fungal resistance and disrupting the body's normal flora. The candidate drugs obtained through screening have higher safety and lower resistance risk, meeting the development needs of novel targeted antifungal drugs.

[0008] 2. Leveraging artificial intelligence for structure prediction to solve the problem of missing target structures. By obtaining protein sequences through PubChem and combining them with Alphafold2 to accurately predict the higher-order structure of proteins, this approach overcomes the technical bottleneck of unresolved crystal structures of some fungal target proteins, which prevents molecular docking screening. It eliminates the need for expensive and complex protein crystallization experiments, enabling accurate modeling of target structures, significantly reducing preliminary experimental costs and shortening the early preparation cycle for drug screening.

[0009] 3. A hierarchical virtual screening system enhances both screening efficiency and accuracy. Employing a two-stage screening model of "broad-spectrum virtual primary screening + precise docking of key amino acids," the system first uses QuickVina 2.1 to perform primary screening of a large number of small molecule compounds, quickly eliminating inactive compounds and reducing the sample size. Then, precise docking verification is performed on the key amino acid sites of the V0c' subunit, identifying highly active candidate drugs from the perspectives of binding site and binding capacity. This significantly reduces the false positive rate and solves the problems of large screening volume and low effective hit rate associated with traditional screening methods.

[0010] 4. A dual-layer validation of phenotypic and molecular mechanisms ensures high reliability of screening results. This method, based on virtual screening, adds validation of fungal phenotypic function and exploration of cellular molecular mechanisms. It cross-validates computer-generated data with real biological activity and cellular mechanisms of action, eliminating data bias from simple molecular docking. This clarifies the mechanism by which candidate drugs inhibit the virulence of Candida albicans, making the screening results more rigorous and reliable, and providing solid experimental and theoretical support for subsequent drug development and clinical translation.

[0011] Preferably, as an improvement, the ideal screening drug is N026, with the following structural formula: .

[0012] Preferably, as an improvement, in step S1, the virtual screening includes the following: using a ligand-free empty protein structure, setting a large-scale docking box with the following parameters: center coordinates center_x = 9.588, center_y = 1.565, center_z = 12.527; box dimensions size_x = 42.0, size_y = 42.0, size_z = 46.0; and selecting the top 100 small molecules based on docking scores to proceed to the next stage.

[0013] Preferably, as an improvement, in step S2, during the fine docking, for molecules that rank highly in the initial screening, the docking box is significantly reduced in size, only enclosing the core binding pocket residues of the VMA11 protein.

[0014] Preferably, as an improvement, in step S2, the binding energy between the key interacting amino acids of the homologous modeling protein of the small molecule obtained by fine docking screening and the template protein is ≤-7.5 kcal / mol.

[0015] Preferably, as an improvement, in step S2, the combination pattern analysis and molecular screening includes extracting compounds that score below -7.5 kcal / mol after fine docking, and then performing three-dimensional conformation and interaction analysis using PyMOL and Discovery Studio (DS) visualization software.

[0016] Preferably, as an improvement, the three-dimensional conformation and interaction analysis includes (1) spatial position verification, which requires that the binding conformation of the small molecule must be located in the correct binding pocket of the c' subunit, and eliminates false positive molecules whose binding sites are shifted due to their small molecular size; (2) interaction verification: the candidate small molecule must form a strong hydrophobic interaction with the key residues of the target protein.

[0017] Preferably, as an improvement, the obtained key residues include Ala 107, Ser 114, Phe 142, Ala 143, Leu 146, Val 145, Tyr 149, and Ile 153.

[0018] Preferably, as an improvement, this solution also provides the use of drug N026 in the preparation of a drug for treating oral candidiasis, acute pseudomembranous candidiasis in immunocompromised patients, denture-associated candidiasis, or removal of biofilms on denture surfaces.

[0019] Preferably, as an improvement, this solution also provides the application of drug N026 in the preparation of drugs for the prevention and treatment of dental caries.

[0020] The principles and advantages of this scheme are: The drug ID N026-0090 (hereinafter referred to as N026) obtained from the virtual screening in this scheme can effectively downregulate the hyphae, biofilm formation, and adhesion ability of Candida albicans, thereby inhibiting its virulence. Specifically, after co-culturing drug N026 with wild-type Candida albicans and its fluconazole-resistant and susceptible strains for 24 hours, microscopic observation revealed a significant reduction in hyphal length compared to the control group. Biofilm formation was measured using a crystal violet assay, showing that N026 significantly reduced biofilm formation in wild-type Candida albicans and also inhibited the amount of biofilm formation in its resistant strains. Furthermore, in a cell adhesion assay, co-culturing Candida albicans treated with N026 with oral mucosal epithelial cells showed that N026 significantly inhibited the adhesion ability of Candida albicans to oral mucosal epithelium. Attached Figure Description

[0021] Figure 1 This is the binding site between the V0c' subunit of the Candida albicans VMA11 protein and the screened drug N026 in Experimental Example 1 of this invention.

[0022] Figure 2 This describes the hydrophobic binding characteristics of the V0c' subunit of the Candida albicans VMA11 protein and the screened drug N026 in Experimental Example 1 of this invention.

[0023] Figure 3 This describes the hydrogen bond binding characteristics between the V0c' subunit of the Candida albicans VMA11 protein and the screened drug N026 in Experimental Example 1 of this invention.

[0024] Figure 4 This is a molecular docking diagram of the remaining small molecule drug N006 in this invention.

[0025] Figure 5 This is a molecular docking diagram of the remaining small molecule drug N040 in this invention.

[0026] Figure 6 This is a molecular docking diagram of the remaining small molecule drug 8013 in this invention.

[0027] Figure 7This is a molecular docking diagram of the remaining small molecule drug G946 in this invention.

[0028] Figure 8 This is a scanning electron microscope image of the antibacterial experiment in Experiment Example 2 of the present invention.

[0029] Figure 9 This is a comparison of the crystal violet staining results in the antibacterial experiment of Experiment Example 2 of the present invention.

[0030] Figure 10 This is the crystal violet OD value in the antibacterial experiment of Experiment Example 2 of the present invention.

[0031] Figure 11 This shows the growth of mycelia in a solid culture medium during the antibacterial experiment in Experiment Example 2 of the present invention.

[0032] Figure 12 This is a diagram showing the infected state of the animal's tongue in Experiment Example 3 of this invention.

[0033] Figure 13 This is a slice of the infected tongue of an animal in Experiment Example 3 of the present invention.

[0034] Figure 14 This is an HE staining image of the infected tongue of an animal in Experiment Example 3 of this invention.

[0035] Figure 15 This is a cell adhesion diagram of the drug N026 of the present invention (left is the control group, right is the N026 group). Detailed Implementation

[0036] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0037] Example 1 This protocol provides a method for screening drugs that target the V-ATPase V0c' subunit of Candida albicans to inhibit its virulence, including: 1. Preparation of target protein and small molecule libraries The protein V0C'(H) encoded by VMA11 of Candida albicans (strain SC5314) was obtained from the NCBI database. + The amino acid sequence of the transporting V0sector ATPase subunit c' (Uniport ID: Q5AH00) was obtained. Its three-dimensional predicted structure was obtained using the AlphaFold2 protein structure database (AlphaFold DB) as a receptor protein model.

[0038] To prepare a small molecule library, this example uses the Chemdiv and Lifechemicals commercial libraries, totaling 6 molecule collections containing approximately 60,000 small molecule compounds as ligand libraries for screening.

[0039] 2. Establishment of a positive reference threshold To verify the accuracy of the docking parameters and establish a screening threshold, this embodiment used the known complex structure 7TAP (a complex of yeast VOV-ATPase and the inhibitor Archazolid A) resolved by cryo-electron microscopy as a template for redocking experiments. The redocking results confirmed the consistency of key interacting amino acids between the homologous modeling protein and the template protein, with a minimum redocking score of -7.5 kcal / mol. Therefore, this invention sets a binding energy ≤ -7.5 kcal / mol as a refined screening threshold for high-affinity molecules.

[0040] 3. Implementation of a multi-level shrinking grid box strategy The docking calculations were performed using qvina2 and AutoDock Vina software. To improve screening efficiency and accuracy, this implementation adopted a three-level docking box setting method from coarse to fine: Level 1: Initial screening and connection across the entire system.

[0041] Using ligand-free empty protein structures, a large-scale docking box was designed with the following parameters: center coordinates center_x = 9.588, center_y = 1.565, center_z = 12.527; box dimensions size_x = 42.0, size_y = 42.0, size_z = 46.0. Based on docking scores, the top 100 small molecules were selected for the next stage.

[0042] Level 2: Precise docking based on key amino acid residues.

[0043] For molecules that rank highly in the initial screening, the docking box is significantly reduced in size, encapsulating only the core binding pocket residues of the VMA11 protein.

[0044] Level 3: Fine-tuned docking based on the dominant conformation (optional optimization).

[0045] 4. Combining pattern analysis with molecular screening criteria Compounds with a score below -7.5 kcal / mol after fine docking were extracted and subjected to 3D conformation and interaction analysis using visualization software such as PyMOL and Discovery Studio (DS). The criteria for determining whether candidate compounds meet the requirements include: (1) Spatial position verification: The binding conformation of small molecules must be located in the correct binding pocket of the c' subunit. False positive molecules (such as some Lifechemicals library molecules) are eliminated because the binding site is shifted due to the small molecular size. (2) Interaction verification: The candidate small molecule must form a strong hydrophobic interaction with the key residues of the target protein (such as Ala107, Phe142, Ile153); Through the above-mentioned rigorous screening across multiple dimensions, a number of lead compounds with the correct binding sites and scores below -7.5 kcal / mol were precisely selected from approximately 60,000 compounds (such as 5 high-potential molecules from the Chemdiv library, detailed citation information for these 5 drugs is shown in Table 1), for subsequent biological activity validation.

[0046] Table 1. Relevant information of the five high-potential molecules selected through screening.

[0047] This plan also provides the application of drug N026 in the preparation of a drug for treating oral candidiasis.

[0048] This protocol also provides the application of drug N026 in the preparation of a drug for treating acute pseudomembranous candidiasis in immunocompromised patients.

[0049] This plan also provides the application of drug N026 in the preparation of a drug for treating denture-associated candidiasis.

[0050] This solution also provides the application of drug N026 in the preparation of a drug for treating the removal of biofilms on the surface of dentures.

[0051] This plan also provides the application of drug N026 in the preparation of drugs for the prevention and treatment of dental caries.

[0052] Experimental Example 1: Analysis of Protein-Ligand Interactions like Figure 1 As shown, the binding site of N026 is characterized by binding within a hydrophobic interface, forming hydrophobic contacts with multiple residues (Ala107, Phe142, Ile153, etc.) (hydrophobic bonding characteristics are as follows). Figure 2 (As shown). Regarding hydrogen bonds, it forms strong hydrogen bonds with the Ser114 side chain and weak hydrogen bonds with the Phe142 main chain (hydrogen bond characteristics are shown in...). Figure 3 (As shown). The binding mode is mainly hydrophobic interaction, with hydrogen bonding as a secondary mechanism. The molecular connection diagrams of the other four drugs are shown below. Figures 4-7 As shown.

[0053] Experiment Example 2: Antibacterial Experiment The experimental steps and procedures are as follows: Solid hyphae were observed; the ability of *Candida albicans* yeast-hyphae phase change under drug intervention was further observed on YPD + 10% FBS solid medium. As shown in the figure, wild-type *Candida albicans* colonies formed dense and thick hyphae on the solid medium, while *Candida albicans* colonies under drug intervention did not fully form hyphae, only showing wrinkled structure changes around the colony center. This indicates that N026 significantly inhibited the *Candida albicans* yeast-hyphae morphological transition, leading to hyphal developmental disorders.

[0054] Electron microscopy: The morphology of Candida albicans cells and the internal structure of the biofilm were observed under a scanning electron microscope. As shown in the figure, the hyphae in the group without drug intervention were intact and well developed. However, after drug treatment, it was found that most Candida albicans remained in the yeast phase, and some of the hyphae structures collapsed and broke, showing obvious growth defects.

[0055] Crystal violet: Candida albicans was activated and cultured in YPD medium to prepare a solution with a concentration of 1×10⁻⁶. 6 CFU / mL bacterial suspension was resuspended in RPMI 1640 medium and inoculated into 96-well cell culture plates. The plates were incubated at 37°C for 90 minutes to allow the bacteria to adhere to the bottom of the wells. Unattached airborne bacteria were then aspirated, and the plates were gently washed with PBS before adding fresh RPMI. 1640 culture medium was cultured at 37°C for 24 hours to form a mature biofilm. Afterwards, the culture medium was aspirated and loose cells were removed by washing with PBS. Culture medium containing different concentrations of the test drug was added to each well. A positive control without drug, a negative control without bacteria, and a solvent control containing the highest concentration of solvent were also included. After incubation at 37°C for 24 hours, the drug solution was aspirated and the cells were gently washed 2 to 3 times with PBS. 0.1% to 1% crystal violet solution was added for staining at room temperature for 15 to 30 minutes. The staining solution was then aspirated and the cells were rinsed with running water or PBS until the washings were colorless. Elution was then performed with 33% glacial acetic acid or 95% ethanol. After standing at room temperature for 15 to 30 minutes, the absorbance was measured using a microplate reader at the corresponding wavelength. Finally, the inhibition rate of the drug on the biofilm was calculated based on the absorbance values ​​of the positive control, drug group, and negative control.

[0056] like Figure 8 As shown in the electron microscopy results, the biofilm formation in the drug-treated group was significantly looser than that in the control group, and the number of proliferating Candida albicans was also much smaller.

[0057] like Figure 9 As shown in the results, the amount of biofilm formed after the addition of crystal violet was significantly reduced.

[0058] like Figure 10 As shown, the analysis of crystal violet OD values ​​also revealed that the amount of biofilm formed by Candida albicans was significantly reduced after the addition of the drug.

[0059] Hyphae are one of the key forms in which Candida albicans express virulence. For example... Figure 11 As shown, the mycelial results indicated that the mycelial length in the drug-treated group was shorter than that in the control group in liquid culture medium. In solid culture medium, the colony mycelia were more prominent, and mycelial formation in the drug-treated group was severely restricted. This experiment demonstrates that the growth of Candida albicans mycelia is severely limited after drug treatment.

[0060] Experiment Example 3: Animal Experiment (1) Preparation of bacterial culture The preserved Candida albicans SC5314 strain was streaked onto YPD agar plates. After recovery, single colonies were selected and inoculated into YPD liquid medium (containing 20.0 g / L peptone, 10.0 g / L yeast extract, 20.0 g / L glucose, pH 6.5 ± 0.2). The culture was incubated overnight at 30°C and 200 rpm with shaking for 16 h. The bacterial suspension was then transferred to fresh YPD medium at a 1:10 ratio and cultured until the logarithmic growth phase. The bacterial suspension was collected, centrifuged at 4000 rpm for 5 min to precipitate the cells, and the supernatant was discarded. The cells were resuspended in sterile physiological saline and washed twice. Finally, the concentration of the bacterial suspension was adjusted to 1 × 10⁻⁶ using a hemocytometer. 7 The cells / mL were prepared as an inoculum for infection.

[0061] (2) Animal grouping and construction of immunosuppression model This animal experimental protocol was reviewed and approved by the Medical Ethics Committee of the Affiliated Stomatological Hospital of Chongqing Medical University (Ethics No.: 2025151). All mice were housed in an SPF-grade environment (Specific Pathogen Free). Eighteen healthy BALB / c mice were selected and, after acclimatization, were randomly divided into three groups (n=6): a blank control group (treated with physiological saline), a model group (infected with the above-mentioned inoculation solution to achieve Candida albicans WT infection), and a drug intervention group (N26y treatment group: applied topically three times a day for three days).

[0062] To simulate the host's immunosuppressed state, 4 mg of cortisone acetate was dissolved in 200 μL of PBS (containing 5% Tween 80 for dissolution) to prepare an immunosuppressive injection. Mice were subcutaneously injected into the back of the neck one day before and one day after fungal infection, with a dosage of 4 mg / mouse / dose.

[0063] (3) Establishment of a mouse oral candidiasis model A mouse model of oral candidiasis was established using the sublingual inoculation method with cotton balls. On the day of infection, sterile cotton balls were immersed in the prepared bacterial suspensions of each group and placed on a shaker for 4 hours to ensure full adsorption of the bacterial suspension. Mice were generally anesthetized by intraperitoneal injection of 5% chloral hydrate (10 mg / kg). After the mice were comatose and their breathing was stable, cotton balls soaked in bacterial suspension were placed under the tongue of the mice using ophthalmic forceps and kept in contact for 75 minutes to complete the inoculation. During anesthesia and inoculation, the mice were placed in a pet incubator to maintain body temperature. If the mice showed signs of awakening during inoculation, an additional 20% of the initial anesthetic dose was administered to maintain the depth of anesthesia. The vital signs of the mice were closely monitored to prevent suffocation or aspiration due to cotton ball displacement.

[0064] (4) Sample collection and processing Two days after infection, all mice were euthanized. The tongue tissue was completely dissected under aseptic conditions, and gross photographs were taken, with the lesion area recorded. The tongue was longitudinally incised along the midline, dividing the tongue tissue in half. The left half was immediately placed in a centrifuge tube containing 4% paraformaldehyde and fixed at 4°C for histopathological analysis. The right half was placed in a sterile centrifuge tube for fungal load determination.

[0065] (5) Determination of fungal load (CFU count) in tongue tissue Accurately weigh the wet weight of the tongue tissue from each group of mice, and homogenize it thoroughly in a tissue homogenizer containing 1 mL of sterile PBS. The original homogenate was then serially diluted 10-fold (10... 1 10 2 10 3 (Multiple dilutions). Take 100 μL of each serial dilution and spread it onto the surface of the Candida albicans chromogenic medium. Incubate the plates at 37°C in the dark for 48 hours, and then select plates with appropriate colony counts for counting. Calculate the colony forming units (CFU) per gram of tissue based on the dilution factor and tissue weight. Set up 3 replicates for each sample.

[0066] (6) Histopathological evaluation Fixed tongue tissue samples were sent to Saive Biotechnology Co., Ltd. for further processing. After gradient dehydration with ethanol, clearing, and paraffin embedding, tissue sections (4-5 μm thick) were prepared. Hematoxylin-eosin (HE) staining and periodic acid-Scheffler (PAS) staining were performed. The stained sections were digitally imaged using an Olympus VS200 whole-slide scanning system to observe epithelial integrity, inflammatory cell infiltration, and fungal hyphae invasion.

[0067] like Figures 12-14As shown, HE staining revealed extensive sheet-like or focal necrosis of mucosal epithelial cells. The lamina propria showed abundant residual cell debris and infiltration of neutrophils and macrophages, indicating significant fungal invasion and immune inflammation. In the N026 treatment group, yeast-like Candida albicans was observed, with continuous and undamaged lingual papillae. PAS staining suggested that WT Candida albicans primarily infiltrated via hyphae, with a deeper penetration into the basal layer. In contrast, the N026 treatment group showed relatively less hyphae formation and a shallower penetration area.

[0068] Experiment Example 4: Cell Adhesion Experiment To evaluate the intervention effect of drug N026 on the ability of Candida albicans to adhere to oral epithelial cells (HOK), standard strains of Candida albicans were inoculated into YPD liquid medium and cultured overnight with shaking at 30°C and 200 rpm. The next day, the cells were transferred to fresh YPD medium at a ratio of 1:10 and cultured until the logarithmic growth phase. The cells were collected by centrifugation at 4000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in DMEM high-glucose medium, and drug N026 was added to adjust the bacterial concentration to 2 × 10⁻⁶. 5 cells / mL were prepared for use; meanwhile, HOK cells with a confluence of approximately 70-80% were trypsinized, centrifuged, resuspended in DMEM complete medium, and the cell density was adjusted to 1×10⁻⁶ cells / mL. 5 Cells / mL, 1 mL of the cell suspension was seeded into a confocal microplate and incubated overnight at 37°C to establish monolayer cell adhesion. At the beginning of the experiment, the old culture medium in the microplate was carefully aspirated, and N026 pretreated bacterial suspension was added. Drug-free bacterial suspension and sterile DMEM high-glucose medium were set up as growth control and negative control, respectively. After co-incubation at 37°C for 1 hour, the supernatant was aspirated and the cells were gently washed 3 times with sterile PBS buffer to fully remove unadhered planktonic fungi. Then, 4% paraformaldehyde was added for fixation at room temperature for 15 minutes. After fixation, Gram staining was performed strictly according to the kit instructions. Finally, the cells were observed under a microscope. 8 to 10 fields of view were randomly selected from each sample to collect images and count the number of fungi adhering to the surface of HOK cells.

[0069] Experimental results are as follows Figure 15 As shown, Candida albicans still adhered to the cells after three rinses, and the adhesion ability of Candida albicans was significantly reduced after drug intervention. The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for screening drugs that target the V-ATPase V0c' subunit of Candida albicans to inhibit its virulence, characterized in that: Includes the following steps: Step S1, Deconstruction and Initial Screening: The protein sequence of V0c' was queried through PubChem, its structure was predicted using Alphafold2, and small molecule compounds that could bind to the protein were virtually screened on the software QuickVina 2.

1. Step S2, Amino Acid Fine Docking: The compounds after initial screening are finely docked with the V0c' subunit using QuickVina 2.1 software; based on the fine docking results, combined with pattern analysis and molecular screening, ideal screening drugs are obtained; Step S3, Verification: Verify the inhibitory effect of the obtained drug on the in vitro and in vivo toxicity of Candida albicans through experiments.

2. The method for screening drugs that target the V-ATPase V0c' subunit of Candida albicans to inhibit its virulence according to claim 1, characterized in that: The desired screening drug is N026, with the following structural formula: 。 3. The method for screening drugs that target the V-ATPase V0c' subunit of Candida albicans to inhibit its virulence according to claim 1, characterized in that: In step S1, the virtual screening includes the following: using ligand-free empty protein structures, setting a large-scale docking box with the following parameters: center coordinates center_x = 9.588, center_y = 1.565, center_z = 12.527; box dimensions size_x = 42.0, size_y = 42.0, size_z = 46.0; and selecting the top 100 small molecules based on docking scores to proceed to the next stage.

4. The method for screening drugs that target the V-ATPase V0c' subunit of Candida albicans to inhibit its virulence according to claim 3, characterized in that: In step S2, during the fine docking process, for the molecules that rank highly in the initial screening, the docking box is significantly reduced in size, encapsulating only the core binding pocket residues of the VMA11 protein.

5. A method for screening drugs that target the V-ATPase V0c' subunit of Candida albicans to inhibit its virulence according to claim 4, characterized in that: In step S2, the binding energy between the homologous modeling protein of the small molecule obtained by fine docking screening and the key interacting amino acids of the template protein is ≤-7.5 kcal / mol.

6. A method for screening drugs that target the V-ATPase V0c' subunit of Candida albicans to inhibit its virulence according to claim 5, characterized in that: In step S2, the binding pattern analysis and molecular screening include extracting compounds that score below -7.5 kcal / mol after fine docking and then performing three-dimensional conformation and interaction analysis using PyMOL and Discovery Studio (DS) visualization software.

7. A method for screening drugs that target the V-ATPase V0c' subunit of Candida albicans to inhibit its virulence according to claim 6, characterized in that: The three-dimensional conformation and interaction analysis includes (1) spatial position verification, which requires that the binding conformation of the small molecule must be located in the correct binding pocket of the c' subunit, and eliminates false positive molecules whose binding sites are shifted due to their small molecular size; (2) interaction verification: the candidate small molecule must form a strong hydrophobic interaction with the key residues of the target protein.

8. A method for screening drugs that target the V-ATPase V0c' subunit of Candida albicans to inhibit its virulence according to claim 7, characterized in that: The key residues obtained include Ala 107, Ser 114, Phe 142, Ala 143, Leu 146, Val 145, Tyr 149, and Ile 153.

9. The use of drug N026 obtained by the method according to any one of claims 1 to 8 in the preparation of a drug for treating oral candidiasis, acute pseudomembranous candidiasis in immunocompromised patients, denture-associated candidiasis, or removal of biofilms on denture surfaces.

10. The use of drug NO26 obtained by the method according to any one of claims 1 to 8 in the preparation of a drug for the prevention and treatment of dental caries.

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