Use of cis-6-octadecenoic acid in the preparation of antifungal drugs

Abstract

The application discloses application of cis-6-octadecenoic acid or a pharmaceutically acceptable salt thereof in preparation of an antifungal drug, and a structure of the cis-6-octadecenoic acid is as shown in the following formula: By using a Candida albicans single allele knockout bacteria library, fructose-1,6-bisphosphate aldolase is screened as an antifungal action target point of PeAc, and PeAc is an inhibitor of fructose-1,6-bisphosphate aldolase. In the application, by using a micro liquid-based dilution method, it is found that PeAc shows good in-vitro antifungal activity by widely screening a natural compound library. In the application, by using mouse and wax moth larva fungal infection experiments, it is found that PeAc shows good in-vivo antifungal activity in animals. In the application, by using a chessboard type micro liquid-based dilution method, a bacteriostatic ring method and a growth curve method, it is found that PeAc and fluconazole show clear synergistic antifungal action on drug-resistant fungi.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology, specifically, it relates to the application of cis-6-octadecenoic acid in the preparation of antifungal drugs. Background Technology

[0002] In recent years, with the increasing number of immunocompromised individuals such as cancer patients, organ transplant recipients, and the elderly, the incidence and mortality rates of invasive fungal infections (IFIs) have been rising. Invasive Candidiasis (IC) is the dominant IFI, with a mortality rate roughly estimated to be over 40%, with Candida albicans being the primary infecting strain. Besides causing superficial and deep fungal infections in the human body through its yeast and hyphal forms, Candida albicans can also adhere to the surfaces of internal organs or biological materials by forming a dense biofilm, making it difficult to eradicate and causing recurrent infections. Currently, commonly used antifungal drugs in clinical practice include azoles, polyenes, echinocandins, and allylamines. However, with the long-term use of traditional drugs, drug resistance in fungi is increasingly common in clinical practice, which has become one of the main reasons for treatment failure in fungal infections. In addition, traditional antifungal drugs have problems such as high toxicity, low efficacy, and high price, which make it difficult to meet the needs of human health. There is an urgent need to develop new antifungal drugs that are highly effective and low in toxicity.

[0003] cis-6-octadecenoic acid (PeAc will be used to represent cis-6-octadecenoic acid in the following text) is an unsaturated fatty acid found in plants such as coriander and carrots. Its structural formula is shown in (I), and its molecular formula is C1. 18 H 34 O2, molecular weight 282.46.

[0004]

[0005] Previous studies have shown that PeAc can regulate the gut microbiota (Genes. 2022; 13(11): 1967.). PeAc is a bactericide against Porphyromonas gingivalis and is a major active ingredient in plant extracts for the prevention and treatment of periodontal disease (Microbiol. 2022; 13: 816047.). PeAc can inhibit the formation of Staphylococcus aureus biofilms, including biofilms of methicillin-resistant Staphylococcus aureus (MRSA), and PeAc can also inhibit the production of Staphylococcus aureus virulence factors (Microbiology Spectrum. 2022; 10(3): e0133022.). PeAc can alleviate intestinal damage and reduce apoptosis in mice with intestinal ischemia / reperfusion injury (British journal of anaesthesia. 2022; 128(3): 501-12.). The kingdom of fungi (eumycetes) is a kingdom of organisms, a large class of higher eukaryotes, which are completely different from bacteria. Most of the above reports are research reports on PeAc's resistance to pathogenic bacteria, while the research on PeAc's resistance to pathogenic fungi involved in this invention has not been reported at home and abroad.

[0006] Haploid deficiency profiling (HIP) is an effective method for identifying antifungal drug targets. Literature reports that diploid fungi exhibit a halved gene dose after the deletion of an allele encoding a drug target, resulting in a significant increase in drug sensitivity in the deleted target gene. This phenomenon is known as the haploid deficiency effect. Researchers can construct gene knockout libraries and use the haploid deficiency effect to screen for drug-sensitive gene knockout bacteria, thereby identifying drug targets. In 2014, Science reported the identification of the fluconazole target Erg11p and the tamoxifen target Neo1p using a Saccharomyces cerevisiae gene knockout library (Science. 2014; 344(6180):208-11). Scientists have also used the haploid deficiency effect to identify that the antifungal target of Turbinmicin is Sec14p (Science. 2020; 370(6519):974-8), the antifungal target of F901318 is dihydrolactic acid dehydrogenase (DHODH) (Proc Natl Acad Sci US A. 2016; 113(45):12809-14), and the antifungal targets of compounds G884 and G365 are Gwt1p (ACS Infect Dis. 2015; 1(1):59-72), etc. Summary of the Invention

[0007] The purpose of this invention is to provide an application of cis-6-octadecenoic acid in the preparation of antifungal drugs.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] The first aspect of the present invention provides the use of cis-6-octadecenoic acid or a pharmaceutical salt thereof in the preparation of antifungal drugs.

[0010] The structure of the cis-6-octadecenoic acid (PeAc) is shown below:

[0011]

[0012] The antifungal target of cis-6-octadecenoic acid is fructose-1,6-bisphosphate aldolase (Fba1p). Cis-6-octadecenoic acid exerts its antifungal effect through fructose-1,6-bisphosphate aldolase (Fba1p).

[0013] The medicinal salts refer to acid addition salts formed by cis-6-octadecenoic acid and the following acids: hydrochloric acid, sulfuric acid, nitric acid, citric acid, maleic acid, etc.

[0014] The fungi are selected from Candida albicans, Candida krusei, Candida tropicalis, Trichophyton mentagrophytes, etc.; specifically: Candida albicans SC5314; Candida krusei 4996, 62588, 10153; Trichophyton mentagrophytes T5b, T5a; Candida tropicalis 409, 8915; Candida albicans 14103, 7654, 9161, 10066, 10060, 10061, 7879, 901, 953.

[0015] The effective concentration of the cis-6-octadecenoic acid or its pharmaceutical salt in anti-Candida albicans drugs is ≥2 μg / ml; the effective concentration of the cis-6-octadecenoic acid or its pharmaceutical salt in anti-Candida krusei drugs is ≥2 μg / ml; the effective concentration of the cis-6-octadecenoic acid or its pharmaceutical salt in anti-Candida tropicalis drugs is ≥32 μg / ml; and the effective concentration of the cis-6-octadecenoic acid or its pharmaceutical salt in anti-Trichophyton mentagrophytes drugs is ≥4 μg / ml.

[0016] A second aspect of the present invention provides the use of cis-6-octadecenoic acid or its pharmaceutical salt in conjunction with commonly used clinical antifungal drugs in the preparation of antifungal drugs.

[0017] The commonly used antifungal drugs mentioned are selected from fluconazole, ketoconazole, itraconazole, amphotericin B, caspofungin, terbinafine, and other commonly used antifungal drugs.

[0018] The medicinal salts refer to acid addition salts formed by cis-6-octadecenoic acid and the following acids: hydrochloric acid, sulfuric acid, nitric acid, citric acid, maleic acid, etc.

[0019] The fungi are selected from Candida albicans, Candida krusei, Candida tropicalis, Trichophyton mentagrophytes, etc.; specifically: Candida albicans SC5314; Candida krusei 4996, 62588, 10153; Trichophyton mentagrophytes T5b, T5a; Candida tropicalis 409, 8915; Candida albicans 14103, 7654, 9161, 10066, 10060, 10061, 7879, 901, 953.

[0020] This invention has discovered that for fluconazole-resistant Candida albicans, the combined use of PeAc and fluconazole can reduce the effective drug concentration, lowering the effective concentration of fluconazole from 64 μg / ml to 0.5 μg / ml. The preferred combination regimen is a PeAc concentration of 2 μg / ml + a fluconazole concentration of 0.5 μg / ml; or a PeAc concentration of 2 μg / ml + a fluconazole concentration of 1 μg / ml.

[0021] This invention has found that PeAc has very low toxicity at effective antifungal concentrations, and only becomes toxic to mammalian cells at concentrations ≥64 μg / ml.

[0022] A third aspect of the present invention provides the use of cis-6-octadecenoic acid or a pharmaceutical salt thereof in the preparation of fructose-1,6-bisphosphate aldolase inhibitor (Fba1p).

[0023] This invention experimentally verifies that PeAc exerts its antifungal effect through Fba1p, an inhibitor of fructose-1,6-bisphosphate aldolase (Fba1p). Specifically, it includes the following steps: constructing a Candida albicans monoallelic knockout library; demonstrating the scientific validity and feasibility of using the haploid deficiency effect for drug target identification; and studying the antifungal drug target of PeAc based on the haploid deficiency effect. Fructose-1,6-bisphosphate aldolase is the drug target of PeAc, and PeAc is an inhibitor of fructose-1,6-bisphosphate aldolase.

[0024] In this invention, when using wax moth larvae (Example 7) or mice (Example 7) as in vivo experimental models of fungal infection, a PeAc concentration of 32 mg / kg can significantly improve the survival rate of wax moth larvae infected with fungi, and 16 mg / kg can significantly improve the survival rate of mice infected with fungi.

[0025] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0026] This invention discovers an effective antifungal compound with pharmaceutical potential: cis-6-octadecenoic acid (PeAc). The compound PeAc of this invention exhibits clear antifungal activity against pathogenic fungi such as Candida albicans, Candida krusei, Candida tropicalis, and Trichophyton mentagrophytes, with a MIC... 80 The concentration of PeAc ranges from 2 to 32 μg / ml. PeAc inhibits the growth of Candida albicans, suppressing both hyphae (2 μg / ml) and biofilm formation (16 μg / ml). PeAc also exhibits good antifungal activity against fluconazole-resistant strains of Candida albicans and Candida krusei (naturally resistant to fluconazole), and the combination of PeAc and fluconazole shows a synergistic effect against drug-resistant fungi. PeAc exhibits low cytotoxicity and effectively improves the survival rate of fungal infections in wax moth larvae and mice. This invention also reveals that fructose-1,6-bisphosphate aldolase inhibitors are the target of PeAc, which is a fructose-1,6-bisphosphate aldolase inhibitor.

[0027] This invention provides the application of cis-6-octadecenoic acid (PeAc) as an antifungal compound and its application as an inhibitor of fructose-1,6-bisphosphate aldolase. In vitro and in vivo experiments have demonstrated that PeAc has a broad antifungal spectrum, significantly inhibiting the formation of Candida albicans hyphae and exhibiting good antifungal activity against fluconazole-resistant bacteria. The combination of PeAc and fluconazole shows a clear synergistic effect against drug-resistant fungi. PeAc can improve the survival rate of fungal-infected animals and has low toxicity to mammalian cells. PeAc is an antifungal compound with drug development potential. Based on haploid deficiency effect analysis and other studies, this invention has found that fructose-1,6-bisphosphate aldolase (Fba1p) is the target of PeAc. Experiments have shown that PeAc exerts its antifungal effect through Fba1p, and cis-6-octadecenoic acid is an inhibitor of fructose-1,6-bisphosphate aldolase.

[0028] This invention utilizes a Candida albicans monoallelic knockout library to screen for Fba1p as the antifungal target of PeAc, which is an inhibitor of Fba1p. In this invention, extensive screening of a natural compound library using a micro-liquid dilution method revealed that PeAc exhibits good antifungal activity. Attached Figure Description

[0029] Figure 1 This is a schematic diagram showing the results of PeAc inhibiting the formation of Candida albicans SC5314 hyphae starting from a concentration of 2 μg / ml in Example 2.

[0030] Figure 2 This is a schematic diagram showing the results of PeAc inhibiting the formation of Candida albicans SC5314 biofilm starting from a concentration of 16 μg / ml in Example 3.

[0031] Figure 3 This is a schematic diagram showing the results of the inhibition zone determination of PeAc combined with fluconazole against drug-resistant fungi in Example 5.

[0032] Figure 4 This is a schematic diagram showing the dose-effect relationship of PeAc and fluconazole in combination against drug-resistant fungi in Example 6.

[0033] Figure 5 This is a schematic diagram showing the results of PeAc effectively improving the survival rate of mice infected with Candida albicans in Example 7.

[0034] Figure 6 This is a schematic diagram of the cytotoxicity results of PeAc in Example 8.

[0035] Figure 7 This is a schematic diagram of the strategy for establishing a Candida albicans monoallelic knockout library in Example 9.

[0036] Figure 8 This is a schematic diagram illustrating the implementation of establishing a Candida albicans monoallelic knockout library in Example 9.

[0037] Figure 9 This is a schematic diagram illustrating the implementation of establishing a Candida albicans monoallelic knockout library in Example 9.

[0038] Figure 10 This is a schematic diagram of the results of the spot plate experiment in the feasibility verification of using the haploid deficiency effect for antifungal drug target identification in Example 10.

[0039] Figure 11 This is a schematic diagram of the MIC experiment results in the feasibility verification of using the haploid deficiency effect for antifungal drug target identification in Example 10.

[0040] Figure 12This is a schematic diagram of the results of the spot plate experiment in the specificity verification of the haploid deficiency effect for antifungal drug target identification in Example 10.

[0041] Figure 13 This is a schematic diagram of the MIC experiment results in the specificity verification of the haploid deficiency effect for antifungal drug target identification in Example 10.

[0042] Figure 14 This is a schematic diagram of the results of the spot plate experiment and MIC experiment for PeAc target screening using the haploid deficiency effect in Example 10.

[0043] Figure 15 This is a schematic diagram of the results of the spot test and MIC experiment for PeAc target screening using the haploid deficiency effect in Example 10.

[0044] Figure 16 This is a schematic diagram showing the results of investigating the interaction between PeAc and Fba1p through molecular docking in Example 10.

[0045] Figure 17 This is a schematic diagram illustrating the verification results of PeAc's antifungal effect through Fba1p in Example 11, specifically its inhibition of hyphal formation.

[0046] Figure 18 This is a schematic diagram illustrating the verification results of PeAc's antifungal effect through Fba1p in Example 11, demonstrating its ability to inhibit biofilm formation.

[0047] Figure 19 This is a schematic diagram illustrating the survival results of wax moth larvae in Example 11, where PeAc exerts its antifungal effect through Fba1p. Detailed Implementation

[0048] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0049] In this invention, it was discovered that the compound PeAc has good antifungal activity and synergistic antifungal activity with fluconazole. PeAc has low cytotoxicity, and its antifungal target is Fba1p.

[0050]

[0051] In this invention, the fungi used can be obtained or cultured by conventional methods, or obtained commercially, such as isolated from somatic cells or tissues of patients with the corresponding disease, or purchased from any institution or company that provides commercially available standard strains, such as ATCC. It should be understood that the strain samples used in the embodiments of this invention are merely exemplary examples, and any fungi capable of forming hyphae or biofilms or resistant to fluconazole can be used to test the antifungal activity of the compounds of this invention, without being limited to the scope of the embodiments. It should be understood that in the embodiments of this invention, only the minimum concentration of the active ingredient is specified for effectively inhibiting fungal hyphae formation and biofilm formation. As is known in the art, increasing the concentration of the active antifungal drug component can improve the antifungal effect. Therefore, any person skilled in the art, after reading this specification and combining it with common knowledge in the art, can use doses higher than the stated minimum concentration to inhibit fungal hyphae formation or biofilm growth; such modifications also fall within the scope of this invention.

[0052] Example 1: Determination of the antifungal spectrum of PeAc

[0053] Materials and methods

[0054] 1. Test Drugs: PeAc: purchased from Shanghai Taoshu Biotechnology Co., Ltd. Fluconazole (FLC): purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Dimethyl sulfoxide (DMSO): purchased from Macklin Biotechnology Co., Ltd. PeAc and fluconazole were prepared into 10 mg / ml solutions using DMSO. The test drugs were stored at -20℃. Before the experiment, the drug stock solutions were removed and thawed in a 35℃ incubator, thoroughly mixed, and then used for subsequent experiments.

[0055] 2. Strains: ATCC standard strain: Candida albicans SC5314 (available from ATCC). Other strains: Candida krusei 4996, 62588, 10153, Trichophyton mentagrophytes T5b, T5a, Candida tropicalis 409, 8915 provided by the Department of Dermatology, Shanghai Changzheng Hospital; Candida albicans 14103, 7654, 9161, 10066, 10060, 10061, 7879, 901, 953 provided by the Department of Pharmaceutical Chemistry, Department of Pharmacy, Naval Medical University.

[0056] All experimental Candida strains were activated by streaking on Saburg dextrose agar (SDA) and incubated at 35°C for 48 h. Single colonies were then picked and reactivated by streaking. The second batch of single colonies was placed on SDA slants and incubated using the same method. The cultured colonies were then stored at 4°C for later use. Trichophyton mentagrophytes were activated on potato dextrose agar (PDA) using the same method.

[0057] 3. Culture medium: RPMI 1640 medium: RPMI 1640 (Gibco BRL) 10g, NaHCO3 2.0g, morpholinepropanesulfonic acid (MOPS) (Sigma) 34.5g (0.165M), dissolved in 900ml triple-distilled water, adjust pH to 7.0 (25℃) with 1N NaOH, bring volume to 1000ml, filter for sterilization, and store at 4℃.

[0058] Saburg Dextrose Agar (SDA) medium: Dissolve 10g peptone, 40g glucose, and 18g agar in 900ml triple-distilled water, add 50ml 2mg / ml chloramphenicol aqueous solution, adjust pH to 7.0, bring volume to 1000ml, autoclave, and store at 4℃.

[0059] YPD liquid culture medium: Dissolve 10g yeast extract, 20g peptone, and 20g glucose in 900ml triple-distilled water, bring the volume up to 1000ml, autoclave, and store at 4℃.

[0060] 4. Instruments: Constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd. MJ-150-I); Shaking incubator (Jiangsu Taicang Experimental Equipment Factory HZ-2111K-B); Multifunctional microplate reader (TECAN Infinite M200).

[0061] 5. Preparation of Antimicrobial Susceptibility Testing Plates: Candida albicans was inoculated into YPD liquid medium and cultured at 30°C for 16 h. The activated bacterial suspension was transferred to a 1.5 ml centrifuge tube, centrifuged at 3000 g for 1 min, the medium was discarded, and the solution was washed once with PBS. After centrifugation, the supernatant was discarded, and the solution was resuspended in 1 ml PBS. After dilution, 10 μl was transferred to a hemocytometer for counting. The bacterial concentration was adjusted to 1 × 10⁻⁶ using RPMI 1640 medium. 3Cells / ml. Transfer the bacterial culture to a disposable reagent tank, mix thoroughly by pipetting repeatedly with a multichannel pipette, then pipette 100 μl of the bacterial culture into columns 3-12 of a pre-prepared 96-well plate. Add 200 μl of the bacterial culture to column 2, and add 100 μl of RPMI 1640 liquid medium to column 1 as a blank control. Prepare stock solutions of PeAc and FLC with DMSO. Add PeAc and FLC to each well in column 2 to a final concentration of 64 μg / ml (note that the DMSO content in each well should be less than 1%), and perform 3 replicates for each drug. Perform 2-fold serial dilutions in wells 2-11 using a multichannel pipette. Do not add any drug to well 12 as a growth control. Incubate the prepared drug sensitivity plate at 35°C. After 24 h and 48 h, shake the drug sensitivity plate to mix thoroughly, and measure the OD value of each well at 630 nm using a microplate reader. The MIC is the drug concentration in the lowest concentration well where the OD value decreased by more than 80% compared to the growth control well. 80 (Drug concentration when fungal growth is inhibited by 80%), the experimental results are shown in Table 1.

[0062] Conclusion: Table 1 shows that PeAc has strong antibacterial activity against common pathogenic fungi and a broad antibacterial spectrum. PeAc exhibited stable antibacterial activity against all 10 tested Candida albicans strains, with MIC... 80 Between 2-16 μg / ml, it exhibited similar antifungal activity to susceptible strains, particularly against fluconazole-resistant Candida albicans. It also showed good antifungal activity against three Candida krusei strains (naturally resistant to fluconazole), with MIC... 80 The concentration was 2 μg / ml. Its antifungal activity against Trichophyton mentagrophytes remained between 4-8 μg / ml, and it also showed some antifungal activity against two strains of Candida tropicalis. The MIC was... 80 It is 32 μg / ml.

[0063] Table 1

[0064]

[0065]

[0066] Example 2: Determination of the inhibitory effect of PeAc on Candida albicans hyphae formation

[0067] Materials and methods

[0068] 1. Test reagents: PeAc: purchased from Shanghai Taoshu Biotechnology Co., Ltd. Dimethyl sulfoxide (DMSO): purchased from Macklin. PeAc was prepared into a 10 mg / ml solution with DMSO, and the test drug was stored at -20℃. Before the experiment, the drug stock solution was taken out and placed in a 35℃ incubator to thaw, and thoroughly mixed before subsequent experiments.

[0069] 2. Strains: ATCC standard strain: Candida albicans SC5314 (available from ATCC). The experimental Candida albicans strain was activated by streaking on Saburg dextrose agar (SDA) medium and cultured at 35°C for 48 hours. Single colonies were picked and activated again by streaking. The second batch of single colonies was placed on SDA slant and cultured using the above method. The cultured colonies were then stored at 4°C for later use.

[0070] 3. Culture medium

[0071] Spider liquid culture medium: Weigh 10g of Nutrient broth, 10g of mannitol, and 2.0g of K2HPO4, dissolve them in double-distilled water, and bring the volume to 1L in a graduated cylinder. After high-temperature autoclaving, store in a refrigerator at 4℃.

[0072] Lee liquid culture medium: Accurately weigh 0.5g alanine, 1.3g leucine, 1.0g lysine, 0.1g methionine, 0.0714g ornithine, 0.5g phenylalanine, 0.5g proline, and 0.5g threonine. Dissolve them in an appropriate amount of triple-distilled water by sonication. Then add 0.5g (NH4)2SO4, 0.357g MgSO4·7H2O, 0.25g K2HPO4, 5.0g NaCl, 0.001g biotin, and 12.5g glucose. Stir thoroughly to dissolve, then add water to bring the volume to 1L. Autoclave and store at 4℃.

[0073] YPD liquid culture medium: Dissolve 10g yeast extract, 20g peptone, and 20g glucose in 900ml triple-distilled water, bring the volume up to 1000ml, autoclave, and store at 4℃.

[0074] 4. Instruments: Constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd. MJ-150-I); Shaking incubator (Jiangsu Taicang Experimental Equipment Factory HZ-2111K-B); Philips XL-30 inverted phase contrast microscope (Amersham Pharmacia AMGEVOS×1).

[0075] 5. Preparation and observation of liquid mycelium

[0076] Candida albicans was inoculated into YPD liquid medium and cultured at 30°C for 16 h. The activated bacterial suspension was transferred to a 1.5 ml centrifuge tube, centrifuged at 3000 g for 1 min, the medium was discarded, and the cells were washed once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1 ml PBS. After dilution, 10 μl was transferred to a hemocytometer for counting. The bacterial concentration was adjusted to 2 × 10⁻⁶ cells / mL using Spider's liquid medium and Lee's liquid medium, respectively. 5Cells / ml: Bacterial culture was transferred to 12-well plates, 2 ml / well. A series of test drug concentrations (0, 2, 4, 8 μg / ml) were then added to each well. The plates were incubated statically at 37℃ for 3 h, and hyphal formation was observed and photographed using an inverted microscope. Experimental results are shown below. Figure 1 As shown, Figure 1 This is a schematic diagram showing the results of PeAc inhibiting the formation of Candida albicans SC5314 hyphae starting from a concentration of 2 μg / ml in Example 2.

[0077] The results showed that PeAc had a clear inhibitory effect on liquid hyphae formation. In the absence of drug treatment, *Candida albicans* formed long, unsegmented hyphae in both hyphae induction media. In Spider liquid medium, 2 μg / ml PeAc significantly inhibited *Candida albicans* hyphae formation; from 4 μg / ml onwards, most *Candida albicans* remained in a yeast-like state, and this inhibition of *Candida albicans* hyphae formation was dose-dependent. In Lee liquid medium, *Candida albicans* hyphae also began to shorten under the action of 2 μg / ml PeAc, and the hyphae gradually shortened with increasing PeAc concentration, reaching a significant shortening at a PeAc concentration of 8 μg / ml.

[0078] Example 3: Determination of the inhibitory effect of PeAc on Candida albicans biofilm

[0079] Materials and methods

[0080] 1. Reagents: PeAc: Purchased from Shanghai Taoshu Biotechnology Co., Ltd. XTT (2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, 3′-[1-[(anilino)-carbonyl]-3,4-tetrazole]-bis(4-methoxy-6-nitro)benzene-sulfonate sodium salt): Purchased from Macklin. Menadione: Purchased from Macklin. Dimethyl sulfoxide (DMSO): Purchased from Macklin. Acetone: Purchased from Sigma. PeAc was prepared into a 10 mg / ml solution with DMSO. The test drug was stored at -20℃. Before the experiment, the drug stock solution was removed and thawed in a 35℃ incubator, thoroughly mixed, and then used for subsequent experiments.

[0081] Dissolve menadione in acetone to prepare a 10 mM stock solution and store it protected from light. Weigh XTT powder and dissolve it in PBS to prepare a 0.5 mg / ml solution. Mix the two solutions at a ratio of 1 μl menadione stock solution to 10 ml XTT solution, and filter through a 0.22 μm microporous membrane for sterilization, protecting the solution from light throughout the process. Store XTT and menadione at -80℃. Before the experiment, remove the drug stock solution and thaw it in a gradient of 4℃ refrigerator and 35℃ incubator, mixing thoroughly before conducting pharmacodynamic experiments.

[0082] 2. Strains: ATCC standard strain: Candida albicans SC5314 (available from ATCC). All experimental strains were activated by streaking on Saburg dextrose agar (SDA). After culturing Candida albicans at 35°C for 48 hours, single colonies were picked and re-activated by streaking. The second batch of single colonies was placed on SDA slant, cultured using the above method, and then stored at 4°C for later use.

[0083] 3. Culture medium

[0084] Spider liquid culture medium: Weigh 10g of Nutrient broth, 10g of mannitol, and 2.0g of K2HPO4, dissolve them in double-distilled water, and bring the volume to 1L in a graduated cylinder. After high-temperature autoclaving, store in a refrigerator at 4℃.

[0085] Saburg Dextrose Agar (SDA) medium: Dissolve 10g peptone, 40g glucose, and 18g agar in 900ml triple-distilled water, add 50ml 2mg / ml chloramphenicol aqueous solution, adjust pH to 7.0, bring volume to 1000ml, autoclave, and store at 4℃.

[0086] YPD liquid culture medium: Dissolve 10g yeast extract, 20g peptone, and 20g glucose in 900ml triple-distilled water, bring the volume up to 1000ml, autoclave, and store at 4℃.

[0087] 4. Instruments: Constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd. MJ-150-I); Shaking incubator (Jiangsu Taicang Experimental Equipment Factory HZ-2111K-B); Multifunctional microplate reader (TECAN Infinite M200).

[0088] 5. Preparation of Candida albicans biofilm

[0089] Candida albicans was inoculated into YPD liquid medium and cultured at 30°C for 16 h to induce the fungus into the late exponential growth phase. The activated bacterial suspension was transferred to a 1.5 ml centrifuge tube, centrifuged at 3000 g for 1 min, the medium was discarded, and the cells were washed once with PBS. After centrifugation, the supernatant was discarded, and the suspension was resuspended in 1 ml PBS. After dilution, 10 μl was transferred to a hemocytometer for counting. The bacterial concentration was adjusted to 1 × 10⁻⁶ cells / mL using Spider's liquid medium. 6 cells / ml. Add 100 μl of bacterial culture to each well of a surface-treated 96-well plate (tissue culture treated, TC). Note that the first well should be left empty with Spider liquid medium as a blank control. Incubate the 96-well plate at 37°C for 90 min to allow the Candida albicans cells to settle and adhere to the surface of the wells.

[0090] 6. Preparation of anti-Candida albicans biofilm drug sensitivity test plates

[0091] Take a new 96-well plate and add 150 μl of Spider liquid medium to each well. Add an additional 150 μl of medium to column 1 (total 300 μl) and 1.92 μl of PeAc (final concentration 64 μg / ml). Mix well with a multichannel pipette and add 150 μl to the corresponding well in column 2. Mix well again, and so on for serial dilution (final concentrations are 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / ml respectively). Do not add any drug to column 12; only add the corresponding volume of DMSO. After the drug-dosing plate is prepared, take out the biofilm plate that has been adhered for 90 minutes, aspirate the upper culture medium, and gently wash with PBS 2-3 times, trying to avoid washing away the adhered cells. Then, add the drug solution from the drug-dosing plate to the corresponding wells in the biofilm plate in sequence, 100 μl per well. Continue to incubate the culture plate in a 37°C constant temperature incubator for 24 h.

[0092] 7. Determination of biofilm activity

[0093] Remove the 96-well plate after 24 hours of culture, discard the culture medium, gently wash three times with PBS, and then add 150 μl of XTT reaction solution to each well (including well 12). Incubate at 37°C in the dark for 3 hours. Remove the 96-well plate and transfer 70 μl of the upper orange liquid from each well to a new 96-well plate. Measure the absorbance (OD490) of each solution at 490 nm using a multi-mode microplate reader. Biofilm formation rate for each treatment group = (OD490 of that group) / (OD490 of that group). 490 -Background comparison OD 490 ) / (Growth control OD 490 -Background comparison OD 490()×100%. The experiment was repeated three times, and statistical analysis was performed using one-way ANOVA and Dunnett's t-test. The experimental results are shown in […]. Figure 2 As shown, Figure 2 This is a schematic diagram illustrating the inhibition of Candida albicans SC5314 biofilm formation by PeAc starting at a concentration of 16 μg / ml in Example 3. The horizontal axis represents PeAc concentration (μg / ml), and the vertical axis represents biofilm formation rate (%). * indicates P < 0.05, *** indicates P < 0.001. The results show that PeAc exhibits an anti-Candida albicans biofilm effect starting at a concentration of 16 μg / ml.

[0094] Example 4: Determination of the partial inhibitory concentration index (FICI) of PeAc combined with fluconazole

[0095] Materials and methods

[0096] 1. Test Drugs: PeAc: purchased from Shanghai Taoshu Biotechnology Co., Ltd. Fluconazole: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Dimethyl sulfoxide (DMSO): purchased from Macklin. PeAc and fluconazole were prepared into 10 mg / ml solutions using DMSO. The test drugs were stored at -20℃. Before the experiment, the drug stock solutions were removed and thawed in a 35℃ incubator, thoroughly mixed, and then used for subsequent experiments.

[0097] 2. Strains: Fluconazole-resistant Candida albicans strains (901, 953) were provided by the Department of Pharmaceutical Chemistry, Department of Pharmacy, Naval Medical University. The experimental Candida albicans strains were activated by streaking on Saburg dextrose agar (SDA) medium. After incubation at 35°C for 48 hours, single colonies were picked and reactivated by streaking. The second batch of single colonies was then placed on SDA slant agar and cultured using the above method before being stored at 4°C for later use.

[0098] 3. Culture medium

[0099] RPMI 1640 medium: 10 g RPMI 1640 (Gibco BRL), 2.0 g NaHCO3, 34.5 g (0.165 M) morpholine propanesulfonic acid (MOPS) (Sigma), dissolved in 900 ml triple-distilled water, adjusted pH to 7.0 (25 °C) with 1 N NaOH, brought to a final volume of 1000 ml, filtered for sterilization, and stored at 4 °C.

[0100] Saburg Dextrose Agar (SDA) medium: Dissolve 10g peptone, 40g glucose, and 18g agar in 900ml triple-distilled water, add 50ml 2mg / ml chloramphenicol aqueous solution, adjust pH to 7.0, bring volume to 1000ml, autoclave, and store at 4℃.

[0101] YPD liquid culture medium: Dissolve 10g yeast extract, 20g peptone, and 20g glucose in 900ml triple-distilled water, bring the volume up to 1000ml, autoclave, and store at 4℃.

[0102] 4. Instruments: Constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd. MJ-150-I); Shaking incubator (Jiangsu Taicang Experimental Equipment Factory HZ-2111K-B); Multifunctional microplate reader (TECAN Infinite M200).

[0103] 5. Determination of FICI in combination with PeAc and fluconazole using the checkerboard micro-liquid dilution method

[0104] Candida albicans was inoculated into YPD liquid medium and cultured at 30°C for 16 h. The activated bacterial suspension was transferred to a 1.5 ml centrifuge tube, centrifuged at 3000 g for 1 min, the medium was discarded, and the cells were washed once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1 ml PBS. After dilution, 10 μl was transferred to a hemocytometer for counting. The bacterial concentration was adjusted to 1 × 10⁻⁶ cells / mL using RPMI 1640 medium. 3 Cells / ml. After mixing the prepared bacterial suspension, transfer it to a 6-well plate. Add 2.6 ml to the first well and 1.3 ml to each of the 2nd to 6th wells. Prepare stock solutions of PeAc and FLC using DMSO. Add a certain volume of PeAc to the first well to achieve a final concentration of 64 μg / ml. Perform 50-fold dilutions sequentially to achieve final concentrations of 64-2 μg / ml in wells 1-6. Transfer the prepared PeAc-containing bacterial suspension to rows AF of a 96-well plate according to drug concentration from highest to lowest: 200 μl / well in the first column and 100 μl / well in columns 2-10. Add 100 μl / well of untreated blank bacterial suspension to wells A11-F11 in column 11 and wells G1-G10 in row G as growth controls. Add the prepared FLC to wells A1-G1 in the first column to a final concentration of 64 μg / ml. Each column was sequentially halved from left to right to achieve a final FLC concentration of 64-0.25 μg / ml in columns 1-9. 100 μl of RPMI 1640 medium was added to each well in column 12 and row H of the 96-well plate as a blank control. The 96-well plate was then incubated at 30℃ for 48 hours before measuring its OD value. 630 The OD of the growth control group was used as an example. 630 The value is 100%. The antibacterial rate (%) of the compound at different drug concentrations for each well is calculated to obtain the MIC of PeAc and FLC used alone or in combination. 80 Then, the synergistic index FICI was calculated. The experimental results are shown in Table 2, which shows the determination results of the partial inhibitory concentration index (FICI) of PeAc combined with fluconazole.

[0105] Table 2

[0106]

[0107] Conclusion: The results in Table 2 show that the combination of PeAc and fluconazole has a synergistic effect against drug-resistant fungi. The fractional inhibitory concentration index (FICI) is the main parameter for evaluating the interaction mode of two drugs in combined medication. The calculation method of FICI is the sum of the ratios of the measured minimum inhibitory concentration (MIC) of the two drugs when used in combination for bacteriostasis to their respective MICs when used alone, that is, FICI = FICA + FICB = (MICAB / MICA) + (MICBA / MICB), where MICAB and MICBA are the minimum inhibitory concentrations of drug A and drug B when the two drugs are used in combination, respectively, and MICA and MICB are the minimum inhibitory concentrations of drug A and drug B when used alone. This experiment adopts the currently internationally recognized standard to evaluate the effect of combined medication: FICI ≤ 0.5 indicates a synergistic effect, and the smaller the FICI index, the stronger the synergistic effect; 0.5 < FICI ≤ 1 indicates an additive effect; 1 < FICI ≤ 4 indicates an indifferent effect; FICI > 4 indicates an antagonistic effect. The results of the checkerboard microbroth dilution test show that for the two drug-resistant strains (fluconazole-resistant Candida albicans strains 901 and 953), the FICI of the combined use of PeAc and fluconazole is less than 0.5, and the combination of the two drugs has a synergistic effect.

[0108] Example 5: Determination of the inhibition zone of the combination of PeAc and fluconazole against drug-resistant fungi

[0109] Materials and Methods

[0110] 1. Test drugs: PeAc: purchased from Shanghai Topscience Co., Ltd. Fluconazole: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Dimethyl sulfoxide (DMSO): purchased from Macklin. PeAc and fluconazole were prepared into 10 mg / ml solutions with DMSO, and the test drugs were stored at -20°C. Before the experiment, the drug stock solutions were taken out and thawed in a 35°C incubator, mixed thoroughly, and then used for subsequent experiments respectively.

[0111] 2. Strains: Fluconazole-resistant Candida albicans strains (901, 953) were provided by the Department of Medicinal Chemistry, School of Pharmacy, Naval Medical University. The Candida albicans strains for the experiment were streak-activated on Sabouraud Dextrose Agar (SDA), cultured at 35°C for 48 h, then single clones were picked for streak activation again. The single clones obtained from the second activation were placed on SDA slants, cultured by the above method, and then stored at 4°C for later use.

[0112] 3. Culture medium

[0113] RPMI 1640 medium: 10 g RPMI 1640 (Gibco BRL), 2.0 g NaHCO3, 34.5 g (0.165 M) morpholine propanesulfonic acid (MOPS) (Sigma), dissolved in 900 ml triple-distilled water, adjusted pH to 7.0 (25 °C) with 1 N NaOH, brought to a final volume of 1000 ml, filtered for sterilization, and stored at 4 °C.

[0114] Saburg Dextrose Agar (SDA) medium: Dissolve 10g peptone, 40g glucose, and 18g agar in 900ml triple-distilled water, add 50ml 2mg / ml chloramphenicol aqueous solution, adjust pH to 7.0, bring volume to 1000ml, autoclave, and store at 4℃.

[0115] YPD liquid culture medium: Dissolve 10g yeast extract, 20g peptone, and 20g glucose in 900ml triple-distilled water, bring the volume up to 1000ml, autoclave, and store at 4℃.

[0116] YPD solid medium containing 64 μg / ml FLC: Add 20 g of agar to the YPD liquid medium, autoclave, and add filtered sterilized FLC liquid when the medium is no longer hot to the touch, so that the final concentration of FLC in the medium is 64 μg / ml. Then pour the medium into 90 mm petri dishes and let it air dry for later use.

[0117] 4. Instruments: Constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd. MJ-150-I); Shaking incubator (Jiangsu Taicang Experimental Equipment Factory HZ-2111K-B).

[0118] 5. Paper diffusion experiment procedure

[0119] Candida albicans was inoculated into YPD liquid medium and cultured at 30°C for 16 h. The activated bacterial suspension was transferred to a 1.5 ml centrifuge tube, centrifuged at 3000 g for 1 min, the medium was discarded, and the cells were washed once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1 ml PBS. After dilution, 10 μl was transferred to a hemocytometer for counting. The bacterial concentration was adjusted to 1 × 10⁻⁶ cells / mL with sterile PBS. 5 Cells / ml. Take 5ml of the diluted bacterial suspension and pour it onto YPD solid medium containing 64μg / ml FLC. Incubate at 30℃ for 0.5–1h to allow the bacteria to settle and adhere to the medium surface. Discard the supernatant and remove the agar after the surface liquid has evaporated. Place sterile paper discs (6mm in diameter) on the solid medium, and add the same volume (5μl) of the test drug at different concentrations to the center of the disc. Invert the agar culture plates and incubate at 30℃ for 48h. Observe the formation of inhibition zones, measure the diameter of the inhibition zones, and photograph them. Experimental results are shown below. Figure 3 As shown, Figure 3 This is a schematic diagram showing the results of the inhibition zone determination of PeAc combined with fluconazole against drug-resistant fungi in Example 5. The results show that PeAc and fluconazole have a synergistic effect against drug-resistant fungi. Compared with the control paper discs, PeAc at concentrations from 8 μg to 256 μg produced obvious inhibition zones on the plates of both types of bacteria. The inhibition zones all showed no clonal growth inside and had clear boundaries, and the inhibition zones increased in size with increasing PeAc concentration.

[0120] Example 6: Determination of the dose-effect relationship between PeAc and fluconazole in combination against drug-resistant fungi

[0121] Materials and methods

[0122] 1. Test reagents: PeAc: purchased from Shanghai Taoshu Biotechnology Co., Ltd. Fluconazole: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Dimethyl sulfoxide (DMSO): purchased from Macklin. PeAc was prepared into a 10 mg / ml solution with DMSO, and the test drugs were stored at -20℃. Before the experiment, the drug stock solution was taken out and placed in a 35℃ incubator to thaw, and thoroughly mixed before subsequent experiments.

[0123] 2. Strains: Fluconazole-resistant Candida albicans strain 901 was provided by the Department of Pharmaceutical Chemistry, Naval Medical University. The experimental Candida albicans strain was activated by streaking on Saburg dextrose agar (SDA) medium and incubated at 35°C for 48 hours. Single colonies were then picked and reactivated by streaking. The second batch of single colonies was placed on SDA slant agar and cultured using the above method before being stored at 4°C for later use.

[0124] 3. Culture medium

[0125] RPMI 1640 medium: 10 g RPMI 1640 (Gibco BRL), 2.0 g NaHCO3, 34.5 g (0.165 M) morpholine propanesulfonic acid (MOPS) (Sigma), dissolved in 900 ml triple-distilled water, adjusted pH to 7.0 (25 °C) with 1 N NaOH, brought to a final volume of 1000 ml, filtered for sterilization, and stored at 4 °C.

[0126] Saburg Dextrose Agar (SDA) medium: Dissolve 10g peptone, 40g glucose, and 18g agar in 900ml triple-distilled water, add 50ml 2mg / ml chloramphenicol aqueous solution, adjust pH to 7.0, bring volume to 1000ml, autoclave, and store at 4℃.

[0127] YPD liquid culture medium: Dissolve 10g yeast extract, 20g peptone, and 20g glucose in 900ml triple-distilled water, bring the volume up to 1000ml, autoclave, and store at 4℃.

[0128] 4. Instruments: Constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd. MJ-150-I); Shaking incubator (Jiangsu Taicang Experimental Equipment Factory HZ-2111K-B); Multifunctional microplate reader (TECAN Infinite M200).

[0129] 5. Determination of fungal growth curves

[0130] Candida albicans was inoculated into YPD liquid medium and cultured at 30°C for 16 h. The activated bacterial culture was transferred to a 1.5 ml centrifuge tube, centrifuged at 3000 g for 1 min, the medium was discarded, and the cells were washed once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1 ml PBS. After dilution, 10 μl was transferred to a hemocytometer for counting. Before starting the culture, 200 μl of undiluted original bacterial culture was added to wells A1-C1 (triple replicates) in the first column of a 96-well plate. 100 μl of RPMI 1640 liquid medium was added to each well in columns 2-10. OD values ​​were measured in each well starting from column 1 to column 10. 630 And according to the different OD of each hole 630 A standard curve was obtained by plotting the values ​​(x) and their corresponding bacterial concentrations (y). The bacterial culture was then diluted with RPMI 1640 liquid medium and the concentration was adjusted to 1 × 10⁻⁶. 6 The bacterial culture was divided into several portions and transferred to 50 ml centrifuge tubes, 10 ml per tube. Different concentrations (2, 4, 8 μg / ml) of the test drug PeAc were added to each tube. Then, different concentrations (4, 16, 64 μg / ml) of FLC were added to the tubes containing 2 μg / ml PeAc, and the same procedure was repeated for the tubes containing 4 and 8 μg / ml PeAc. Finally, three tubes remained: one containing only 8 μg / ml PeAc, one containing only 64 μg / ml FLC, and one without any drug. All centrifuge tubes containing bacterial culture were incubated at 30°C with shaking at 200 rpm. At 0, 2, 4, 8, and 12 h, 100 μl of each bacterial culture was aspirated into a 96-well plate (after vortexing the bacterial culture from each tube beforehand) and the OD of each well was measured. 630 The bacterial concentration at different culture time points in each group was calculated using the equation fitted to the standard curve, and a curve was plotted against time using log10 CFU / ml. The experimental results are shown below. Figure 4 As shown, Figure 4 This is a schematic diagram showing the dose-effect relationship of PeAc and fluconazole in combination against drug-resistant fungi in Example 6.

[0131] The results showed that fluconazole (64 μg / ml) or PeAc (8 μg / ml) alone exhibited a certain inhibitory effect on the growth of Candida albicans after 8 hours, but the inhibitory effect rapidly weakened after 12 hours, and the growth of Candida albicans was similar to that of the control group. When the PeAc concentration was fixed at 2 μg / ml, the combined use with fluconazole at concentrations of 4, 16, and 64 μg / ml showed that the synergistic inhibitory effect of the two drugs on drug-resistant bacteria increased with increasing fluconazole concentration, but the difference in inhibitory effect within the groups was not significant. However, with the increase of PeAc concentration, the ability of the two drugs to inhibit the growth of drug-resistant bacteria gradually strengthened. Especially when the PeAc concentration reached 8 μg / ml, the combined use with fluconazole at concentrations of 4, 16, and 64 μg / ml showed a significant inhibitory effect on the growth of drug-resistant bacteria, with significant differences in inhibitory effect within the groups. In particular, the combination of "PeAc 8 μg / ml + FLC 64 μg / ml" maintained the growth inhibition of drug-resistant bacteria at a low level. Therefore, the synergistic antifungal effect of the two drugs combined is more clearly related to the dose-response relationship of PeAc.

[0132] Example 7: Determination of the in vivo antifungal activity of PeAc

[0133] Materials and methods

[0134] 1. Reagents and main reagents: PeAc: purchased from Shanghai Taoshu Biotechnology Co., Ltd.; Dimethyl sulfoxide (DMSO): purchased from Macklin Biotechnology Co., Ltd.; PEG300: purchased from Shanghai Titan Biotechnology Co., Ltd.; Tween-80: purchased from Shanghai Titan Biotechnology Co., Ltd.; Physiological saline: purchased from Shandong Qidu Pharmaceutical Co., Ltd.; Cyclophosphamide: purchased from Baxter Oncology GmbH; Levofloxacin lactate: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; PBS: purchased from Shanghai Sangon Biotech Co., Ltd.

[0135] PeAc was prepared as a 200 mg / ml stock solution using DMSO, and the test drug was stored at -20°C. Before the experiment, the drug stock solution was removed and thawed in a 35°C incubator, thoroughly mixed, diluted, and then used for in vivo experiments.

[0136] The drug solution administered to mice was prepared to the target dosage concentration according to the ratio of 10% DMSO, 40% PEG300, 5% Tween-80, and 45% physiological saline per milliliter.

[0137] The drug solution administered to wax moth larvae was diluted with physiological saline to the appropriate concentration (DMSO content not exceeding 5%).

[0138] 2. Strains: ATCC standard strain: Candida albicans SC5314 (available from ATCC). The experimental Candida albicans strain was activated by streaking on Saburg dextrose agar (SDA) medium and cultured at 35°C for 48 hours. Single colonies were picked and activated again by streaking. The second batch of single colonies was placed on SDA slant and cultured using the above method. The cultured colonies were then stored at 4°C for later use.

[0139] 3. Culture medium

[0140] Saburg Dextrose Agar (SDA) medium: Dissolve 10g peptone, 40g glucose, and 18g agar in 900ml triple-distilled water, add 50ml 2mg / ml chloramphenicol aqueous solution, adjust pH to 7.0, bring volume to 1000ml, autoclave, and store at 4℃.

[0141] YPD liquid culture medium: Dissolve 10g yeast extract, 20g peptone, and 20g glucose in 900ml triple-distilled water, bring the volume up to 1000ml, autoclave, and store at 4℃.

[0142] 4. Animals: The mice used in the experiment were clean-grade ICR mice, purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd., all of which were 4-6 weeks old and weighed 18-22g at the time of purchase.

[0143] The wax moth larvae used in the experiment were purchased from Crawling World Company and weighed approximately 0.5g.

[0144] 5. Instruments: Constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd. MJ-150-I); Shaking incubator (Jiangsu Taicang Experimental Equipment Factory HZ-2111K-B).

[0145] 6. In vivo experiments

[0146] A mouse model of Candida albicans gastrointestinal infection: Healthy female ICR mice aged 4-6 weeks and weighing 18-22g were used and acclimatized for 3 days before the experiment. Two days before modeling, mice were intraperitoneally injected with cyclophosphamide 100mg / kg to induce immunosuppression, and their drinking water was replaced with an aqueous solution containing 0.4mg / ml levofloxacin. Candida albicans was inoculated into YPD liquid medium and cultured at 30℃ for 16h. The activated bacterial suspension was transferred to a 1.5ml centrifuge tube, centrifuged at 3000g for 1min, the medium was discarded, and the mice were washed once with PBS. The supernatant was discarded after centrifugation, and the suspension was resuspended in 1ml PBS. After dilution, 10μl was taken for counting on a hemocytometer, and the bacterial concentration was adjusted to 1×10⁻⁶ with physiological saline injection. 9 On the day of modeling, mice were given 0.4 ml of bacterial culture per gavage (4 × 10⁻⁶ cells / ml). 8(cells / mouse), recorded as day 0 of the observation period. Two hours after bacterial administration, mice in each group were injected intraperitoneally with cyclophosphamide again. Subsequently, mice in each group were administered 0.2 ml / mouse of different concentrations of the drug by gavage, while the control group was administered physiological saline containing 1% DMSO by gavage. Cyclophosphamide was administered again on days 3 and 6 of the observation period. The drug administration lasted for 10 days, and the survival status of mice was recorded daily. Survival curves were plotted, and Log-rank statistical analysis was performed using GraphPadPrism software. The experimental results are shown below. Figure 5 As shown, Figure 5 This is a schematic diagram illustrating the results of Example 7 showing that PeAc effectively improved the survival rate of mice infected with Candida albicans. * represents P < 0.05, ** represents P < 0.01. The results show that PeAc at 16 mg / kg (*P ≤ 0.05) and 32 mg / kg (**P ≤ 0.01) significantly improved the survival rate of mice. This indicates that PeAc can protect mice infected with Candida albicans and exhibits in vivo antifungal activity.

[0147] Candida albicans infection model in wax moth: Candida albicans (WT and FBA1 monoallelic knockout strains) were inoculated into YPD liquid medium and cultured at 30°C for 16 h. The activated bacterial suspension was transferred to a 1.5 ml centrifuge tube, centrifuged at 3000 g for 1 min, the medium was discarded, and the cells were washed once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1 ml PBS. After dilution, 10 μl was transferred to a hemocytometer for counting. The bacterial concentration was adjusted to 1 × 10⁻⁶ cells / mL with sterile PBS. 8 Cells / ml. Sixteen healthy wax moth larvae of similar size were placed in the same petri dish, and the average weight of the larvae was calculated. 5 μl of bacterial suspension was injected into the last leg on the left side of each larva. The drug-treated group was then injected with 5 μl of different concentrations (8, 16, 32 mg / kg) of the drug (DMSO ≤ 1.5 μl) into the last leg on the right side. Syringes were cleaned with ethanol and water before and after each use. After injection, the wax moth larvae were incubated at 37℃. Mortality was observed daily for 10 consecutive days, and survival curves were plotted. Log-rank statistical analysis was performed using GraphPad Prism software. Experimental results are shown below. Figure 19 As shown in the figure, compared with wild-type (WT), the survival time of wax moth larvae infected with fba1Δ / FBA1 was prolonged, but the mortality rate still exceeded 80% on day 2 of the observation period. PeAc 32 mg / kg significantly improved the survival rate of both WT and fba1Δ / FBA1-infected larvae. However, in terms of the final number of surviving larvae, 32 mg / kg PeAc treatment resulted in a 50% survival rate for fba1Δ / FBA1-infected wax moth larvae, while the survival rate for WT-infected larvae was only 25%. This indicates that PeAc treatment is more effective in improving the survival rate of fba1Δ / FBA1-infected wax moth larvae than in treating wild-type fungal-infected larvae.

[0148] Example 8: Cytotoxicity assay of PeAc

[0149] Materials and methods

[0150] 1. Reagents and main reagents: PeAc: purchased from Shanghai Taoshu Biotechnology Co., Ltd. Dimethyl sulfoxide (DMSO): purchased from Macklin Biotechnology Co., Ltd. CCK-8: purchased from Shanghai Taoshu Biotechnology Co., Ltd. PeAc was prepared into a 10 mg / ml solution with DMSO, and the test drug was stored at -20℃. Before the experiment, the drug stock solution was taken out and placed in a 35℃ incubator to thaw, and thoroughly mixed before subsequent experiments.

[0151] 2. Cell line: Human umbilical vein endothelial cells (HUVEC) (available from ATCC). HUVEC cells were cultured in complete medium (DMEM + 10% fetal bovine serum) at 37°C.

[0152] 3. Culture medium: DMEM high glucose medium: purchased from Shanghai Titan Technology Co., Ltd. Fetal bovine serum: purchased from Zhejiang Tianhang Biotechnology Co., Ltd. Trypsin: purchased from Shanghai Titan Technology Co., Ltd.

[0153] 4. Instruments: Constant temperature incubator (Shanghai Yiheng Scientific Instruments Co., Ltd. MJ-150-I); Multifunctional microplate reader (TECAN Infinite M200).

[0154] 5. Preparation of cell drug delivery plates

[0155] Cultured human umbilical vein endothelial cells (HUVECs) were harvested, digested with trypsin, centrifuged to collect the cells, and resuspended in complete culture medium. Cells were counted using a hemocytometer and the cell density was adjusted to 5 × 10⁻⁶ cells / mL. 4 Cells / ml. Divide a 96-well plate into experimental, control, and cell-free zones. Add 100 μl of cell culture to each well in the experimental and control zones, and 100 μl of complete culture medium to each well in the cell-free zone. Ensure uniform cell distribution within the wells. Incubate cells overnight at 37°C. The next day, use sterile EP tubes to serially dilute the test drug with complete culture medium to a final concentration of 64-8 μg / ml. Remove the 96-well plate and discard the original culture medium from the experimental and control zones. Add 100 μl of culture medium containing different drug concentrations (triple replicates for each concentration) to each well and incubate at 37°C for 24 hours. Remove the 96-well plate, discard the original culture medium from the experimental, control, and cell-free zones, and add 110 μl of complete culture medium containing CCK-8 to each well (CCK-8:complete culture medium = 1:10). Incubate at 37°C for 2 hours. Measure the OD of each well. 450When processing the results, the OD value of the cell-free area was subtracted from the OD value of the experimental and control areas, and the Cell Viability of the control area was set to 1.0 to calculate the Cell Viability of each group. The experiment was repeated three times, and graphs were plotted and statistically analyzed using GraphPad. The experimental results are shown below. Figure 6 As shown, Figure 6 This is a schematic diagram of the cytotoxicity results of PeAc in Example 8. The horizontal axis represents PeAc concentration (μg / ml), and the vertical axis represents cell viability (%). **** represents P < 0.0001. The results show that significant toxicity was only observed when the PeAc concentration reached as high as 64 μg / ml, indicating that the antifungal effect of PeAc has a certain degree of specificity and relatively low toxicity to human cells.

[0156] Example 9: Establishment of a Candida albicans monoallelic knockout library

[0157] This invention independently constructed a Candida albicans gene knockout library, utilizing a well-established gene knockout method: the SAT1 Flipper gene knockout method (Future Microbiol. 2018, 13: 1141-1156.). This method uses nourseothricin resistance markers to screen positive clones (the nourseothricin resistance gene can be completely knocked out in subsequent experiments), avoiding interference from auxotrophic marker genes in Candida albicans. The gene knockout strains are reliable for subsequent phenotypic studies. This method achieves gene knockout based on the principle of homologous recombination, thus avoiding off-target effects.

[0158] Materials and methods

[0159] 1. Reagents and Key Test Kits: SanPrep column plasmid extraction kit: purchased from Shanghai Sangon Biotech Co., Ltd. Ampicillin trihydrate (AMP): purchased from Shanghai Sangon Biotech Co., Ltd. DNA molecular weight standard marker: purchased from Shanghai Sangon Biotech Co., Ltd. Restriction endonuclease SacⅠ: purchased from NEB New England Biolabs. Restriction endonuclease ApaⅠ: purchased from NEB New England Biolabs. Yeast transformation kit: purchased from Shanghai Maokang Biotechnology Co., Ltd. Norilskine: purchased from Shanghai Maokang Biotechnology Co., Ltd. 10×TE Buffer: purchased from Beijing Tianenze Biotechnology Co., Ltd. Ex Taq: purchased from TaKaRa Biotechnology (Dalian) Co., Ltd. Low-electroosmotic agarose: purchased from Shanghai Camon Biotechnology Co., Ltd. Nucleic acid dye Super Red: purchased from biosharp. 50×TAE: purchased from biosharp. DNA Loading Baffer: purchased from Vazyme Biotech Co., Ltd.

[0160] 2. Strains: ATCC standard strain: Candida albicans SC5314 (available from ATCC). All experimental strains were activated by streaking on Saburg dextrose agar (SDA). After culturing Candida albicans at 35°C for 48 hours, single colonies were picked and re-activated by streaking. The second batch of single colonies was placed on SDA slant, cultured using the above method, and then stored at 4°C for later use.

[0161] 3. Culture medium

[0162] Saburg Dextrose Agar (SDA) medium: Dissolve 10g peptone, 40g glucose, and 18g agar in 900ml triple-distilled water, add 50ml 2mg / ml chloramphenicol aqueous solution, adjust pH to 7.0, bring volume to 1000ml, autoclave, and store at 4℃.

[0163] YPD liquid culture medium: Dissolve 10g yeast extract, 20g peptone, and 20g glucose in 900ml triple-distilled water, bring the volume up to 1000ml, autoclave, and store at 4℃.

[0164] Norilskine solid agar plates: Add 20g of agar to YPD liquid medium, autoclave, and cool the medium to about 50°C. Add filtered sterilized Norilskine solution to make the final concentration of Norilskine in the medium 200μg / ml. Then pour into 90mm petri dishes and let them air dry for later use.

[0165] LB liquid medium: Weigh 20g of LB medium powder, add triple-distilled water to a final volume of 1L, autoclave, and store at 4℃.

[0166] 4. Instruments: Clean bench (Jiangsu Antai Air Technology Co., Ltd. SW-CJ-1FD); Constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd. MJ-150-I); Mini centrifuge (Hitachi CT15RE); Electric hot water bath (Hualian Medical Instrument Co., Ltd. DK-8D); Shaking incubator (Jiangsu Taicang Experimental Equipment Factory HZ-2111K-B); Gel imaging system (Shanghai Tianneng Tanon 2500R); Horizontal electrophoresis apparatus (Shanghai Titan Technology Co., Ltd. HE-130).

[0167] 5. Construction of monoallelic knockout bacteria using the SAT1 flipper knockout method

[0168] Using wild-type Candida albicans SC5314 as the parent bacterium, the target gene was replaced by a cassette (containing the noroside resistance marker caSAT1) through two homologous recombinations. The cassette contained the Candida albicans adaptive FLP fragment, which could be completely deleted, ultimately resulting in a knockout bacterium with only the target gene missing. Specifically, 250 bases (500 bases total) were selected from the inner and outer sides of the ORF of the target gene upstream, and cloned upstream of the MAL2p-CaFLP-CaSAT1 cassette in the pSFS2 plasmid. Similarly, 250 bases (500 bases total) were selected from the inner and outer sides of the ORF of the target gene downstream, and cloned downstream of the MAL2p-CaFLP-CaSAT1 cassette, thus constructing the pSFS2-"target gene" updwn plasmid. These two homologous arms facilitate homologous recombination between the knockout plasmid and the target gene. After obtaining the pSFS2-"target gene" updwn plasmid, it was double-digested with both ApaI and SacI restriction endonucleases. The digested fragment was then transformed into Candida albicans SC5314 using the lithium acetate method. This fragment then replaced one allele of the target gene through homologous recombination. Transformants resistant to norsocrine were identified using colony polymerase chain reaction (PCR) with multiple primers. The norsocrine resistance marker (CaSAT1) was deleted from the correct transformants to obtain a monoallelic knockout strain of the target gene.

[0169] The experimental results are shown in Figure 7 , 8 As shown in Figure 9; Figure 7This is a schematic diagram illustrating the strategy for establishing a Candida albicans monoallelic knockout library in Example 9. This diagram demonstrates the use of the norsinoxin resistance gene SAT1 and the principle of homologous recombination in gene knockout. Nested PCR was used to identify whether the gene knockout was successful; the nested PCR primer design concept is shown in this diagram.

[0170] Figure 8 This is a schematic diagram illustrating the implementation of establishing a Candida albicans monoallelic knockout library in Example 9. Taking the Candida albicans ARO1 gene as an example, the composite diagram from left to right shows: primer information, predicted band sizes, and agarose gel electrophoresis results for nested PCR verification after Candida albicans ARO1 monoallelic knockout. Based on the agarose gel electrophoresis results on the right side of the composite diagram, the sizes of the five bands in the verified ARO1 monoallelic knockout bacteria match their corresponding predicted band sizes, proving that the Candida albicans ARO1 monoallelic gene was successfully knocked out.

[0171] Figure 9 This is a schematic diagram illustrating the implementation of the Candida albicans monoallele knockout library in Example 9. Taking the Candida albicans FBA1 gene as an example, the composite diagram from left to right shows: primer information, predicted band sizes, and agarose gel electrophoresis results for nested PCR verification after the Candida albicans FBA1 monoallele knockout. Based on the agarose gel electrophoresis results on the right side of the composite diagram, the sizes of the five bands in the verified FBA1 monoallele knockout bacteria match their corresponding predicted band sizes, proving that the Candida albicans FBA1 monoallele was successfully knocked out. The results show that the SAT1 flipper knockout method can successfully construct a Candida albicans monoallele knockout bacterium.

[0172] Example 10: Compound target screening based on haploid deficiency effect analysis

[0173] This invention first confirms that the haploid deficiency effect can be used for the identification of antifungal drug targets. It is known that azole drugs such as fluconazole, itraconazole, and voriconazole target ERG11. Using Candida albicans with a single allele knockout of ERG11 (erg11Δ / ERG11) and its wild-type parent SC5314 (WildType, WT), spot plate experiments and MIC experiments were performed. The results consistently showed that strains lacking one allele of ERG11 exhibited significantly increased sensitivity to fluconazole, itraconazole, and voriconazole, perfectly consistent with the haploid deficiency effect. The results are as follows... Figure 10 , 11 As shown.

[0174] This invention further investigates the specificity of the haploid deficiency effect in identifying antifungal drug targets. It is known that azole drugs such as fluconazole, itraconazole, and voriconazole target ERG11. Therefore, monoallelic knockouts of genes other than ERG11 should not show particular sensitivity to azole drugs. Multiple gene knockouts (FBA1, ARO1, etc.) were selected to examine their sensitivity to azole drugs. Experiments included plate tests and MIC assays. The study found that strains lacking one FBA1 (or ARO1, etc.) allele showed no change in sensitivity to azole drugs, demonstrating the specificity of the haploid deficiency effect in identifying drug targets. The results are as follows: Figure 12 , 13 As shown.

[0175] Given that the haploid deficiency effect is both sensitive and specific for identifying drug targets, this invention utilizes a self-constructed Candida albicans gene knockout library to study the target of PeAc. Extensive screening of strains with single allele deletions for PeAc sensitivity (micro-liquid dilution and spot plate assays) revealed that only strains lacking one FBA1 allele showed increased sensitivity to PeAc, suggesting that Fba1p is a target of PeAc (results are shown in Figure 1). Figure 14 (As shown) and is a specific target of PeAc (results as shown) Figure 15 (As shown).

[0176] Materials and methods

[0177] 1. Reagents and main reagents: PeAc: purchased from Shanghai Taoshu Biotechnology Co., Ltd. Fluconazole: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Dimethyl sulfoxide (DMSO): purchased from Macklin. PeAc was prepared into a 10 mg / ml solution with DMSO, and the test drug was stored at -20℃. Before the experiment, the drug stock solution was taken out and placed in a 35℃ incubator to thaw, and thoroughly mixed before subsequent experiments.

[0178] 2. Strains: ATCC standard strain: Candida albicans SC5314 (available from ATCC). All experimental Candida albicans monoallelic knockout strains used SC5314 as the parent strain. All experimental strains were activated by streaking on Saburg dextrose agar (SDA). After culturing Candida albicans at 35°C for 48 hours, single colonies were picked and re-activated by streaking. The second batch of single colonies was placed on SDA slant, cultured using the above method, and then stored at 4°C for later use.

[0179] 3. Culture medium

[0180] RPMI 1640 medium: 10 g RPMI 1640 (Gibco BRL), 2.0 g NaHCO3, 34.5 g (0.165 M) morpholine propanesulfonic acid (MOPS) (Sigma), dissolved in 900 ml triple-distilled water, adjusted pH to 7.0 (25 °C) with 1 N NaOH, brought to a final volume of 1000 ml, filtered for sterilization, and stored at 4 °C.

[0181] Saburg Dextrose Agar (SDA) medium: Dissolve 10g peptone, 40g glucose, and 18g agar in 900ml triple-distilled water, add 50ml 2mg / ml chloramphenicol aqueous solution, adjust pH to 7.0, bring volume to 1000ml, autoclave, and store at 4℃.

[0182] YPD liquid culture medium: Dissolve 10g yeast extract, 20g peptone, and 20g glucose in 900ml triple-distilled water, bring the volume up to 1000ml, autoclave, and store at 4℃.

[0183] YPD solid medium is prepared by adding 20g of agar to the liquid medium, autoclaving, pouring into 90mm petri dishes, and air-drying for later use.

[0184] 4. Instruments: Constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd. MJ-150-I); Shaking incubator (Jiangsu Taicang Experimental Equipment Factory HZ-2111K-B); Multifunctional microplate reader (TECAN Infinite M200).

[0185] 5. Experimental Procedure

[0186] Micro-liquid dilution method: The method is the same as in Example 1.

[0187] Spot test: Candida albicans was inoculated into YPD liquid medium and cultured at 30°C for 16 h. The activated bacterial suspension was transferred to a 1.5 ml centrifuge tube, centrifuged at 3000 g for 1 min, the medium was discarded, and the solution was washed once with PBS. After centrifugation, the supernatant was discarded, and the solution was resuspended in 1 ml PBS. After dilution, 10 μl was transferred to a hemocytometer for counting. The bacterial concentration was adjusted to 3 × 10⁻⁶ cells / mL with PBS. 6 cells / ml. A series of dilutions of WT and various haploid knockout bacteria were prepared, serially diluted 10-fold to five concentrations. 5 μl of each Candida albicans series concentration dilution was spotted onto YPD agar plates pre-added with different concentrations of the drug to be investigated. The plates were inverted in a constant temperature incubator and incubated at 35℃ for 24-48 h. During this period, the differences in growth among the strains were observed and photographed.

[0188] Molecular docking: Computer analysis of the interaction mechanism between PeAc and Fba1p.

[0189] The experimental results are shown in Figures 10-16 As shown, Figure 10 This is a schematic diagram of the results of the spot plate experiment in the feasibility verification of using the haploid deficiency effect for antifungal drug target identification in Example 10. It is known that the target of azole drugs such as fluconazole, itraconazole, and voriconazole is ERG11. The results show that Candida albicans with ERG11 monoallelic knockout (erg11Δ / ERG11) has significantly increased sensitivity to fluconazole, itraconazole, and voriconazole compared with its wild-type parent SC5314 (Wild Type, WT), which is completely consistent with the haploid deficiency effect.

[0190] Figure 11 This is a schematic diagram of the MIC experiment results in the feasibility verification of using the haploid deficiency effect for antifungal drug target identification in Example 10. The results are presented in the form of a heatmap. The results also show that the ERG11 monoallelic knockout Candida albicans (erg11Δ / ERG11) has increased sensitivity to fluconazole, itraconazole and voriconazole compared with its wild-type parent SC5314 (Wild Type, WT), which is also consistent with the haploid deficiency effect. Figure 10 , 11 The results collectively demonstrate that the haploid deficiency effect is feasible and effective for identifying antifungal drug targets.

[0191] Figure 12 This is a schematic diagram of the results of the spot plate experiment in Example 10, where the haploid deficiency effect was used to verify the specificity of antifungal drug target identification. It is known that azole drugs such as fluconazole, itraconazole, and voriconazole target ERG11. Therefore, monoallelic knockout bacteria of genes other than ERG11 should not show particular sensitivity to azole drugs. The results showed no significant difference in sensitivity to fluconazole, itraconazole, and voriconazole between other monoallelic knockout Candida albicans (such as FBA1, ARO1, KGD1, and ENO1) and their wild-type parent SC5314 (Wild Type, WT).

[0192] Figure 13 This is a schematic diagram of the MIC experiment results in the specificity verification of the haploid deficiency effect for antifungal drug target identification in Example 10. The results also show that there is no significant difference in the sensitivity of Candida albicans with other monoallelic knockouts (such as FBA1, ARO1, KGD1, ENO1) to fluconazole, itraconazole, and voriconazole compared with its wild-type parent SC5314 (Wild Type, WT). Figure 12 , 13 The results collectively demonstrate that strains with alleles other than ERG11 showed no change in sensitivity to azole drugs, proving that the haploid deficiency effect has specificity in identifying drug targets.

[0193] Figure 14 This is a schematic diagram of the results of the spot test and MIC experiment for screening PeAc targets using the haploid deficiency effect in Example 10. The results show that the Candida albicans with FBA1 monoalle knockout (fba1Δ / FBA1) has increased sensitivity to PeAc compared with its wild-type parent SC5314 (Wild Type, WT), suggesting that Fba1p is a target of PeAc.

[0194] Figure 15 This is a schematic diagram of the results of the spot test and MIC experiment for screening PeAc targets using the haploid deficiency effect in Example 10. The results show that there is no significant difference in the sensitivity of other (such as ARO1, KGD1, ENO1) Candida albicans with single allelic knockout and its wild-type parent SC5314 (Wild Type, WT) to PeAc, suggesting that Fba1p is a specific target of PeAc.

[0195] Figure 16 This is a schematic diagram showing the results of investigating the interaction between PeAc and Fba1p through molecular docking in Example 10. Figure 16 The results showed that the carboxyl group of cis-6-octadecenoic acid (PeAc) formed three hydrogen bonds with the surrounding amino acid residues Ser267, Thr288, and Asp289, respectively, and the alkane chain formed hydrophobic interactions with the surrounding amino acid residues such as Asn35, His109, Gly265, and His225.

[0196] Example 11: Study on the antifungal effect of PeAc through Fba1p

[0197] This invention verifies that PeAc exerts its antifungal effect through Fba1p by examining liquid hyphae formation (same as Example 2), biofilm formation (same as Example 3), and in vivo survival rate (same as Example 7).

[0198] The experimental results are shown in Figures 17-19 As shown: Figure 17This is a schematic diagram illustrating the results of PeAc's antifungal effect through Fba1p in inhibiting hyphal formation, as demonstrated in Example 11. The results show that, in the absence of drug intervention, both Candida albicans species (WT and fba1Δ / FBA1) could form hyphae in the hyphal induction medium. PeAc at a concentration of 1 μg / ml significantly inhibited the formation of fba1Δ / FBA1 hyphae, and even yeast-like cells were observed. However, for the wild-type (WT) strain, 1 μg / ml of PeAc did not shorten the hyphae. This indicates that the FBA1 monoallelic knockout Candida albicans (fba1Δ / FBA1) showed increased sensitivity to PeAc compared to its wild-type parent SC5314 (Wild Type, WT), suggesting that Fba1p is the target of PeAc.

[0199] Figure 18 This is a schematic diagram illustrating the results of PeAc's antifungal effect through Fba1p in inhibiting biofilm formation, as demonstrated in Example 11. The results show that PeAc can dose-dependently reduce the biofilm formation rate of Candida albicans. 16 μg / ml PeAc inhibited biofilm formation of WT bacteria by approximately 34.12% (#P<0.05). For fba1Δ / FBA1, 8 μg / ml PeAc achieved the same inhibitory effect as 16 μg / ml PeAc against WT bacteria, with an inhibition rate of approximately 34.07% (*P<0.05). When the PeAc concentration increased to 16 μg / ml, the biofilm inhibition rate against fba1Δ / FBA1 bacteria reached 50.43% (***P<0.001). The results indicate that the FBA1 monoallelic knockout Candida albicans (fba1Δ / FBA1) is more sensitive to PeAc than its wild-type parent SC5314 (Wild Type, WT), suggesting that Fba1p is a target of PeAc.

[0200] Figure 19 This is a schematic diagram illustrating the survival results of wax moth larvae in Example 11, where PeAc exerts its antifungal effect through Fba1p. Figure 19The results showed that compared with WT, the survival time of fba1Δ / FBA1-infected wax moth larvae was prolonged, but the mortality rate still exceeded 80% on the second day of the observation period. PeAc 32 mg / kg significantly improved the survival rate of both WT and fba1Δ / FBA1-infected larvae. However, in terms of the final number of surviving larvae, 32 mg / kg PeAc treatment resulted in a 50% survival rate for fba1Δ / FBA1-infected wax moth larvae, while the survival rate for WT-infected larvae was only 25%. This indicates that the FBA1 monoallelic knockout Candida albicans (fba1Δ / FBA1) is more sensitive to PeAc than its wild-type parent SC5314 (Wild Type, WT), suggesting that Fba1p is a target of PeAc.

[0201] All the above results demonstrate that PeAc exerts its antifungal effect through Fba1p. Fructose-1,6-bisphosphate aldolase (Fba1p) is the target of PeAc, and PeAc is an inhibitor of fructose-1,6-bisphosphate aldolase.

[0202] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The application of cis-6-octadecenoic acid or its pharmaceutical salt in the preparation of antifungal drugs in conjunction with commonly used clinical antifungal drugs, characterized in that, The commonly used antifungal drug in clinical practice is fluconazole; The fungi are selected from Candida albicans, Candida krusei, Candida tropicalis, and Trichophyton mentagrophytes.

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