A polyene macrolide natural product pac-g5 and its use

Abstract

The application provides a polyene macrolide natural product PAC-G5 or a pharmaceutically acceptable salt thereof, and the compound structural formula of the natural product PAC-G5 is as shown in the following formula, and the natural product PAC-G5 can target ergosterol in a fungal cell membrane, so as to cause death of the fungal cell. The natural product PAC-G5 has strong in-vitro antibacterial activity and a broad antibacterial spectrum on various WHO published multi-drug resistant fungal key pathogens including candida, aspergillus, cryptococcus and the like, and can be used for preparing an antifungal drug.

Description

Technical Field

[0001] This invention belongs to the field of microbial natural products, and particularly relates to a polyene macrolide natural product PAC-G5 and its applications. Background Technology

[0002] Fungal infections are a major global public health challenge. In recent years, with the widespread use of immunosuppressants and the development of medical technologies such as organ transplantation and intensive care, the number of immunocompromised patients has been increasing, leading to a significant rise in the incidence and mortality rates of fungal infections. Currently, the main first-line antifungal drugs used clinically include four types: echinocandins (such as cabobfenac), polyene macrolides (such as amphotericin B), azoles (such as fluconazole), and 5-fluorocytosine (Nature Reviews Microbiology 2022; 20:9 557-571). Polyene macrolide antibiotics are an important class of antifungal drugs widely used to treat local and systemic fungal infections. Their structures are mainly macrocyclic structures with 26, 28, 36, and 38 rings, and contain 3 to 7 double bonds and 1-2 deoxysugar substituents (Mol Phylogenet Evol. 2018; 127:239-247). Its mechanism of action is mainly through the interaction with ergosterol, a steroid molecule on the fungal cell membrane, to form a transmembrane channel, which exposes important substances such as potassium ions, nucleotides, and amino acids from the cell, thereby inhibiting fungal growth and exerting strong antifungal activity (Proc Natl Acad Sci US A.2012; 109(7):2234-9; Proc Natl Acad Sci US A.2011; 108(17):6733-8).

[0003] Although polyunsaturated natural products, such as amphotericin B, have potent antifungal activity, they also have strong toxic side effects. Clinical use often requires extremely low doses and prolonged infusion time to reduce these side effects, which severely limits the effectiveness of this type of drug in treating invasive fungal infections. Therefore, there is an urgent need to develop antifungal drugs with potent antifungal activity and low toxicity. Summary of the Invention

[0004] Purpose of the invention: In order to solve the problems of high toxicity of polyene natural products such as amphotericin B and nystatin, this invention aims to provide a novel polyene macrocyclic lactone natural product, PAC-G5, which has strong antifungal activity and low toxicity. It is expected to solve the technical problems of high toxic side effects and low oral bioavailability of current antifungal drugs, which limit their clinical application.

[0005] This invention also provides a method for preparing the polyene macrocyclic lactone natural product PAC-G5 and its application.

[0006] Technical solution: To achieve the above objectives, the present invention provides a polyene macrolide natural product PAC-G5 or a pharmaceutically acceptable salt thereof, wherein the structural formula of the natural product PAC-G5 compound is shown in Formula I:

[0007]

[0008] The core parent ring of the polyene macrolide natural product PAC-G5 is a 38-membered macrolide, wherein: C20-C29 are conjugated pentaene structures, C32 is a monoene structure; C19 is connected to a carboxylic acid; C3, C5, C7, C10, C11, and C13 are connected to a β-hydroxyl group; C15 is connected to an α-hydroxyl group; C13 and C15 are connected by an oxygen bridge; C1 is an ester group; C34, C36, and C37 are connected to an α-methyl group; and C16 is connected to a formic acid group.

[0009] The method for preparing the polyene macrolide natural product PAC-G5 or its pharmaceutically acceptable salt according to the present invention includes the following steps:

[0010] The strain containing the PAC-G5 biosynthetic gene cluster was prepared into a seed culture, cultured, fermented, extracted, separated and purified to obtain the natural product PAC-G5.

[0011] The strain containing the PAC-G5 biosynthetic gene cluster is Streptomyces fulvorobeus DSM41455.

[0012] The application of the polyene macrolide natural product PAC-G5 or its pharmaceutically acceptable salt in the preparation of antifungal drugs.

[0013] The fungus is any one or more of Candida, Aspergillus, and Cryptococcus.

[0014] The Candida species mentioned are Candida albicans, Candida auris, Candida glabrata, Candida tropicalis, or Candida subglabrata.

[0015] The pharmaceutical composition of the antifungal drug of the present invention comprises the polyene macrolide natural product PAC-G5 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0016] The pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhaler, ointment, suppository or patch.

[0017] The application of the antifungal drug composition described in this invention in the preparation of antifungal drugs.

[0018] The fungus is any one or more of Candida, Aspergillus, and Cryptococcus.

[0019] This invention discovered a strain, Streptomyces fulvorobeus DSM 41455, expressing PAC-G5, from the Microbial Secondary Metabolite Database (MiSM) through sequence tag-directed mining; combinatorial biosynthesis revealed a natural product, PAC-G5, with potent broad-spectrum activity against multidrug-resistant fungi. PAC-G5 has a unique 38-membered ring chemical structure with mycosamine at C19, a pentene structural unit at C20, and a monoene structural unit at C32.

[0020] The PAC-G5 compound of this invention is a natural product produced by fermentation of Streptomyces fulvorobeus DSM 41455. The screening of this Streptomyces utilized a targeted mining technique combining big data analysis, phylogenetic analysis, and cluster analysis: using conserved carbamoyl glycosyltransferases as sequence tags, a Hidden Markov Model was used to mine all biosynthetic gene clusters expressing carbamoyl glycosyltransferase sequence tags from the Microbial Secondary Metabolite Database (MiSM). Then, combined with phylogenetic analysis, targeted mining identified a strain expressing PAC-G5, Streptomyces fulvorobeus DSM 41455.

[0021] In investigating the mechanism of action of this compound, feeding experiments, isothermal calorimetric titration, and UV-Vis were used. The preparation of PAC-G5 requires exploring fermentation conditions to remove fermentation byproducts and extracting, separating, and purifying the crude product.

[0022] The PAC-G5 of this invention induces fungal cell death by targeting ergosterol in fungal cells. PAC-G5 exhibits potent activity against strains resistant to clinical antifungal drugs.

[0023] This invention discovered a strain, *Streptomyces fulvorobeus* DSM 41455, expressing a novel polyskeletal biosynthetic gene cluster through pan-genome mining. Further fermentation and purification confirmed that its expression product is PAC-G5. PAC-G5 exhibits potent and broad-spectrum antifungal activity, showing strong activity against various key fungal pathogens, including *Candida*, *Aspergillus*, and *Cryptococcus*.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0025] This invention is the first to propose a polyene macrocyclic lactone natural product, PAC-G5. The natural product PAC-G5 is obtained by preparing a seed culture of a strain containing the PAC-G5 biosynthetic gene cluster, followed by cultivation, fermentation, extraction, separation and purification.

[0026] The natural product PAC-G5 obtained in this invention exhibits potent antifungal activity and a broad-spectrum antifungal spectrum against various WHO-listed priority multidrug-resistant pathogenic microorganisms, including Candida, Aspergillus, and Cryptococcus. The minimum inhibitory concentration (MIC) of the compound against Candida, particularly against multidrug-resistant strains including Candida albicans and Candida auris, is 1-2 times that of clinically used nystatin and comparable to amphotericin B, ranging from 0.25-2 μg / mL. Its activity against multidrug-resistant Cryptococcus neoformans reaches 0.125 μg / mL, which is 32 times that of cabofenin, 64 times that of fluconazole, and 128 times that of 5-fluorocytosine. Its activity against multidrug-resistant Aspergillus fumigatus reaches 2 μg / mL, which is 32 times that of fluconazole and 16 times that of 5-fluorocytosine. This demonstrates that PAC-G5 possesses broad-spectrum antifungal activity and potent antifungal activity against clinically antifungal drug-resistant strains. Meanwhile, the cytotoxicity test of the present invention showed that PAC-G5 was significantly less cytotoxic to Hep-G2 and HK2 than amphotericin B; the hemolysis test showed that the hemolytic activity of PAC-G5 was also significantly less than that of amphotericin B. Attached Figure Description

[0027] Figure 1 The HPLC detection chromatogram and UV spectrum of PAC-G5 are shown below.

[0028] Figure 2 The spectrum is that of PAC-G5 HR-ESI-MS.

[0029] Figure 3 PAC-G5 1 H spectrum;

[0030] Figure 4 PAC-G5 13 C spectrum;

[0031] Figure 5 The spectrum of PAC-G5 HSQC;

[0032] Figure 6 The spectrum is that of PAC-G5 COSY.

[0033] Figure 7 The spectrum is PAC-G5 HMBC.

[0034] Figure 8 The relationship between the PAC-G5 structure and two-dimensional NMR signals;

[0035] Figure 9To predict the chirality of the hydroxyl C in PAC-G5 using bioinformatics;

[0036] Figure 10 PAC-G5 cytotoxicity;

[0037] Figure 11 PAC-G5 hemolysis;

[0038] Figure 12 This is a PAC-G5 feed assay.

[0039] Figure 13 UV-vis;

[0040] Figure 14 ITC is the interaction between PAC-G5 and sterols. Detailed Implementation

[0041] The technical solution of the present invention will be further described below with reference to the accompanying drawings. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or according to the manufacturer's recommendations.

[0042] Among them, strain Streptomyces fulvorobeus DSM 41455 was purchased from the DSMZ platform.

[0043] TSB premix (purchased from Guangdong Huankai Microbial Technology Co., Ltd., item number 024048). Yeast extract (purchased from Beijing Aoboxing Biotechnology Co., Ltd., item number 01-012).

[0044] Amphotericin B (Macklin, CAS: 1397-89-3, Product No.: A6061).

[0045] Nystatin (Macklin, CAS: 1400-61-9, Product No.: N814558).

[0046] Example 1

[0047] Discovery of the PAC-G5 biosynthetic gene cluster

[0048] Based on the known carboxysyltransferase sequences of polyene macrolide natural products, conserved protein structural sequences were obtained through analysis. A unique hidden Markov model for polyene carboxysyltransferases was constructed. This model was used to scan the sequence similarity of microbial secondary metabolite databases. Protein sequences with a similarity value less than 2e-145 were considered as polyene-related carboxysyltransferase sequences, and their corresponding functional gene clusters were identified as potential novel polyene-encoding gene clusters. This method yielded 531 candidate sequences. A phylogenetic tree was constructed for these sequences, and the phylogenetic relationships between different sequences were analyzed. The results revealed a new evolutionary branch, with the strain *Streptomycesfulvorobeus* DSM 41455 encoding a novel polyene natural product, named PAC-G5.

[0049] Example 2

[0050] PAC-G5 bio-fermentation

[0051] (1) Preparation of spore suspension

[0052] Spores were prepared by spreading Streptomyces fulvorobeus DSM 41455 containing the PAC-G5 biosynthetic gene cluster on ISP4 solid medium (containing 10.0 g soluble starch, 1.0 g dipotassium hydrogen phosphate, 1.0 g magnesium sulfate, 1.0 g sodium chloride, 2.0 g ammonium sulfate, 2.0 g calcium sulfate, 0.001 g ferrous sulfate, 0.001 g manganese chloride, 0.001 g zinc sulfate, 15.0 g agar per liter, pH = 7.2) and culturing at 30°C for 5 days.

[0053] (2) Preparation of seed solution:

[0054] Add 50 mL of TSB medium (containing 17.0 g casein pancreatic digest, 3.0 g soybean digest, 5.0 g sodium chloride, 2.5 g dipotassium hydrogen phosphate, 2.5 g glucose monohydrate, pH 7.3 per liter of ddH2O) to a triangular flask. Inoculate Streptomyces fulvorobeus DSM 41455 strain, cultured on ISP4 agar plates (containing 10.0 g soluble starch, 1.0 g dipotassium hydrogen phosphate, 1.0 g magnesium sulfate, 1.0 g sodium chloride, 2.0 g ammonium sulfate, 2.0 g calcium sulfate, 0.001 g ferrous sulfate, 0.001 g manganese chloride, 0.001 g zinc sulfate, 15.0 g agar, pH 7.2 per liter of ddH2O), onto the TSB medium and incubate on a shaker (200 rpm, 30°C) for 2 days to prepare the seed culture.

[0055] (3) Preparation of fermentation broth:

[0056] 0.5 mL of seed solution was transferred to a 50 mL (250 mL Erlenmeyer flask) FM20 fermentation medium (containing 10.0 g yeast extract, 2.0 g K2HPO4, 1.0 g MgSO4-7H2O, 20 g mannitol, 0.0001 g FeSO4-2H2O, 0.0001 g MnCl2-4H2O, 0.0001 g ZnSO4-7H2O, 0.0001 g CuSO4-5H2O, and 0.0001 g CoCl2-6H2O per liter of ddH2O) and cultured for 10 days (shaking at 200 rpm, 30°C). After fermentation, n-butanol was added to the fermentation flask at a 1:1 volume ratio and stirred overnight (100 rpm) for extraction. The n-butanol extract was evaporated to dryness using a rotary evaporator and then dissolved in methanol.

[0057] Example 3

[0058] Isolation and purification of PAC-G5

[0059] Example 2: A methanol solution was obtained and separated by column chromatography using a YMC-GEL C18 powder (12nm × 50μm) packed column. The column was equilibrated with 2 cv 10% methanol (H2O:CH2OH = 9:1). The methanol solution was added, and different concentrations of methanol-water solutions (10%, 30%, 50%, 70%, 90%, 100%) were used as eluents, one 100mL vial per vial. The components were detected by UPLC-MS. The eluent containing PAC-G5 (90% methanol-water solution) was collected and evaporated to dryness. The UPLC-MS conditions were as follows: C18 column (Waters, T3-1.8μm, 2.1 × 100mm), mobile phase: phase A - deionized (0.1% formic acid), phase B - acetonitrile (0.1% formic acid), flow rate 1mL / min, gradient of mobile phase B 30%-90%, incrementing by 6% per minute. MS detection range 200-2000, simultaneous positive and negative modes. Samples containing PAC-G5 were further purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 column (Shimadzu, ShimNet HE C18-AQ, 5μm OBD, 19×250mm column). RP-HPLC conditions were as follows: solvent A, deionized water (0.1% formic acid); solvent B, acetonitrile (0.1% formic acid). Flow rate was 3 mL / min, solvent B gradient was 30%-90%, increasing by 1.5% per minute, and the PAC-G5 fraction with a purity of 87.5% (334 nm) was collected. Figure 1 Place at -80℃ for 6-10 hours, then freeze-dry in a freeze dryer for 2-3 days to obtain a light yellow powder.

[0060] Example 4

[0061] Structural identification of PAC-G5

[0062] Example 3: The purified PAC-G5 was a yellow powder, Q-TOF high resolution (Q-TOF high resolution) Figure 2 Analysis showed that its protonated ion was [M+H] with an m / z of 926.5128. + ; indicating its molecular formula C 47 H 75 NO 17 (Δppm-2.16), containing 11 double bond equivalents (DBE). The purified PAC-G5 from Example 3 was dissolved in deuterated CD3OD, and H2 spectra were obtained using a 600M NMR instrument. Figure 3 ), C spectrum ( Figure 4 ) and HSQC ( Figure 5 COSY Figure 6 ), HMBC ( Figure 7 The structure of PAC-G5 was determined by using two-dimensional spectra such as [missing information]. 1 H and 13 C10 NMR spectroscopy data revealed 12 allyl protons (δ1000- ... H 5.31-6.41, δ C 130.9-135.1), multiple oxymethylene (δ) C 67.7-79.3) and 2 carbonyl groups (δ) C The NMR signals at 172.8 and 179.3 nm indicate that PAC-G5 possesses a highly oxidized polyene macrocyclic ketone skeleton. A 38-membered macrocyclic ketone skeleton was identified using a series of 2D NMR techniques (HSQC, HMBC, COSY).

[0063] Through corresponding 1 acetal carbon (δ) H -1′4.61,δ C The presence of a single glycosyl group in the PAC-G5 structure was confirmed by an NMR signal of H-1′ (99.3). COSY and HMBC spectral analyses revealed the presence of a mycosamine in PAC-G5. H 4.61) to C-19 (δ) C HMBC correlation observation (79.1) confirmed the C-19 link between mycosamine and macrocyclic ketones. 1 H and 13 The C-spectrum (CD3OD) NMR data are shown in Table 2. Further, based on the H-spectrum, C-spectrum, and two-dimensional spectra such as HSQC, HMBC, and COSY... Figures 3-8 ) and the chiral bioinformation of PAC-G5 ( Figure 9Analysis revealed that the specific configurations of the product PAC-G5 of this invention are 2R, 10R, 11S, 12S, 13R, 14R, 16R, and 18R. The natural product PAC-G5 is shown in Formula I:

[0064]

[0065] Table 2 PAC-G5 1 H and 13 C-spectrum (CD3OD) NMR data

[0066]

[0067] *The assignment of some carbon signals was supported by HSQC and HMBCcorrelations. # The coupling constants for proton signals were not provided asmost signals are highly overlapped or broad.

[0068] Example 5

[0069] Bioactivity analysis of PAC-G5

[0070] (1) In vitro detection of anti-multidrug-resistant bacteria activity

[0071] Using the CLSI standard, the antifungal activity of PAC-G5 against priority fungal pathogens published by the WHO was determined. The results are shown in Table 1 below. PAC-G5 has potent and broad-spectrum antifungal activity.

[0072] Table 1 Antifungal activity of PAC-G5

[0073]

[0074] (2) Cytotoxicity of PAC-G5

[0075] The cytotoxicity of PAC-G5 was determined using the MTT assay (2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Hep-G2 and HK2 cells cultured in DMEM (containing 10% fetal bovine serum) were seeded into 96-well flat-bottom microplates (2,500 cells per well, depending on cell type) and cultured at 37°C and 5% CO2. After 24 hours, the medium was removed, and 100 μL of fresh medium containing serially concentrated PAC-G5 (maximum DMSO concentration less than 0.25%) was added. After incubation at 37°C for 48 hours, the medium was removed, and 110 μL of MTT solution (10 μL 5 mg / ml MTT premixed with 100 μL DMEM in PAC-G5) was added to each well. After incubation at 37°C and 5% CO2 for 3 hours, 100 μl of dissolving solution (40% DMF, 16% SDS, and 2% acetic acid aqueous solution) was added to dissolve the precipitate. The absorbance of each well was then measured at OD570 nm using a microplate reader (Epoch microplate spectrophotometer, BioTek). Amphotericin B was used as a positive control. IC50 50 The calculation of the value (Prism 7.0) refers to the concentration of each compound required to inhibit cell growth by 50% relative to the no-compound control. The IC50 of PAC-G5 on Hep-G2 cells... 50 =74.16μM, IC50 for HK2 cells 50 =84.08 μM; IC50 of amphotericin B on Hep-G2 cells 50 =7.59μM, IC50 for HK2 cells 50 =4.14μM ( Figure 10 PAC-G5 has significantly lower cytotoxicity than amphotericin B.

[0076] (3) Hemolytic activity of PAC-G5

[0077] PAC-G5 was tested for hemolytic activity according to a previously reported method (Kelvin JY Wu et al., 2024, Science, 383(6684), 721-726). Fresh, sterile, deproteinized sheep blood was centrifuged at 3,000 rpm for 10 minutes at 4°C to separate the precipitated blood cells, which were then resuspended in PBS solution (pH 7.4) to prepare a concentration of 1×10⁻⁶. 9Cells / mL suspension. Test compounds were prepared at concentrations ranging from 0.39 μM to 100 μM and mixed with blood cells to a final volume of 500 μL. 0.5% DMSO and 1% Triton X100 served as negative (0% hemolysis) and positive (100% hemolysis) controls, respectively. After incubation at 37°C for 3 hours, the supernatant was collected by centrifugation at 3,000 rpm for 20 minutes and transferred to 96-well polypropylene plates. The absorbance of the supernatant at OD540 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader to determine the degree of hemolysis. Figure 11 The results showed that PAC-G5 had almost no hemolytic activity at a high concentration of 100 μM, while amphotericin B showed severe hemolysis at 17.38 μM, indicating that PAC-G5 has better safety.

[0078] Example 6

[0079] Verification of the mechanism of action of PAC-G5

[0080] Since known polyunsaturated antifungal natural products exert their antifungal activity by binding to ergosterol on the fungal cell membrane, this study will verify whether PAC-G5 also functions by binding to ergosterol.

[0081] (1) Study on the inhibitory effect of ergosterol on PAC-G5

[0082] The effect of different concentrations of ergosterol on the antibacterial activity of PAC-G5 was evaluated using Candida albicans BNCC186382 and the broth dilution method. All components were dissolved in 10% DMSO to prepare different concentrations required for the experiment, and then added to the PAC-G5 MIC assay medium to observe the effect of ergosterol on the PAC-G5 MIC. Results are as follows: Figure 12 As shown, similar to known polyene macrolide antifungal antibiotics (amphotericidal B), ergosterol also significantly inhibits the antibacterial activity of PAC-G5. At a concentration of 0.8 mg / mL, the antifungal activity of PAC-G5 can be completely inhibited, indicating that PAC-G5 may have the same target as the known amphotericin B - ergosterol.

[0083] (3) UV-vis determines the target site of PAC-G5

[0084] To further verify whether the target of PAC-G5 is ergosterol, this experiment used UV-vis to detect whether a spectral shift would occur when PAC-G5 and ergosterol were mixed at different concentrations. First, an Erg / Chol solution was prepared: a stock solution of Erg recrystallized from chloroform (approximately 100 mM) was diluted to 5 mM with DMSO. For the composite solution: a stock solution (≥10 mM) of PAC-G5 (approximately 10 mg) was prepared by dissolving it in dimethyl sulfoxide (0.2–0.5 mL). After incubating the complex at room temperature for 0.5 h, 200 μL was pipetted into a 96-well plate. The UV absorption shift of the compound was observed using the spectral function (200 nm–500 nm) of a microplate reader, and the data were exported and plotted using Origin software.

[0085] See results Figure 13 When different concentrations of sterols (ergosterol or cholesterol) were mixed with PAC-G5, the UV absorbance of PAC-G5 changed significantly, proving that PAC-G5 can bind with ergosterol to form a new complex that causes a deflection of the UV spectrum. This verifies that PAC-G5 can bind with ergosterol, which is the target of PAC-G5.

[0086] (3) Determination of the target site of PAC-G5 by isothermal calorimetric titration

[0087] 20 mM PAC-G5 was diluted to 1 mM with 5.0 mM HEPES (pH = 7.4) containing 5% DMSO. Ergosterol was dissolved in 5.0 mM HEPES (pH = 7.4) containing 5% DMSO (the solution was 12 mM DUPC) to prepare a 500 μM composition. The purchased liposome membrane was then hydrated with 5.0 mM HEPES (pH = 7.4), and the 500 μM ergosterol suspension was passed through a 100 nm polycarbonate filter six times using an Avanti Mini extruder to prepare LUV.

[0088] The exothermic relationship between PAC-G5 and sterol binding was determined using a PEAQ-ITC isothermal titration calorimeter. At 25°C, a 1 mM (40 μl) PAC-G5 solution was placed in an automated syringe, and a 500 μM LUV (250 μl) suspension was placed in the sample cell. The initial injection volume was 0.23 μl, followed by 18 subsequent injections of 2 μl each. Each injection was spaced 80 seconds apart to ensure the instrument returned to a stable baseline before the next injection. The stirring speed was 500 rpm for each experiment.

[0089] To test the binding strength between PAC-G5 and ergosterol, this experiment employed isothermal titration to immobilize ergosterol on a membrane-like structure, and the binding affinity between the two was then determined. The results are shown below. Figure 14 The results showed that PAC-G5 has a high binding capacity for ergosterol (K). d The value reached 32.1±4.1 μM; while the binding ability of amphotericin B to ergosterol was 24.6±2.1 μM; PAC-G5 and amphotericin B had similar binding abilities to ergosterol.

Claims

1. A polyene macrolide natural product PAC-G5 or a pharmaceutically acceptable salt thereof, characterized in that, The structural formula of the natural product PAC-G5 compound is shown in Formula I:

2. A method for preparing the polyene macrocyclic lactone natural product PAC-G5 as described in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, Includes the following steps: The strain containing the PAC-G5 biosynthetic gene cluster was prepared into a seed culture, cultured, fermented, extracted, separated and purified to obtain the natural product PAC-G5.

3. The preparation method according to claim 2, characterized in that, The strain containing the PAC-G5 biosynthetic gene cluster is preferably Streptomyces fulvorobeus DSM 41455.

4. The use of the polyene macrolide natural product PAC-G5 of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an antifungal drug.

5. The application according to claim 4, characterized in that, The fungus is any one or more of Candida, Aspergillus, and Cryptococcus.

6. The application according to claim 5, characterized in that, The Candida species mentioned are Candida albicans, Candida auris, Candida glabrata, Candida tropicalis, or Candida subglabrata.

7. A pharmaceutical composition of an antifungal drug, characterized in that, It comprises the polyene macrolide natural product PAC-G5 as described in claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhaler, ointment, suppository or patch.

9. Use of a pharmaceutical composition of the antifungal drug according to claim 8 in the preparation of an antifungal drug.

10. The application according to claim 9, characterized in that, The fungus is any one or more of Candida, Aspergillus, and Cryptococcus.

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