Anti-Candida Activities And Mechanisms Of Heteropolytungstates

Invasive mycosis has been on the increase in recent years,and its rate of rise has far exceeded that of other pathogens.The incidence of deep fungal infection in the immunocompromised population is approximately 11%to 40%,and the mortality rate exceeds 40%.Among them,the incidence of fungal infections in recipients of organ transplants ranges from 20%to 40%,while the incidence of fungal infections in AIDS patients is as high as 90%.The mortality rate of patients with systemic fungal infections is very high.Second only to patients with hematological malignancies is the main cause of death in immunocompromised patients.The one-year overall survival rate is very low,generally less than 20%.This is closely related to the increasing number of immunocompromised populations such as AIDS,diabetes,and cancer patients,the extensive use of broad-spectrum antibiotics,corticosteroids,and chemotherapeutic agents,organ and bone marrow transplantation,and the development of invasive techniques.The existing drugs for the treatment of deep fungal infections mainly include azoles,polyenes,echinocandins,and the like,and there are problems such as fewer types and large toxic and side effects,and drug resistance caused by the widespread use of certain drugs is increasing.The fungi are inherently resistant to existing drugs,such as Candida krusei,part of Candida glabrata,and other natural resistance to fluconazole.Therefore,the development of new,highly effective,and low-toxic antifungal drugs is very important for the effective treatment of fungal infections and saving the lives of patients.Polyoxometalates（POMs,abbreviated as polyacids）are a kind of metal-oxygen clusters,which are formed by self-assembly of a transition metal and an oxygen atom and form an inorganic compound having a spatial network structure,with many different biological activities than mononuclear complexes.As a new candidate drug,these compounds have the advantages of simple synthesis,low cost,flexible design and modification of molecules,low side effects,and excellent physical and chemical properties（surface charge distribution,molecular shape,pH,solubility,etc.）.Up to now,more than 200 kinds of polyacids with various drug activities have been reported,mainly focusing on antiviral and antitumor^[19-21].The study of antibacterial activity of polyacids was relatively rare.Only Yamata group in Japan conducted relevant research.They found that decavovanadate can selectively inhibit Streptococcus pneumoniae,polytungstates have synergistic effects with beta-lactam antibiotics,and can increase its antibacterial activity against methicillin-resistant Staphylococcus aureus.Few studies on antifungal activity have been carried out.In China,a few agricultural anti-fungal studies have been conducted.There is no systemic study of poly-acid human pathogenic fungi at home and abroad.In this study,heteropoly compounds were selected as the antifungal research system for the first time.The pharmaceutical,in vitro and in vivo antifungal pharmacodynamics,cytotoxicity and acute toxicity of heteropolytungstates were systematically studied,and their antifungal mechanisms were explored at the cellular and molecular levels..Inpharmaceuticalresearch,theheteropolytungstatesα-1,2,3-K₆H[SiW₉V₃O₄₀]（codenamed SiW-3）,K₆PV₃W₉O₄₀（codenamed PW-6）,α-K₄PVW₁₁O₄₀（codenamed PW-8）,K₁₃[Ce(SiW₁₁O₃₉)₂]·17H₂O（codenamed SiW-5）,K₁₃[Eu(SiW₁₁O₃₉)₂]·25H₂O（codenamed SiW-10）.Using infrared spectroscopy,UV spectroscopy and other methods,the structure of the test compounds were characterized.In the study of antifungal activity in vitro,according to the microdilution method（M27-A protocol）promulgated by the National Clinical Laboratory Standardization Committee（CLSI）,six species of 34 human pathogenic fungi were selected for in vitro antifungal drug sensitivity screening.It shows that the five tested compounds all have different degrees of antifungal activity.Silicotungstate Si W-5 has the highest antifungal activity,showing good activity against all five Candida species and Cryptococcus neoformans.In combination with the structural analysis of polyacids,tungstates,especially highly negative sandwich-type Keggin-structured tungstates,have higher antifungal activity.After in vitro pharmacodynamic screening,the test compound SiW-5 was selected for cytotoxicity,acute toxicity,antifungal activity in vivo and its mechanism of action.In the cytotoxicity experiment,human kidney epithelial cell line 293T was selected as the test cell,and the toxicity of Si W-5 to the cells was initially observed using the MTS method.Results within the range of 25-800μM showed that the cell inhibition rate did not reach 50%at 24 h,the IC₅₀0 at 21 h was 216.2μM,and the IC₅₀0 at 72 h was 99.5μM.It can be seen that the dose of IC₅₀0 of SiW-5 is much higher than its antifungal MIC dose（<0.2-10.2μM）,which proves its safety to human cells.Therefore,SiW-5 can be used for further in vivo antifungal study.The oral acute toxicity evaluation results showed that the LD₅₀0 of SiW-5 was 1651.5mg/kg,and the 95%confidence limit of LD₅₀0 ranged from 1539.6 mg/kg to 1926.5 mg/kg.According to the WHO acute toxicity grading standard,it was judged as Low-toxic compounds.In the study of anti-fungal effects in vivo,BALB/c mice were first immunosuppressed,and clinical isolates of Candida albicans HL27 were used as infected strains.A systemic candida infection mouse model was successfully established.The general state of mice and survival rate,kidney fungus colony counts,renal gross morphology and histopathological changes were observed to evaluate the efficacy of SiW-5.The results showed that the survival rate of mice treated with SiW-5 was significantly improved,the survival condition was improved,the burden of renal fungal burden was reduced,and gross morphology observation of the kidney and pathological staining of PAS showed that the fungal infiltration was reduced.This shows that SiW-5 has a good therapeutic effect on systemic Candida infection.With regard to the mechanism study,our group had previously confirmed by cell X-ray microscopy and nano-CT imaging techniques that the target of antiviral activity of heteropolytungstate Cs₂K₄Na[SiW₉Nb₃O₄₀]·H₂O was on the cell surface rather than on the cell interior.Fukuda N et al.reported that polytungsten tungstate acts preferentially on the cell membrane after it acts on bacteria,thus affecting bacterial morphology and substance transport.Therefore,this study suggests that the fungal cell membrane may be one of the target sites for silicotungstic acid SiW-5.In the mechanism study of this study,TEM results showed that the membrane of Candida albicans was indeed destroyed after the action of Si W-5,resulting in the change of cell morphology from a regular round or oval to a smaller irregular shape,as well as leakage of contents and structural disorders within the cytoplasm.Ergosterol is an important component of fungal cell membrane.It plays an important role in maintaining biological functions such as cell structural integrity,regulation of cell membrane fluidity,and cell cycle metabolism.Therefore,the biosynthesis of ergosterol is the target of most existing antifungal drugs（such as fluconazole,itraconazole,ketoconazole,amphotericin B,terbinafine,etc.）and new drug development.The drug destroys the cell membrane by binding to ergosterol or interfering with ergosterol biosynthetic pathways,thereby inhibiting the synthesis of ergosterol and leading to the death of fungi.In this study,thin-layer chromatography and high performance liquid chromatography were used to detect Candida ergosterol levels in different concentrations of drug treatment.The results showed that the inhibition rate of SiW-5 on ergosterol increased with the increase of drug concentration.The inhibition of ergosterol biosynthesis may be one of the mechanisms of antibacterial action of SiW-5.In order to further study the molecular mechanism of polyacid interference in ergosterol biosynthesis,five essential genes on the ergosterol synthesis pathway were selected for real-time PCR detection.The results showed that in addition to the expression of one of the genes,the expression levels of the other four genes,ERG1,ERG7,ERG11,and ERG28,were upregulated.Because SiW-5 was also found to have good activity against fluconazole-resistant strains in the earlier period,it was suggested that it acts on the ergosterol pathway different from the targets of azole drugs.Based on the above studies,we analyzed the possible mechanisms of SiW-5 inhibition of ergosterol as follows:（1）SiW-5 acts on the post-transcriptional level of genes involved in ergosterol synthesis;（2）SiW-5 combine with one or several intermediate sterols to interfere with the ergosterol synthesis pathway;（3）SiW-5 binds directly to the final product ergosterol.In this study,the antifungal activity of heteropoly compounds on human pathogenic fungi was systematically studied for the first time.In vitro and in vivo experiments demonstrated that polyacids have inhibitory effects on fungi,and they are low-toxic compounds.Their action mechanisms are explored to provide new drug candidates to treat fungal infections,and may find new targets of action that are different from existing antifungal drugs.The research results,on the one hand,have made breakthroughs in the antifungal theory of polyacid,enriched the application of polyacids in the field of pharmaceutical research,and on the other hand provided theoretical and experimental evidence for the development of new highly effective antifungal drugs with China’s independent intellectual property rights.