Study On The Screening And Mechanism Of Antifungal Compounds Synergistically With Fluconazole Against Candida Albicans

Objectives:As the widely use of immunosuppressor and the growing number of patients with severe infection,the incidence of fungal infections has been increasing around the world.In addition,the increase of surgical patients and the increase of hospitalization days also lead to severe challenges in the prevention and treatment of nosocomial fungal infection.Clinically,most of the strains isolated from blood,sputum and urine samples of patients with fungal infection are Candida,among which Candida albicans accounts for more than 40%.Therefore,it’s of great importance to prevent against the infections of Candida albicans,especially the invasive Candidiasis.Up to now,the number of effective anti-fungal agents available clinically is very limited compared with other anti-bacterial or anti-viral drugs.Also,the long period use of anti-fungal agents can cause the development of anti-fungal resistance.As eukaryotes,fungi are similar to mammalian cells,antifungal drugs are easy to have toxic and side effects,which also makes the research progress of antifungal drugs slow,and there is an urgent need to develop new and effective drugs against drug-resistant fungi.In order to solve the problems of side effects and the situation of drug resistance,our research group has been devoted to the research of antifungal synergists to enhance the sensitivity of existing antifungal drugs,including chemical small molecules and antibodies.This project continued the previous direction of our research.Through the screening of compound library and the structural modification of active compounds in the early stage,we acquired the compound CZ-66.This compound could kill fungi synergistically with fluconazole.In addition,the mechanism of destruction of fungal cell wall and cell membrane by the combination of CZ-66 and fluconazole was discussed,which provided a new idea for the study of the combination of drugs against drug-resistant fungi.Methods and results:1.In this project,we examined the efficacy of CZ-66 when it’s combined with different types of anti-fungal drugs.The anti-fungal spectrum of CZ-66 combined with fluconazole was also determined.Firstly,we investigated the antifungal activity of CZ-65、CZ-66 and phenazine derivatives by in vitro screening.It turns out that CZ-65、CZ-66 or phenazine derivatives showed no significant antifungal activity against Candida albicans.Antifungal spectrum of CZ-65、CZ-66、 phenazine derivatives glyceryl monostearate combined with fluconazole and caspofungin against common clinical Candida was detected by microdilution.The results showed that the combination of CZ-65 and fluconazole had no obvious antifungal activity against Candida.However,CZ-66,FQ-12,FQ-17 or FQ-18 had obvious antifungal activity when they’re combined with fluconazole.Glyceryl monostearate showed showed a synergistic antifungal effect with caspofungin in Candida.After that,we used the microdilution method to screen more clinical fungi.It was shown that CZ-66 had synergistic antifungal effect on more than 80% of clinical drugresistant fungi when cooperated with fluconazole.In addition,the clinical commonly used anti-fungal agents such as caspofungin,amphotericin B and 5-fluorocytosine were selected.The results showed that they all had synergistic anti-fungal activity when combined with CZ-66.2.In this study,Candida albicans was selected as the representative fungi.We investigated the antifungal circles of CZ-66,fluconazole and CZ-66 combined with fluconazole.The results showed that there was no fungal growth around the paper containing both CZ-66 and fluconazole,which indicated that CZ-66 may have fungicidal effect when cooperate with fluconazole.3.The growth curves and fungicidal curves of CZ-66 combined with fluconazole were drawn.The results showed that 0.5 μg/m L CZ-66 combined with 0.5 μg/m L fluconazole still had significant fungicidal effect.4.The effect of CZ-66 on the hyphae formation of Candida albicans was investigated.When CZ-66,fluconazole or the mixture of these two drugs were added into different hyphae induction mediums,the results showed that none of them could inhibit the hyphae formation of Candida albicans.5.Using the fluorescence probe DCFH-DA,we measured the effects of CZ-66,fluconazole and the mixture of these two drugs on the production of intracellular reactive oxygen species(ROS).The results showed that the intracellular reactive oxygen species were significantly higher in the cells treated with CZ-66 and fluconazole.The antifungal effect of the mixture of these two drugs was weakened after the addition of antioxidant Vitamin C and Glutathione,which showed that the antifungal effect of CZ-66 combined with fluconazole was partially dependent on the increase of ROS.6.The hydrophobicity of Candida albicans cell wall changed significantly under the treatment of CZ-66 and fluconazole in combination.The transmission electron microscope results showed that the fungal cell wall and cell membrane were seriously damaged after the treatment of CZ-66 and fluconazole,which indicated an important mechanism for the synergistic fungicidal effect of CZ-66 and fluconazole.7.In order to further study on the drug target,we used DARTS method to investigate the proteins that might interact with CZ-66.We explored the experimental conditions of interaction between CZ-66 and total protein of Candida albicans,including the concentration of protease,the interaction time between protein and drug,the concentration of gel and the concentration of CZ-66.Finally,a different band appeared between 55 k D and60 k D in the 10 m M CZ-66 treated group.We identified the proteins by mass spectrometry.Through the analysis of peak area,molecular weight,protein bioinformatics and protein function,we preliminarily concluded that Cdc19 p might be a potential target of CZ-66.Then,we tested the antifungal sensitivity of CZ-66 and fluconazole in liquid mediums containing fermentation carbon sources(glucose,sucrose and galactose)and non-fermentation carbon source(glycerin).We found that the synergistic antifungal effect of CZ-66 and fluconazole decreased significantly in medium containing non-fermentation carbon source.It is preliminary verified that CZ-66 may exert antifungal effect by affecting the function of Cdc19 p,and the specific target and mechanism need to be further studied.Conclusions:In this study,compounds CZ-66,FQ-12,FQ-17 and FQ-18,which are synergistic with fluconazole,were obtained by screening of phenazine compounds library and of structural modification of prophase compounds.With the micro-dilution method,we chose a variety of commonly used clinical anti-fungal agents to cooperate with CZ-66.It was found that azoles had synergistic fungicidal activity in combination with CZ-66.In addition,glyceryl monostearate showed showed a synergistic antifungal effect with caspofungin in Candida.To further investigate the antifungal mechanisms of CZ-66,we found that the combination of CZ-66 and fluconazole could increase intracellular ROS,which caused obvious damage to the cell wall and changed the hydrophobicity of the cell surface.Otherwise,in order to further study the drug target of CZ-66,the DARTS method was successfully established and the different bands were found.After mass spectrometry identification,it was preliminarily concluded that Cdc19 p might be the potential target of CZ-66,but the specific mechanism still needed to be further studied.