Study On Antifungal Activity And Mechanism Of Novel Compound NT-89

Invasive fungal infections threat to human health more seriously in recent years,of which the main treatment is drug therapy.Antifungal agents include azoles,polyenes and echinocandins are widely used in clinic.However,defects such as increased drug resistance,severe toxic side effects and short half-life period are gradually limiting the clinical use of existing antifungal drugs,and the emergence of multi-drug resistant fungi in recent years has made it urgent to develop novel agents and research for new antifungal targets.Fungal cell wall is the structure distinguished from mammalian cell and plays an important role in the life activities of fungi.The fungal cell wall is composed of a polysaccharide layer mainly constructed with glucan skeleton in the inner layer and an outer glycoprotein layer,which are connected covalently.Major proteins in outer layer are anchored by a glycosylphosphatidylinositol?GPI?remnant to the inner layer of cell wall,called the GPI-anchored cell wall protein?GPI-CWP?,which plays an important role in the life activities of fungi.Thus destruction of fungal cell walls by inhibiting the synthesis of GPI anchors has become a hot targets in the research of antifungal drugs.Our group have previously designed and synthesized a novel pyridine amide derivative NT-89?formerly known as GPI-89?with better antifungal activity based on the structure of GPI biosynthesis inhibitors such as 10b,E1210 and G884,which all target at the Gwp1 protein that perform as an inositol acyltransferase in the early step of GPI biosynthesis pathway.NT-89 has been tested for destroying the cell wall structure of Candida albicans.Based on the previous research,we will further investigat the antifungal activity of NT-89 in vitro and in vivo and the mechanism of action in this study,and then provide reference for the development of new drugs.We first investigated the sensitivity of NT-89 against 41 strains of C.albicans and other fungi by examining the minimum inhibitory concentration?MIC?,in comparison with azoles.The interaction between NT-89 and azole was also examined.The results showed that NT-89 effectively inhibited the growth of C.albicans and was not affected by fluconazole-resistance,with the MIC₈₀ ranged from 0.0625 to 0.5?g/mL.It also showed good activity against other fungi,except dermatophytes.Besides,the combination of NT-89 with fluconazole and other azole drugs mainly exhibited synergistic or additive effects in vitro.In the investigation of the inhibitory effect of NT-89 against virulence factors of C.albicans,we found that low concentrations of NT-89 can significantly inhibit the adhesion,hyphae and biofilm formation.We then examined the in vivo activity of NT-89 alone and in combination with fluconazole by using a murine disseminated candidiasis model.The results showed that NT-89 at a certain therapeutic concentration prolonged the survival time of mice infected with C.albicans,and,it shows synergistic effect when in combination with low concentration of fluconazole in vivo.The therapeutic effects of different routes of administration are different.Next,we verified the target of NT-89 by constructing the GWT1 gene heterozygous mutation?GWT1/gwt1??strain.The results showed that the GWT1/gwt1?strain was more sensitive to NT-89 than the parent strain SN152,and the hyphae growth is more susceptibly inhibited by NT-89,suggesting that GWT1 expression product is the target of NT-89 possibly.By using GC-MS to analyze the sterol component and RT-qPCR to detect the gene expression levels of key enzymes in the sterol biosynthesis pathway of C.albicans,we found that NT-89 also affected the synthesis pathway of ergosterol.The key enzymes are directly or indirectly inhibited,resulting in an increase of sterol intermediates and a decrease in ergosterol biosynthesis.This mechanism contributes to the antifungal effect of NT-89,and may be the reason of synergistic effect between NT-89 and azoles.Finally,we used the quantitative proteomics technique iTRAQ to detect and analyze the changes in the content of C.albicans proteome after NT-89 treatment.The results showed that the content of GPI-anchored protein was decreased after NT-89 treatment,and the differential proteins related to fungal invasion and virulence such as Ywp1p and Pga10p were most significantly reduced.In addition,the content of GPI-anchored protein related to cell wall repair was increased,which suggested that the cell wall structure was destroyed by the loss of glycoprotein on the cell wall surface that triggered the enhancement in the repairing of cell wall through feedback regulation in C.albicans,and the increase of protein synthesis related proteins also indicated that the protein synthesis was enhanced for cell wall repairing.In summary,NT-89 is an effective new antifungal compound with good antifungal activity in vitro and in vivo,as well as the inhibition of virulence factors such as adhesion and hyphae.It can reduce the content of GPI-anchored protein in cell wall surface and inhibit the growth of fungi by inhibiting Gwt1 protein in GPI anchor synthesis pathway.Furthermore,NT-89 can also affect the ergosterol biosynthesis of C.albicans.The specific antifungal mechanism of NT-89 remains to be further studied.