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. 2015 Dec 15;7(12):2589-602.
eCollection 2015.

The non-Geldanamycin Hsp90 inhibitors enhanced the antifungal activity of fluconazole

Liping Li  1 , Maomao An  1 , Hui Shen  2 , Xin Huang  1 , Xueya Yao  3 , Jian Liu  1 , Fang Zhu  1 , Shiqun Zhang  1 , Simin Chen  1 , Lijuan He  4 , Jundong Zhang  1 , Zui Zou  3 , Yuanying Jiang  5
Affiliations

The non-Geldanamycin Hsp90 inhibitors enhanced the antifungal activity of fluconazole

Liping Li et al. Am J Transl Res. .

Abstract

The molecular chaperone heat shock protein 90 (Hsp90) is highly conserved in eukaryotes and facilitates the correct folding, productive assembly and maturation of a diverse cellular proteins. In fungi, especially the most prevalent human fungal pathogen Candida albicans, Hsp90 influences development and modulates drug resistance. Here, we mainly explore the effect of non-Geldanamycin Hsp90 inhibitor HSP990 on the activity of fluconazole (FLC) against Candida albicans and investigate the underlying mechanism. We demonstrate that HSP990 has potent synergistic antifungal activity with FLC against FLC-resistant C. albicans through the checkerboard microdilution assay,agar diffusion tests and time-kill curves, and shows low cytotoxicity to human umbilical vein endothelial cells. Further study shows that the activity of FLC against C. albicans biofilm formation in vitro is significantly enhanced when used in combination with HSP990. In a murine model of disseminated candidiasis, the therapeutic efficacy of FLC is also enhanced by the pharmacological inhibition of C. albicans Hsp90 function with HSP990. Thus, the combined use of small molecule compound and existing antifungal drugs may provide a potential therapeutic strategy for fungal infectious disease.

Keywords: Hsp90 inhibitors; fluconazole; fungal pathogens; synergistic antifungal activity.

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Figures

Figure 1
Figure 1
Agar disk diffusion assay of different Hsp90 inhibitors (AUY922, E973, HSP990, STA9090) combined with FLC against C. albicans 103. (A and C) show agar plates, and (B) shows an agar plate containing 1 μg/ml FLC. (D) describes the images for (A and B), containing 2 μg of different Hsp90 inhibitors and DMSO as control per disc. (E) describes the image for (C), the combination of different Hsp90 inhibitors (2 μg) with FLC (1 μg) or FLC (1 μg) alone as control were contained in each disc.
Figure 2
Figure 2
Agar disk diffusion assay of different concentrations of HSP990 combined with FLC against C. albicans 103. (A and C) show agar plates, and (B) shows an agar plate containing 1 μg/ml FLC. (D) Describes the images for (A and B), which contain 0.5, 1, 2, 4 μg of HSP990 and just DMSO as control per disc. (E) Describes the image for (C), the combination of HSP990 (0.5, 1, 2, 4 μg) with FLC (1 μg) and DMSO as control were contained in each disc.
Figure 3
Figure 3
Time killing curves of C. albicans 103 treated with HSP990 and FLC. FLC-resistant C. albicans 103 were treated with FLC (4 μg/ml), HSP990 (8 μg/ml and FLC+HSP990 (4+8) μg/ml by using initial inoculums of 105 CFU/ml. Aliquots were obtained at the indicated time points and serially dilutions were spreaded on SDA agar plates. Colony counts were determined after 48 h incubation.
Figure 4
Figure 4
The in vitro cytotoxicity evaluation of Hsp90 inhibitors using the XTT assay. The cytotoxic effect of Hsp90 inhibitors (AUY922, E973, HSP990 and STA9090) towards HUVEC viability was assessed by an XTT test following a 4-h treatment, when compared to that of FLC. **, p<0.01; ***, p<0.001.
Figure 5
Figure 5
The combination of Hsp90 inhibitor HSP990 and FLC inhibits C. albicans biofilm formation in vitro. Effects of different concentrations of HSP990 alone and in combination with 16 μg/ml FLC on C. albicans biofilm formation were evaluated by the XTT reduction assay through calculating the percent of viable C. albicans cell relative the control cells without drug treatment. Data are shown as means ± standard deviation. **, p<0.01; ***, p<0.001 compared with the value of the group treated with FLC alone.
Figure 6
Figure 6
Pharmacological inhibition of Hsp90 inhibitor HSP990 enhances the therapeutic efficacy of FLC in a murine model of disseminated disease. C57BL/6 mice were infected with an inoculum of 200 μl of a 1×106 CFU/ml of FLC-resistant C. albicans 103. 0.5 mg/kg FLC was administered IP and 0.5 mg/kg HSP990 was administered by oral gavages at 1 h after infection (day 1) and then repeated on day 3.
Figure 7
Figure 7
The proteomics of C. albicans 103 treated with Hsp90 inhibitor HSP990 and FLC using LC-MS/MS. (A) Cluster analysis of protein level based on LC-MS/MS data, depicting the differentiation of protein level among samples treated by different drugs (Con: the untreated Control; F16: samples treated by 16 μg/ml FLC; FH: samples treated by 16 μg/ml FLC and 64 μg/ml HSP990). (B) Venn diagram for differential proteins in paired comparisons. Comparison 1: differential proteins identified in FLC-treated sample versus Control sample; Comparison 2: differential proteins identified in FLC+HSP990-treated sample versus Control sample; Comparison 3: differential proteins identified in FLC+HSP990-treated sample versus FLC-treated sample. (C) The interaction network of differential proteins associated with C. albicans Hsp90. Network symbols indicate the classification of these differential proteins from Comparison 3 (FLC+HSP990-treated sample versus FLC-treated sample). (D) The relative protein level of differential proteins compared to Control, corresponding to proteins shown in (C).
Figure 8
Figure 8
The scheme of a proposed model of fluconazole resistance by the activation of diverse signaling pathways. Solid and dashed arrow lines indicate known pathways and putative pathways predicted in the present study, respectively. Under fluconazole exposure, upregulation of CaMsi3 level might have activated the calcineurin signaling pathway or other unknown pathways required for azole resistance. The inhibition of Hsp90 function by HSP990 leaded to significantly reduced levels of CaMsi3 and CaCmd1 which is a component of the calcineurin signaling pathway. we propose that Msi3 functions in azole resistance cooperatively with Hsp90 as a cochaperone through the activation of the calcineurin signaling pathway or some unknown mechanisms.
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References

    1. Gudlaugsson O, Gillespie S, Lee K, Vande Berg J, Hu J, Messer S, Herwaldt L, Pfaller M, Diekema D. Attributable mortality of nosocomial candidemia, revisited. Clin Infect Dis. 2003;37:1172–1177. - PubMed
    1. Horn DL, Neofytos D, Anaissie EJ, Fishman JA, Steinbach WJ, Olyaei AJ, Marr KA, Pfaller MA, Chang CH, Webster KM. Epidemiology and outcomes of candidemia in 2019 patients: data from the prospective antifungal the rapy alliance registry. Clin Infect Dis. 2009;48:1695–1703. - PubMed
    1. Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis. 2004;39:309–317. - PubMed
    1. Morace G, Borghi E. Fungal infections in ICU patients: epidemiology and the role of diagnostics. Minerva Anestesiol. 2010;76:950–956. - PubMed
    1. Lehrnbecher T, Frank C, Engels K, Kriener S, Groll AH, Schwabe D. Trends in the postmortem epidemiology of invasive fungal infections at a university hospital. J Infect. 2010;61:259–265. - PubMed

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