Abstract
Background and Purpose:
The high morbidity and mortality caused by invasive mold infections require new and effective treatment strategies. Heat shock proteins, which are found in all living organisms, play a role in the homeostatic control of the cell and the stress response mediated by calcineurin. Their release increases especially under stress conditions, and they play a role in ensuring the stability of cellular proteins. Therefore, inhibition of Hsp90 or calcineurin may be an effective method in antifungal therapy. This study aimed to evaluate the in vitro activity of four different antifungal agents (caspofungin, amphotericin B, itraconazole, and voriconazole) in combination with fungal stress response regulators, Hsp90 inhibitors, and calcineurin inhibitors, against clinical isolates of Aspergillus, Rhizopus, and Fusarium.
Materials and Methods:
In this study, the effectiveness of Hsp90 inhibitors geldanamycin, 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), radicicol, novobiocin (NOV), and calcineurin inhibitors cyclosporine, tacrolimus (TAC), and rapamycin (RAP) combined with common antifungals itraconazole (ITRA), voriconazole (VOR), caspofungin (CAS), and amphotericin B (AMB) were investigated against four Aspergillus, three Rhizopus, and three Fusarium isolates using checkerboard method.
Results:
The minimum inhibitory concentration (MIC)/minimum effective concentration (MEC) values of ITRA, VOR, CAS, and AMB were ≤ 0.25, ≤ 0.06-0.125, ≤ 0.03-> 4, and 1-4 µg/mL for Aspergillus spp.; 2-8, > 4, > 4, and 2 µg/mL for Rhizopus spp.; 8- > 16, 1-4, > 4, and 2-4 µg/mL for Fusarium spp., respectively. Although tacrolimus was found to have generally low MIC values (≤0.03 µg/mL) for Aspergillus and Rhizopus isolates, NOV, and 17-AAG did not exhibit antifungal activity (MICs>128 and ≥16 µg/mL, respectively) against almost all of the isolates. In combination testing against Aspergillus and Rhizopus spp., synergistic interactions were prevalent (≥75%) for the combinations of ITRA and all inhibitory substances, except for TAC. The effects of CAS and TAC in combination tests were weak. Moreover, synergistic interactions were not frequent in all combinations against Fusarium spp. However, antagonistic interaction was observed only in one ITRA and RAP combination throughout this study.
Conclusion:
The Hsp90 and calcineurin inhibitors did not have significant antifungal activity alone. Moreover, they did not show a significant antagonistic effect in combination and even increased the efficacy of antifungals at some concentrations.
Keywords: Antifungal combination, Aspergillus, Fusarium, Hsp90/calcineurin inhibitors, Rhizopus
Introduction
Invasive mold infections are less common than superficial fungal infections, but their incidence is increasing due to the limited availability of treatment options and the growing problem of resistance [ 1 , 2 ]. Aspergillus fumigatus, followed by Aspergillus flavus are the most common causes of invasive aspergillosis, which has a high mortality rate. Genera of Fusarium and Rhizopus, which include species intrinsically resistant to most antifungal agents, are opportunistic fungal pathogens in immunocompromised individuals [ 3 - 5 ]. In particular, F. oxysporum is one of the most common species in human fusariosis [ 6 ]. Mucorales infections (mucormycosis) are rare but serious fungal infections with a fulminant course and high mortality rates. Their most common agent is Rhizopus oryzae.
Successful management of these infections relies on early diagnosis and appropriate antifungal therapy. Currently, there are three main groups of antifungal agents for the treatment of invasive fungal infections. However, emerging resistance in Aspergillus species and intrinsically antifungal resistance in species of Rhizopus and Fusarium have further limited antifungal treatment options. Therefore, there is an increasing need to investigate new treatment approaches for the treatment of invasive mold infections [ 7 - 9 ].
Heat shock proteins (Hsps), which can be found everywhere in the cell and are classified according to their molecular weight, facilitate the survival of the organism by responding to stress-related changes [ 10 , 11 ]. Hsp90 is a highly conserved molecular chaperone and stimulates the development of drug resistance and antifungal drug tolerance in Candida and Aspergillus species by multiple molecular mechanisms. Therefore, Hsp90 is necessary for all eukaryotes and regulates the form and function of various client proteins that are key signal transducers. Various animal model studies have reported that the inhibition of Hsp90 function restores the sensitivity in drug-resistant fungal pathogens and prevents the development of drug resistance [ 12 , 13 ]. Hence, targeting a highly conserved protein that plays a vital role in many cellular signaling pathways reduces the resistance to Hsp inhibitors [ 14 ]. Therefore, signal transduction mechanisms, including key secondary messenger calcium, which is important for the adaptation and survival of fungi to the environment, have garnered significant attention.
Calcineurin is one of the important regulators of intracellular calcium homeostasis in some fungi, and the calcineurin signal cascade is associated with antifungal resistance. A combination of antifungal agents and calcineurin inhibitors has shown inhibitory effects on the growth of drug-resistant fungal strains. It is reported that the combined use of calcineurin inhibitor tacrolimus (FK506) with caspofungin shows a synergistic fungicidal activity against echinocandin-resistant C. dubliniensis. In addition, the combination of tacrolimus and azole has shown synergistic antifungal activity against azole-resistant C. albicans [ 15 - 17 ]. The present study aimed to evaluate the in vitro activity of the combinations of four different antifungals with fungal stress response regulators, Hsp90 inhibitors, and calcineurin inhibitors, against clinical isolates of Aspergillus, Rhizopus and Fusarium.
Materials and Methods
Isolates and media
In total, 10 clinical mold isolates, namely Aspergillus fumigatus (n=3), A. flavus (n=1), Fusarium oxysporum (n=3), and Rhizopus oryzae (n=3) isolated from respiratory and biopsy specimens were included. These isolates have been previously identified by conventional and molecular methods [ 18 ]. Candida krusei ATCC 6258 and C. parapsilosis ATCC 22019 quality control strains recommended by CLSI M38-A3 were used to determine the antifungal susceptibility of filamentous fungi. In this protocol, the minimum inhibitory concentration (MIC) range of quality control (QC) strains is defined for each drug. Since the accuracy of the applied test is evaluated according to the response of each drug to QC strains, yeast-like fungi can also be used as QC in the susceptibility of filamentous fungi [ 19 , 20 ]. Sabouraud dextrose agar (SDA, Merck, Darmstadt, Germany) was used for the pure culture of isolates, and RPMI 1640 medium (Sigma Chemical Co, St Louis, MO, USA) buffered by MOPS (3-N-morpholinopropanesulfonic acid was used for broth microdilution testing and checkerboard dilution method. Before testing, fresh cultures of all isolates were provided using potato dextrose agar (PDA, Merck, Darmstadt, Germany) at 35 °C.
Antifungal agents and inhibitory substances
Stock solutions of voriconazole (VOR, Sigma Chemical Co, St Louis, MO, USA), itraconazole (ITRA, Sigma), and amphotericin B (AMB, Sigma) were prepared in dimethyl sulfoxide (DMSO) at 1,600 μg/mL concentrations while caspofungin (CAS, Sigma) was prepared in distilled water at 1,600 μg/mL concentrations. Based on our previous studies and the literature data presented below, non-antifungal drug concentrations were determined [ 21 - 24 ]. The Hsp90 inhibitors geldanamycin (GELD), 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), radicicol (RDC), novobiocin (NOV), and calcineurin inhibitors cyclosporine (CICA), tacrolimus (TAC), and rapamycin (RAP) were used as stress response regulators. Stock solutions of TAC, 17-AAG, GELD, RAP (InvivoGen, California, USA), CICA, and RDC (Sigma) were prepared in DMSO at 1,600 µg/mL concentrations. Moreover, NOV (Sigma) was prepared in distilled water at 5,120 μg/mL concentration. All stock solutions were portioned and stored at -70 °C until use.
Antifungal susceptibility testing
Antifungal susceptibility testing was performed by broth microdilution method according to Clinical and Laboratory Standards Institute M38-A3 documents [ 25 - 27 ]. Briefly, conidial inoculum suspensions were prepared from 3-5-day-old sporulated cultures on PDA by using sterile 0.85% saline. The final inoculum concentration was adjusted to 0.4-5 × 104 CFU/mL in RPMI 1640. Sterile, U-bottom, 96-well microplates were used for microdilution testing. Final concentrations of antifungals ranged from 0.0313 to 16 µg/mL for AMB, ITRA, and VOR, and 0.015 - 8 μg/mL for CAS. Final concentrations of inhibitor substances were within the range of 0.03-16 µg/mL for TAC, 17-AAG, GELD, and CICA; 0.008-4 μg/mL for RDC and RAP; and 0.25-128 μg/mL for NOV. The first column, including the drug-free medium, was designed as the growth control wells. The microplates were covered with sterile parafilm and incubated at 35°C. Results were evaluated visually after 24 h incubation for R. oryzae and 48 h incubation for other strains. The MIC values were read as the first well in which visible growth was completely inhibited for AMB, ITRA, and VOR. Minimum effective concentration (MEC) value for caspofungin was evaluated microscopically as the lowest drug concentration causing significant shortening and blunting at hyphae. For other substances, the presence of any inhibition in growth (50% or more) was evaluated.
Combination testing: checkerboard method
The concentration ranges in the checkerboard combination method were determined according to MIC results obtained from susceptibility tests; the concentrations ranged from 1/32 to 8x MIC for antifungal agents and from 1/8 to 8x MIC for inhibitor substances. Dilutions of drugs and substances were prepared in RPMI 1640 medium at four times the final concentrations in the microplates. A total of 50 µL of each solution was dispensed, with antifungals placed in the rows and inhibitor substances in the columns of a 96-well microplate. Fungal inoculums were prepared and inoculated to all wells as described above and incubated at 35°C. The results were visually read after 24 h for R. oryzae and 48 h for other strains. When the absence of adequate growth in the growth control well was observed, the incubation was extended more than 24 h [ 28 ]. In order to evaluate the activity of the combinations, the fractional inhibitor concentration index (FICI) was calculated as follows: MICA in combination/MICA alone + MICB in combination/MICB alone. Drug interactions were defined as synergistic (FICI≤0.5), no interaction/indifference (0.5<FICI < 4), or antagonistic (FICI≥4), [ 29 , 13 ]. Off-scale MIC values were converted to the next highest two-fold concentration.
Results
The MIC/MEC results of drugs and substances for each isolate are presented in Table 1. The lowest MIC/MEC values for antifungals were exhibited against Aspergillus spp. While the lowest MIC ranges for isolates of R. oryzae were observed with AMB (2.0 µg/mL) as expected, the highest MIC/MEC ranges for all drugs were detected against F. oxysporum. The MIC values of the reference isolates were acceptable. Among the inhibitors, the most apparent effect was observed in TAC, especially against Aspergillus and Rhizopus isolates (≤0.03 µg/mL). Although RAP and CICA had MIC values against almost all isolates, GELD, 17-AAG, and RDC had limited activity, and NOV did not exhibit any antifungal activity.
Table 1.
In vitro antifungal activities of antifungals and Hsp90/calcineurin inhibitors used alone against clinical isolates of Aspergillus, Rhizopus, and Fusarium
| Isolates | MIC/MEC values of drugs (µg/mL) | MIC values of inhibitory substances (µg/mL) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| ITRA | VOR | CAS | AMB | RAP | TAC | CICA | GELD | RDC | NOV | AAG | |
| A. fumigatus 1342 | ≤0.25 | ≤0.06 | ≤0.03 | 2 | 1 | ≤0.03 | 1 | 16 | >4 | >128 | >16 |
| A. fumigatus 4373 | ≤0.25 | ≤0.06 | >4 | 1 | 1 | ≤0.03 | 2 | 16 | >4 | >128 | >16 |
| A. fumigatus 1345 | ≤0.25 | ≤0.06 | ≤0.03 | 4 | >4 | >16 | 4 | >16 | 4 | >128 | >16 |
| A. flavus 1343 | ≤0.25 | 0.125 | ≤0.03 | 2 | 1 | ≤0.03 | >16 | 16 | >4 | >128 | >16 |
| R.oryzae YDH | 8 | >4 | >4 | 2 | >4 | ≤0.03 | 4 | 16 | 4 | >128 | 16 |
| R.oryzae 1373 | 4 | >4 | >4 | 2 | 4 | ≤0.03 | 1 | >16 | 4 | >128 | >16 |
| R.oryzae 8204 | 2 | >4 | >4 | 2 | 0.5 | ≤0.03 | 2 | 8 | 0.125 | >128 | 4 |
| F.oxysporum-5028 | >16 | 4 | >4 | 2 | 2 | 4 | >16 | 8 | >4 | >128 | >16 |
| F.oxysporum 1384 | >16 | 1 | >4 | 4 | 64 | 8 | >16 | 8 | >4 | >128 | >16 |
| F.oxysporum 6683 | 8 | 1 | >4 | 2 | 1 | >16 | 8 | 16 | 4 | >128 | >16 |
MIC: minimum inhibitory concentration, MEC: minimum effective concentration, ITRA, itraconazole; VOR, voriconazole; CAS, caspofungin; AMB, amphotericin B; RAP, rapamycin; TAC, tacrolimus; CICA, cyclosporin A; GEL, geldanamycin; RDC, radicicol; NOV, novobiocin; AAG, 17-allylamino-17-demethoxygeldanamycin.
The checkerboard testing results are summarized in Table 2 and Supplementary Table 1. A total of 280 combination tests (28 different combination tests for each isolate) were studied. Antagonistic interaction was observed for only one combination (RAP+ITRA) against A. fumigatus-4373 isolate. Moreover, 187 (67%) combinations resulted in indifference and 92 (33%) showed synergistic interaction. Especially, the combination of RAP + ITRA resulted in synergistic interaction against eight isolates (Supplementary Table 1). In general, the most promising results were observed for the combinations of inhibitory substances with ITRA, followed by their combinations with AMB (Table 2). While MICs of ITRA alone were high (2->16 µg/mL) for almost all isolates (except Aspergillus spp.), significant decreases were observed in combination with inhibitory substances (except for Fusarium spp.), often resulting in synergy (Supplementary Table 1). It should be mentioned that similar results were obtained for AMB. However, synergistic interactions were not apparent for the combinations of inhibitory substances with CAS. A noteworthy observation was that while CAS MICs were > 4 µg/mL for Rhizopus oryzae isolates in combination with TAC, it resulted in synergistic interactions for all three isolates.
Table 2.
Rate of isolates resulting in synergistic interaction according to checkerboard combination tests (number of isolates resulting in synergistic interaction/total number of isolates).
| Inhibitory substances | Aspergillus species (n=4) | Rhizopus oryzae (n=3) | Fusarium oxysporum (n=3) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ITRA | VOR | CAS | AMB | ITRA | VOR | CAS | AMB | ITRA | VOR | CAS | AMB | |
| RAP | 0.75 | 0.25 | 0 | 0.50 | 1.00 | 0.33 | 0 | 0.66 | 0.66 | 0.33 | 0 | 0 |
| TAC | 0 | 0 | 0 | 0.25 | 0 | 0 | 1.00 | 0.33 | 0 | 0.33 | 0.33 | 0 |
| CICA | 0.75 | 0.25 | 0 | 0.25 | 0.66 | 0 | 0 | 1.00 | 0 | 0.33 | 0.33 | 0.33 |
| GELD | 1.00 | 0.50 | 0 | 1.00 | 0.66 | 0.33 | 0 | 0.66 | 0 | 0.33 | 0.33 | 0.33 |
| RDC | 0.75 | 0.25 | 0 | 0.5 | 1.00 | 0 | 0 | 0.33 | 0 | 0 | 0.33 | 0 |
| NOV | 0.75 | 0.50 | 0 | 0.75 | 0.33 | 0 | 0 | 0.33 | 0.33 | 0.33 | 0.33 | 0 |
| 17-AAG | 1.00 | 0.50 | 0.25 | 0.25 | 0.66 | 0 | 0 | 0.66 | 0 | 0.33 | 0 | 0.66 |
ITRA: itraconazole; VOR: voriconazole; CAS: caspofungin; AMB: amphotericin B; RAP: rapamycin; TAC: tacrolimus; CICA: cyclosporin A; GEL: geldanamycin; RDC: radicicol; NOV: novobiocin; 17-AAG: 17-allylamino-17-demethoxygeldanamycin.
In addition, the rate of synergistic interactions for all combinations was the lowest against F. oxysporum isolates.
Discussion
Increasing frequency of invasive fungal infections and the emerging problem of antifungal resistance have increased interest in the studies on new treatment alternatives and combination therapy [ 30 , 31 ]. In particular, combinations of antifungal agents with Hsp and calcineurin inhibitors are promising. In this study, combinations of some Hsp90 calcineurin inhibitors with commonly used antifungals were evaluated against Aspergillus, Rhizopus, and Fusarium isolates.
In literature, it is reported that compounds acting on Hsp90 and calcineurin have different activities on different fungal species. Lamoth et al. investigated the antifungal activity of some components targeting the Hsp90-calcineurin pathway against 62 mold species. In their study, TAC showed variable activities on different Aspergillus species (MIC range: 0.025-0.4 μg/mL for A. fumigatus and A. flavus isolates) and was effective on Mucor species (MIC range: 0.012->0.4 μg/mL) while GELD was found to be effective on Fusarium species [ 32 ]. Similarly, in the present study, the most prominent effect was obtained with TAC. This effect is very pronounced, especially in Aspergillus and Rhizopus isolates and data are consistent with literature.
According to the combination test results in the present study, synergistic interaction was frequently observed in the combinations of GELD and 17-AAG with ITRA or AMB, especially against Aspergillus and Rhizopus isolates. In addition, CICA-AMB, TAC-CAS, and RDC-ITRA showed synergistic interaction against all R. oryzae isolates, and no antagonism was found in any of the combinations. These results are in accordance with the literature [ 33 ]. In addition, a synergistic interaction was detected against all Aspergillus isolates (n=4) in the combination of GELD and AMB, GELD and ITRA, as well as 17-AAG and ITRA. However, no combinations showing synergistic interactions against all Fusarium isolates were observed. Although it has been reported in the literature that RAP shows synergy with various antifungals, this combination was generally found to be indifferent in the present study. Moreover, the RAP-ITRA combination for one isolate (A. flavus) resulted in an antagonistic interaction.
The main goal of echinocandin drugs is to disrupt the stability of the cell wall. Especially in A. fumigatus, it is reported that calcineurin inhibitors increase echinocandin activity and transform the fungistatic activity of caspofungin into fungicidal activity. In addition, according to previous studies, these inhibitors show potent activity against azole and echinocandin-resistant A. fumigatus isolates [ 34 , 35 ]. These findings are consistent with those of the present study as CAS-TAC resulted in synergy in all isolates. These data may provide insights for further studies.
The RDC also binds to Hsp90 with strong affinity and inhibits ATP-dependent chaperone function. In the present study, when RDC was used alone, it showed the most significant antifungal effect against R. oryzae isolates (MIC range: 0.125-4 µg/ml). There was no significant antifungal effect against other mold isolates at the studied concentrations. Although the majority of combinations of RDC with antifungal agents showed indifferent efficacy, it is noteworthy that none of the 40 evaluated combinations showed antagonism. It was also promising that all combinations of RDC+ITRA, two combinations of RDC+AMB, and one combination of VOR+RDC showed synergy against isolates of A. fumigatus and R. oryzae.
The NOV is an effective coumarin antibiotic against Gram-positive bacteria, and it binds to the C terminal of Hsp90, causing these proteins to degrade [ 36 ]. As with the other Hsp90 inhibitors, it is thought that NOV can enhance the effectiveness of antifungals. Although the antifungal activity of a chemical derivative of NOV alone, which is 1,000 times more potent, has been demonstrated [ 37 ], the contribution of the original NOV to antifungals has not been fully elucidated yet. In the present study, NOV alone did not show any activity against isolates (MIC>128 µg/ml for all isolates). However, synergistic interactions observed in NOV-antifungal combinations were most prominent, especially in NOV-ITRA combinations.
Substances that act through the inhibition of Hsp90 and calcineurin enhance the effectiveness of antifungals, possibly by reducing the stress response that occurs in fungal cells exposed to antifungal agents, thereby decreasing resistance to antifungals. Therefore, they can create a promising combination strategy against mold isolates. However, since these inhibitors do not have fungal specificity, their use as antifungals is limited.
Conclusion
Considering all 28 combination tests for each isolate, a negligible number of antagonistic interactions were observed. The synergistic interaction obtained for many combinations may be promising in the management of invasive fungal infections, especially considering the known resistance issues for the fungi included in this study. The most important limitation of this study was the small number of isolates. However, due to the inclusion of a large number of inhibitory substances, the obtained results can serve as a guide for planning future studies.
Acknowledgments
This work was supported by Türkiye Bilimsel ve Teknolojik Araştirma Kurumu (TUBITAK) with Project Number 112S503).
Authors’ contributions
All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by D. T., A. B., and S. Ş. First draft of the manuscript was written by İ. D., Y. Ö., and N. K. All authors commented on previous versions of the manuscript and also read and approved the final manuscript.
Conflicts of interest
The authors declare that there are no conflicts of interest.
Financial disclosure
No financial disclosure was provided.
References
- 1.Whitesell L, Robbins N, Huang DS, McLellan CA, Shekhar-Guturja T, LeBlanc EV, et al. Structural basis for species-selective targeting of Hsp90 in a pathogenic fungus. Nat Commun. 2019; 10(1):402. doi: 10.1038/s41467-018-08248-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Lee SO. Diagnosis and treatment of invasive mold diseases. Infect Chemother. 2023; 55(1): 10–21. doi: 10.3947/ic.2022.0151. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Rudramurthy SM, Paul RA, Chakrabarti A, Mouton JW, Meis JF. Invasive aspergillosis by Aspergillus flavus: epidemiology, diagnosis, antifungal resistance, and management. J Fungi (Basel). 2019; 5(3):55. doi: 10.3390/jof5030055. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Ahmadi B, Hashemi SJ, Zaini F, Shidfar MR, Moazeni M, Mousavi B, et al. A case of onychomycosis caused by Aspergillus candidus. Med Mycol Case Rep. 2012; 1(1):45–48. doi: 10.1016/j.mmcr.2012.06.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Martínez-Herrera EO, Arroyo-Camarena S, Tejada-García DL, Porras-López CF, Arenas R. Onychomycosis due to opportunistic molds. An Bras Dermatol. 2015; 90(3):334–337. doi: 10.1590/abd1806-4841.20153521. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Batista BG, Chaves MA, Reginatto P, Saraiva OJ, Fuentefria AM. Human fusariosis: an emerging infection that is difficult to treat. Rev Soc Bras Med Trop. 2020; 53:e20200013. doi: 10.1590/0037-8682-0013-2020. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Rojas R, Molina JR, Jarque I, Montes C, Serrano J, Sanz J, et al. Outcome of antifungal combination tTherapy for invasive mold infections in hematological patients is independent of the chosen combination. Mediterr J Hematol Infect Dis. 2012; 4(1):e2012011. doi: 10.4084/MJHID.2012.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Peri AM, Verna M, Biffi S, Alagna L, Longhi B, Migliorino GM, et al. A. Combination antifungal therapy for invasive mold Infections among pediatric patients with hematological malignancies: data from A real-life case-series. Pathog Immun. 2019; 4(2):180–194. doi: 10.20411/pai.v4i2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Wattier RL, Dvorak CC, Hoffman JA, Brozovich AA, Bin-Hussain I, Groll AH, et al. A prospective, international cohort study of invasive mold infections in children. J Pediatric Infect Dis Soc. 2015; 4(4):313–322. doi: 10.1093/jpids/piu074. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Tiwari S, Thakur R, Shankar J. Role of heat-shock proteins in cellular function and in the biology of fungi. Biotechnol Res Int. 2015; 2015 doi: 10.1155/2015/132635. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Åkerfelt M, Morimoto RI, Sistonen L. Heat shock factors: integrators of cell stress, development and lifespan. Nat. Rev. Mol. Cell Biol. 2010; 11(8): 545–555. doi: 10.1038/nrm2938. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Cowen LE, Singh SD, Köhler JR, Collins C, Zaas AK, Schell WA, et al. Harnessing Hsp90 function as a powerful, broadly effective therapeutic strategy for fungal infectious disease. Proc Natl Acad Sci U S A. 2009; 106(8):2818–2823. doi: 10.1073/pnas.0813394106. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Singh SD, Robbins N, Zaas AK, Schell WA, Perfect JR, Cowen LE. Hsp90 governs echinocandin resistance in the pathogenic yeast Candida albicans via calcineurin. PLoS Pathog. 2009; 5(7):e1000532. doi: 10.1371/journal.ppat.1000532. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Wirk B. Heat shock protein inhibitors for the treatment of fungal infections. Recent Pat Antiinfect Drug Discov. 2011; 6(1):38–44. doi: 10.2174/157489111794407840. [DOI] [PubMed] [Google Scholar]
- 15.Sun S, Li Y, Guo Q, Shi C, Yu J, Ma L. In vitro interactions between tacrolimus and azoles against Candida albicans determined by different methods. Antimicrob Agents Chemother. 2008; 52(2):409–417. doi: 10.1128/AAC.01070-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Zhang J, Heitman J, Chen YL. Comparative analysis of calcineurin signaling between Candida dubliniensis and Candida albicans. Commun Integr Biol. 2012; 5(2):122–126. doi: 10.4161/cib.18833. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Zhang M, Yang X, Wang D, Yu C, Sun S. Antifungal activity of immunosuppressants used alone or in combination with fluconazole. J Appl Microbiol. 2019;126(5):1304–1317. doi: 10.1111/jam.14126. [DOI] [PubMed] [Google Scholar]
- 18.Kiraz N, Oz Y, Aslan H, Erturan Z, Ener B, Akdagli SA, Muslumanoglu H, Cetinkaya Z. Is the extraction by Whatman FTA filter matrix technology and sequencing of large ribosomal subunit D1-D2 region sufficient for identification of clinical fungi? Mycoses. 2015; 58(10):588–597. doi: 10.1111/myc.12365. [DOI] [PubMed] [Google Scholar]
- 19.Ghazanfari M, Abastabar M, Haghani I, Moazeni M, Hedayati S, Yaalimadad S, et al. Azole-containing agar plates and antifungal susceptibility testing for the detection of azole-resistant Aspergillus species in hospital environmental samples. Microb Drug Resist. 2023; 29(12): 561–567. doi: 10.1089/mdr.2023.0002. [DOI] [PubMed] [Google Scholar]
- 20.Yang X, Chen W, Liang T, Tan J, Liu W, Sun Y, et al. A 20-year antifungal susceptibility surveillance (from 1999 to 2019) for Aspergillus spp. and proposed epidemiological cutoff values for Aspergillus fumigatus and Aspergillus flavus: a study in a tertiary hospital in China. Front Microbiol. 2021; 12: 680884. doi: 10.3389/fmicb.2021.680884. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Zhang J, Tan J, Yang L, He Y. Tacrolimus, not triamcinolone acetonide, interacts synergistically with itraconazole, terbinafine, bifonazole, and amorolfine against clinical dermatophyteisolates. J Mycol Med. 2018;28(4):612–616. doi: 10.1016/j.mycmed.2018.09.003. [DOI] [PubMed] [Google Scholar]
- 22.Méndez Galomo KS. Evaluación de la actividad antifúngica de la sertralina en un modelo murino de aspergilosis pulmonar invasiva (Master's thesis), Universidad Autónoma de Nuevo León. 2018 [Google Scholar]
- 23.Kiraz N, Şen Kaya S, Öz Y, Dağ İ. Evaluation of the efficacy of heat shock protein inhibitors and antifungal drug combinations against Candida spp. Rendiconti Lincei. Scienze Fisiche e Naturali. 2023; 34(1): 179–188. [Google Scholar]
- 24.Khan MSA, Ahmad I. Antifungal activity of essential oils and their synergy with fluconazole against drug-resistant strains of Aspergillus fumigatus and Trichophyton rubrum. Appl Microbiol Biotechnol. 2011; 90(3): 1083–1094. doi: 10.1007/s00253-011-3152-3. [DOI] [PubMed] [Google Scholar]
- 25.CLSI. Reference method For broth dilution antifungal susceptibility testing of filamentous fungi; 3rd edition. CLSI standard M38. Wayne, PA: Clinical and Laboratory Standards Institute; 2017. [Google Scholar]
- 26.Borman AM, Fraser M, Palmer MD, Szekely A, Houldsworth M, Patterson Z, et al. MIC distributions and evaluation of fungicidal activity for amphotericin B, itraconazole, voriconazole, posaconazole and caspofungin and 20 species of pathogenic filamentous fungi determined using the CLSI broth microdilution method. J Fungi. 2017; 3(2):27. doi: 10.3390/jof3020027. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Lamoth F, Juvvadi PR, Gehrke C, Steinbach WJ. In vitro activity of calcineurin and heat shock protein 90 inhibitors against Aspergillus fumigatus azole-and echinocandin-resistant strains. Antimicrob Agents Chemother. 2013; 57(2): 1035–1039. doi: 10.1128/AAC.01857-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Chaturvedi V, Ramani R, Ghannoum MA, Killian SB, Holliday N, Knapp C, et al. Multilaboratory testing of antifungal combinations against a quality control isolate of Candida krusei. Antimicrob Agents Chemother. 2008; 52(4): 1500–1502. doi: 10.1128/AAC.00574-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Şen Kaya S, Kiraz N, Bariş A, Turan D, Öz Y, Dağ İ, et al. Effects of calcineurin inhibitors, cyclosporine A and tacrolimus (FK506), on the activity of antifungal drugs against Candida spp. J Med Microbiol. 2021; 70(4): 001354. doi: 10.1099/jmm.0.001354. [DOI] [PubMed] [Google Scholar]
- 30.Douglas AP, Chen SC, Slavin MA. Emerging infections caused by non-aspergillus filamentous fungi. Clin Microbiol Infect. 2016; 8:670–680. doi: 10.1016/j.cmi.2016.01.011. [DOI] [PubMed] [Google Scholar]
- 31.McCarthy MW, Petraitis V, Walsh TJ. Combination therapy for the treatment of pulmonary mold infections. Expert Rev Respir Med. 2017; 11(6):481–489. doi: 10.1080/17476348.2017.1325322. [DOI] [PubMed] [Google Scholar]
- 32.Lamoth F, Alexander BD, Juvvadi PR, Steinbach WJ. Antifungal activity of compounds targeting the Hsp90-calcineurin pathway against various mould species. J Antimicrob Chemother. 2015; 70(5):1408–1411. doi: 10.1093/jac/dku549. [DOI] [PubMed] [Google Scholar]
- 33.Mahmoudi S, Rezaie S, Daie Ghazvini R, Hashemi SJ, Badali H, Foroumadi A, et al. In vitro interaction of geldanamycin with triazoles and echinocandins against common and emerging Candida species. Mycopathologia. 2019; 184:607–613. doi: 10.1007/s11046-019-00370-7. [DOI] [PubMed] [Google Scholar]
- 34.Johnson MD, Perfect JR. Use of antifungal combination therapy: agents, order, and timing. Curr Fungal Infect Rep. 2010; 4(2):87–95. doi: 10.1007/s12281-010-0018-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Spitzer M, Robbins N, Wright GD. Combinatorial strategies for combating invasive fungal infections. Virulence. 2017; 8(2):169–185. doi: 10.1080/21505594.2016.1196300. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Donnelly A, Blagg BS. Novobiocin and additional inhibitors of the Hsp90 C-terminal nucleotide-binding pocket. Curr Med Chem. 2008; 15(26):2702–2717. doi: 10.2174/092986708786242895. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Tian SZ, Pu X, Luo G, Zhao LX, Xu LH, Li WJ, et al. Isolation and characterization of new p-Terphenyls with antifungal, antibacterial, and antioxidant activities from halophilic actinomycete Nocardiopsis gilva YIM 90087. J Agric Food Chem. 2013; 61(12):3006–3012. doi: 10.1021/jf400718w. [DOI] [PubMed] [Google Scholar]