Abstract
Invasive fungal infections, including those caused by yeasts, moulds and endemic organisms, can be significant causes of morbidity and mortality in immunocompromised hosts, those with multiple comorbidities and occasionally immunocompetent hosts. Current antifungal agents are often limited by drug toxicities, drug interactions or the development of resistance. VT-1598 is a novel tetrazole that has greater specificity for fungal Cyp51 than currently available triazoles and thus the potential for clinically significant drug interactions is reduced. We measured the in vitro activity of VT-1598 against clinical isolates of Candida and Cryptococcus species, endemic fungi, including Coccidioides, Blastomyces and Histoplasma, Aspergillus species and Rhizopus arrhizus.
Antifungal susceptibility testing was performed by broth microdilution or macrodilution methods per CLSI standards. Clinical isolates of each species were used and clinically available antifungal agents were tested against each isolate.
VT-1598 demonstrated in vitro activity against yeasts and moulds that was similar to or greater than that of clinically available antifungal agents, including amphotericin B, fluconazole, caspofungin, voriconazole and posaconazole. The in vitro activity of VT-1598 was also maintained against resistant isolates, including fluconazole-resistant Candida isolates. In vitro activity was also observed against endemic fungi, including Blastomyces, Histoplasma and both Coccidioides immitis and Coccidioides posadasii.
VT-1598 demonstrated in vitro activity against yeasts, moulds and endemic fungi, which was maintained against isolates that had reduced susceptibility to other antifungals. Further studies are warranted to evaluate the in vivo efficacy of VT-1598 against various fungal pathogens.
Introduction
The long-term use of clinically available antifungal agents may be limited by several factors, including drug–drug interactions and adverse reactions/toxicities.1–3 VT-1598 is a novel tetrazole-based inhibitor of fungal Cyp514,5 that disrupts the ergosterol biosynthetic pathway by preventing the conversion of lanosterol to ergosterol, similar to the imidazole- and triazole-based Cyp51 inhibitors. However, due to its unique chemical structure, this agent is significantly more specific for fungal Cyp51 compared with mammalian cytochrome P450 enzymes.5 Thus, the potential for drug–drug interactions, which can be clinically significant for the triazoles, such as fluconazole, voriconazole, posaconazole and isavuconazole, may be avoided. Our objective was to evaluate the in vitro activity of VT-1598 against different yeast and mould species, as well as endemic fungi.
Materials and methods
Isolates
Clinical isolates of Candida species (Candida albicans, Candida glabrata, Candida parapsilosis and Candida krusei), Cryptococcus species (Cryptococcus neoformans and Cryptococcus gattii), Aspergillus species (Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus), Rhizopus arrhizus, Coccidioides species (Coccidioides immitis and Coccidioides posadasii), Blastomyces dermatitidis and Histoplasma capsulatum received by the Fungus Testing Laboratory at the University of Texas Health Science Center at San Antonio were used in this study. The species identification of each isolate had been previously confirmed by DNA sequence analysis of various loci (ITS and D1/D2 regions of rRNA for Candida, Cryptococcus and Rhizopus species, and β-tubulin and calmodulin genes for Aspergillus species) or via a luminescent DNA probe for the dimorphic fungi (GenProbe, Hologics). Coccidioides isolates were determined to the species level (C. immitis or C. posadasii) by ITS barcode analysis.6 Isolates were subcultured onto Sabouraud dextrose agar (yeasts) or potato flake agar (moulds and endemic fungi) prior to susceptibility testing.7
Antifungals and susceptibility testing
VT-1598 was provided by Viamet Pharmaceuticals, Inc. (Durham, NC, USA) and amphotericin B, fluconazole, voriconazole, posaconazole and caspofungin were purchased from Sigma–Aldrich (St Louis, MO, USA). Stock solutions were made in DMSO and stored at −80 °C and further dilutions in RPMI with 0.165 M MOPS (pH 7.0) such that the final DMSO concentration following inoculation was 1% (v/v). Antifungal susceptibility testing was performed by broth microdilution or macrodilution methods according to the CLSI M27-A3 or M38-A2 reference standards for yeasts and moulds, respectively.8,9 The concentration ranges tested for VT-1598 were 0.002–1 mg/L against C. albicans and Cryptococcus species, 0.015–8 mg/L against other Candida species and 0.03–16 mg/L against moulds and endemic fungi. The concentrations for the other antifungals were 0.125–64 mg/L for fluconazole, 0.015–8 mg/L for caspofungin and 0.03–16 mg/L for amphotericin B, posaconazole and voriconazole. For Candida and Cryptococcus species, the MICs of VT-1598, fluconazole and caspofungin (Candida only) were measured as the lowest concentration that resulted in 50% inhibition of growth compared with the growth controls after 24 and 72 h of growth, respectively. For further characterization, MICs of VT-1598 for Candida and Cryptococcus were also measured at 100% inhibition of growth after 24 h. For Aspergillus species and R. arrhizus, VT-1598 and posaconazole MICs were read as 100% inhibition after 48 and 24 h of growth, respectively. For B. dermatitidis, Coccidioides species and H. capsulatum MICs were read at 80% inhibition of growth after 48–168 h of incubation. Amphotericin B MICs were read as 100% inhibition of growth for all isolates tested.
Data analysis
The MIC ranges and geometric mean (GM) MIC values were determined. MIC values greater than the highest concentration tested were assigned a value one dilution higher for statistical purposes. Differences in GM MICs, calculated following log2 transformation of individual MIC values, were assessed for significance by ANOVA with Tukey’s post-test for multiple comparisons. A P value of <0.05 was considered statistically significant. The correlation between VT-1598 MICs and those of the other antifungals was also assessed by Pearson correlation using log2-transformed MIC values.
Results
Candida and Cryptococcus species
VT-1598 demonstrated in vitro activity against Candida and Cryptococcus species. As shown in Figure 1, VT-1598 MICs using the 50% inhibition endpoint were towards the lower end of the concentration ranges tested for the majority of isolates. The VT-1598 GM MICs were also significantly lower than those of fluconazole for all Candida species (Table 1) and this activity was maintained against fluconazole-resistant isolates (VT-1598 GM MICs of 0.124 and 1.19 mg/L for resistant C. albicans and C. glabrata, respectively). There was a strong correlation between VT-1598 and fluconazole MICs (Pearson correlation coefficients of 0.8228 and 0.8484 for C. albicans and C. glabrata, respectively). VT-1598 GM MICs were also lower than those of caspofungin for C. albicans and C. parapsilosis isolates, although the activities of VT-1598 and caspofungin were similar against the fluconazole-resistant isolates. In vitro, as is observed with some fungal Cyp51 inhibitors, VT-1598 did not completely inhibit the growth of Candida species, as evident by the elevated MICs observed using the 100% inhibition endpoint. VT-1598 also demonstrated activity against both C. neoformans and C. gattii, with GM MICs significantly lower than those observed for fluconazole (Table 1). For Cryptococcus, VT-1598 MICs were also low when the 100% inhibition of growth endpoint was used (GM MICs of 0.159 and 0.118 mg/L for C. neoformans and C. gattii, respectively).
GM MICs (mg/L) of VT-1598 (all species), fluconazole (Candida and Cryptococcus spp.), caspofungin (Candida spp.), amphotericin B (Cryptococcus spp. and R. arrhizus), posaconazole (Aspergillus spp., R. arrhizus, B. dermatitidis, Coccidioides spp. and H. capsulatum) and voriconazole (Aspergillus spp.)
| Species | VT-1598 50% | VT-1598 100% | Fluconazole 50% | Caspofungin 50% | Amphotericin B 100% | Posaconazole 100% | Voriconazole 100% |
|---|---|---|---|---|---|---|---|
| C. albicans (all isolates n = 72) | 0.012 | >1 | 1.40a | 0.091a | – | – | – |
| C. albicans (azole susceptible n = 48) | 0.004 | >1 | 0.277a | 0.083a | – | – | – |
| C. albicans (azole resistant n = 21) | 0.124 | >1 | 49.1a | 0.112 | – | – | – |
| C. glabrata (all isolates n = 32) | 0.248 | 6.04 | 6.73a | 0.261 | – | – | – |
| C. glabrata (azole SDD n = 24) | 0.147 | 4.36 | 3.00a | 0.236 | – | – | – |
| C. glabrata (azole resistant n = 8) | 1.19 | >8 | 76.1a | 0.35 | – | – | – |
| C. parapsilosis (n = 10) | ≤0.015 | 0.049 | 0.406a | 0.758a | – | – | – |
| C. tropicalis (n = 9) | 0.019 | >8 | 2.00a | 0.146 | – | – | – |
| C. neoformans (n = 36) | 0.016 | 0.159 | 1.89a | – | 0.463 | – | – |
| C. gattii (n = 16) | 0.039 | 0.118 | 2.71a | – | 0.738 | – | – |
| A. fumigatus (all isolates n = 50) | – | 1.67 | – | – | – | 0.551a | 0.727a |
| A. fumigatus (WT n = 35) | – | 0.686 | – | – | – | 0.357a | 0.427a |
| A. fumigatus (elevated azole MICs n = 15) | – | 13.3 | – | – | – | 1.52a | 2.52a |
| A. flavus (n = 11) | – | 0.685 | – | – | – | 0.567 | 0.828 |
| A. niger (n = 12) | – | 1.78 | – | – | – | 1.00 | 1.26 |
| A. terreus (n = 11) | – | 0.533 | – | – | – | 0.302a | 0.567 |
| R. arrhizus (n = 11) | – | 3.53 | – | – | 0.500a | 1.13a | – |
| B. dermatitidis (n = 12) | – | 0.057 | 13.5a | – | – | 0.081 | – |
| Coccidioides spp. (n = 40) | – | 0.217 | 7.59a | – | – | 0.167 | – |
| H. capsulatum (n = 13) | – | 0.089 | 23.2a | – | – | 0.049 | – |
| Species | VT-1598 50% | VT-1598 100% | Fluconazole 50% | Caspofungin 50% | Amphotericin B 100% | Posaconazole 100% | Voriconazole 100% |
|---|---|---|---|---|---|---|---|
| C. albicans (all isolates n = 72) | 0.012 | >1 | 1.40a | 0.091a | – | – | – |
| C. albicans (azole susceptible n = 48) | 0.004 | >1 | 0.277a | 0.083a | – | – | – |
| C. albicans (azole resistant n = 21) | 0.124 | >1 | 49.1a | 0.112 | – | – | – |
| C. glabrata (all isolates n = 32) | 0.248 | 6.04 | 6.73a | 0.261 | – | – | – |
| C. glabrata (azole SDD n = 24) | 0.147 | 4.36 | 3.00a | 0.236 | – | – | – |
| C. glabrata (azole resistant n = 8) | 1.19 | >8 | 76.1a | 0.35 | – | – | – |
| C. parapsilosis (n = 10) | ≤0.015 | 0.049 | 0.406a | 0.758a | – | – | – |
| C. tropicalis (n = 9) | 0.019 | >8 | 2.00a | 0.146 | – | – | – |
| C. neoformans (n = 36) | 0.016 | 0.159 | 1.89a | – | 0.463 | – | – |
| C. gattii (n = 16) | 0.039 | 0.118 | 2.71a | – | 0.738 | – | – |
| A. fumigatus (all isolates n = 50) | – | 1.67 | – | – | – | 0.551a | 0.727a |
| A. fumigatus (WT n = 35) | – | 0.686 | – | – | – | 0.357a | 0.427a |
| A. fumigatus (elevated azole MICs n = 15) | – | 13.3 | – | – | – | 1.52a | 2.52a |
| A. flavus (n = 11) | – | 0.685 | – | – | – | 0.567 | 0.828 |
| A. niger (n = 12) | – | 1.78 | – | – | – | 1.00 | 1.26 |
| A. terreus (n = 11) | – | 0.533 | – | – | – | 0.302a | 0.567 |
| R. arrhizus (n = 11) | – | 3.53 | – | – | 0.500a | 1.13a | – |
| B. dermatitidis (n = 12) | – | 0.057 | 13.5a | – | – | 0.081 | – |
| Coccidioides spp. (n = 40) | – | 0.217 | 7.59a | – | – | 0.167 | – |
| H. capsulatum (n = 13) | – | 0.089 | 23.2a | – | – | 0.049 | – |
SDD, susceptible dose dependent.
The percentage inhibition endpoint is noted for each antifungal. For B. dermatitidis, Coccidioides spp. and H. capsulatum, 80% inhibition was the endpoint used.
P value <0.05 versus VT-1598.
GM MICs (mg/L) of VT-1598 (all species), fluconazole (Candida and Cryptococcus spp.), caspofungin (Candida spp.), amphotericin B (Cryptococcus spp. and R. arrhizus), posaconazole (Aspergillus spp., R. arrhizus, B. dermatitidis, Coccidioides spp. and H. capsulatum) and voriconazole (Aspergillus spp.)
| Species | VT-1598 50% | VT-1598 100% | Fluconazole 50% | Caspofungin 50% | Amphotericin B 100% | Posaconazole 100% | Voriconazole 100% |
|---|---|---|---|---|---|---|---|
| C. albicans (all isolates n = 72) | 0.012 | >1 | 1.40a | 0.091a | – | – | – |
| C. albicans (azole susceptible n = 48) | 0.004 | >1 | 0.277a | 0.083a | – | – | – |
| C. albicans (azole resistant n = 21) | 0.124 | >1 | 49.1a | 0.112 | – | – | – |
| C. glabrata (all isolates n = 32) | 0.248 | 6.04 | 6.73a | 0.261 | – | – | – |
| C. glabrata (azole SDD n = 24) | 0.147 | 4.36 | 3.00a | 0.236 | – | – | – |
| C. glabrata (azole resistant n = 8) | 1.19 | >8 | 76.1a | 0.35 | – | – | – |
| C. parapsilosis (n = 10) | ≤0.015 | 0.049 | 0.406a | 0.758a | – | – | – |
| C. tropicalis (n = 9) | 0.019 | >8 | 2.00a | 0.146 | – | – | – |
| C. neoformans (n = 36) | 0.016 | 0.159 | 1.89a | – | 0.463 | – | – |
| C. gattii (n = 16) | 0.039 | 0.118 | 2.71a | – | 0.738 | – | – |
| A. fumigatus (all isolates n = 50) | – | 1.67 | – | – | – | 0.551a | 0.727a |
| A. fumigatus (WT n = 35) | – | 0.686 | – | – | – | 0.357a | 0.427a |
| A. fumigatus (elevated azole MICs n = 15) | – | 13.3 | – | – | – | 1.52a | 2.52a |
| A. flavus (n = 11) | – | 0.685 | – | – | – | 0.567 | 0.828 |
| A. niger (n = 12) | – | 1.78 | – | – | – | 1.00 | 1.26 |
| A. terreus (n = 11) | – | 0.533 | – | – | – | 0.302a | 0.567 |
| R. arrhizus (n = 11) | – | 3.53 | – | – | 0.500a | 1.13a | – |
| B. dermatitidis (n = 12) | – | 0.057 | 13.5a | – | – | 0.081 | – |
| Coccidioides spp. (n = 40) | – | 0.217 | 7.59a | – | – | 0.167 | – |
| H. capsulatum (n = 13) | – | 0.089 | 23.2a | – | – | 0.049 | – |
| Species | VT-1598 50% | VT-1598 100% | Fluconazole 50% | Caspofungin 50% | Amphotericin B 100% | Posaconazole 100% | Voriconazole 100% |
|---|---|---|---|---|---|---|---|
| C. albicans (all isolates n = 72) | 0.012 | >1 | 1.40a | 0.091a | – | – | – |
| C. albicans (azole susceptible n = 48) | 0.004 | >1 | 0.277a | 0.083a | – | – | – |
| C. albicans (azole resistant n = 21) | 0.124 | >1 | 49.1a | 0.112 | – | – | – |
| C. glabrata (all isolates n = 32) | 0.248 | 6.04 | 6.73a | 0.261 | – | – | – |
| C. glabrata (azole SDD n = 24) | 0.147 | 4.36 | 3.00a | 0.236 | – | – | – |
| C. glabrata (azole resistant n = 8) | 1.19 | >8 | 76.1a | 0.35 | – | – | – |
| C. parapsilosis (n = 10) | ≤0.015 | 0.049 | 0.406a | 0.758a | – | – | – |
| C. tropicalis (n = 9) | 0.019 | >8 | 2.00a | 0.146 | – | – | – |
| C. neoformans (n = 36) | 0.016 | 0.159 | 1.89a | – | 0.463 | – | – |
| C. gattii (n = 16) | 0.039 | 0.118 | 2.71a | – | 0.738 | – | – |
| A. fumigatus (all isolates n = 50) | – | 1.67 | – | – | – | 0.551a | 0.727a |
| A. fumigatus (WT n = 35) | – | 0.686 | – | – | – | 0.357a | 0.427a |
| A. fumigatus (elevated azole MICs n = 15) | – | 13.3 | – | – | – | 1.52a | 2.52a |
| A. flavus (n = 11) | – | 0.685 | – | – | – | 0.567 | 0.828 |
| A. niger (n = 12) | – | 1.78 | – | – | – | 1.00 | 1.26 |
| A. terreus (n = 11) | – | 0.533 | – | – | – | 0.302a | 0.567 |
| R. arrhizus (n = 11) | – | 3.53 | – | – | 0.500a | 1.13a | – |
| B. dermatitidis (n = 12) | – | 0.057 | 13.5a | – | – | 0.081 | – |
| Coccidioides spp. (n = 40) | – | 0.217 | 7.59a | – | – | 0.167 | – |
| H. capsulatum (n = 13) | – | 0.089 | 23.2a | – | – | 0.049 | – |
SDD, susceptible dose dependent.
The percentage inhibition endpoint is noted for each antifungal. For B. dermatitidis, Coccidioides spp. and H. capsulatum, 80% inhibition was the endpoint used.
P value <0.05 versus VT-1598.
MIC distributions of VT-1598, fluconazole, caspofungin, amphotericin B, posaconazole and voriconazole for C. albicans (a), C. glabrata (b), C. parapsilosis and C. tropicalis (c) C. neoformans and C. gattii (d), A. fumigatus (e), A. flavus, A. niger and A. terreus (f), R. arrhizus (g), B. dermatitidis (h), Coccidioides spp. (i) and H. capsulatum (j). MICs were measured after a period of growth (24–168 h) appropriate for the fungal species. Black symbols represent MICs higher than the concentration range tested.
Endemic fungi
VT-1598 demonstrated in vitro activity against the endemic fungi tested in this study. As shown in Figure 1 and Table 1, the GM MICs for B. dermatitidis, Coccidioides species and H. capsulatum were very low (range 0.057–0.217 mg/L) and MIC values were ≤0.5 mg/L for all isolates. The activity of VT-1598 was similar to that of posaconazole, as demonstrated by GM MICs (VT-1598 range 0.057–0.219 mg/L, posaconazole range 0.049–0.167 mg/L), and VT-1598 GM MICs were significantly lower than those of fluconazole for each species (GM MIC range 7.59–23.2 mg/L). VT-1598 also demonstrated similar activity against the different Coccidioides species, C. immitis and C. posadasii (GM MICs of 0.180 and 0.250 mg/L, respectively).
Aspergillus species and R. arrhizus
VT-1598 also demonstrated activity against Aspergillus species, with GM MICs similar to those of posaconazole and voriconazole for A. flavus, A. niger and A. terreus isolates. The VT-1598 MICs were somewhat higher than those of posaconazole and voriconazole for A. fumigatus isolates, with MICs ranging between 0.25 and 2 mg/L for WT isolates (GM MIC 0.686 mg/L). In contrast, the activity of VT-1598 was markedly reduced (GM MIC of 13.3 mg/L) against A. fumigatus isolates with elevated posaconazole and voriconazole MICs, including those with known CYP51A mutations.10 VT-1598 also demonstrated activity against several R. arrhizus isolates, with MIC values ranging from 0.5 to >16 mg/L. Four of the isolates tested had VT-1598 MICs >16 mg/L and three of these were confirmed to be R. arrhizus var. delemar by ITS barcode analysis.11
Discussion
VT-1598 is a novel antifungal that prevents the biosynthesis of ergosterol within fungal cell membranes through inhibition of Cyp51.4,5 This tetrazole-based agent has been shown to be more specific for fungal Cyp51 than mammalian CYP450 enzymes, thus the potential for clinically relevant drug interactions is reduced compared with the triazole-based inhibitors that are approved for use in humans. As shown in the current study, VT-1598 has potent in vitro activity against yeasts, moulds and endemic fungi. This activity was also maintained against several Candida isolates with resistance to fluconazole. VT-1598 also demonstrated potent activity against endemic fungi, including B. dermatitidis, H. capsulatum and Coccidioides species, with equal potency against both C. immitis and C. posadasii. Several of the Coccidioides isolates previously used to test the in vitro activity of a related compound, VT-1161, were also included in this study.12 Although not tested together, the in vitro activity of VT-1598 was between 2 and 16 times more potent than that of VT-1161 for every isolate tested against both agents (data not shown). VT-1598 GM MICs were also significantly lower than those of fluconazole, which may have clinical significance as reduced susceptibility to fluconazole has been reported for Coccidioides.13 VT-1598 also demonstrated activity against Aspergillus species, although this was reduced against A. fumigatus isolates with elevated posaconazole and voriconazole MICs. Activity was also observed against R. arrhizus isolates. However, as observed with VT-1161, the in vitro potency was reduced against R. arrhizus var. delemar strains.14 Overall, these results show promise for the fungal-specific Cyp51 inhibitor VT-1598 and further studies exploring its in vivo efficacy are warranted.
Funding
Funding for this study and VT-1598 powder were provided by Viamet Pharmaceuticals, Inc.
Transparency declarations
N. P. W. has received research support to the University of Texas Health Science Center at San Antonio from Astellas, bioMérieux, F2G, Merck, Revolution Medicines and Viamet Pharmaceuticals, Inc., and has served on advisory boards for Merck, Astellas, Toyama and Viamet Pharmaceuticals, Inc. C. M. Y., R. J. S. and E. P. G. are employees of Viamet Pharmaceuticals, Inc., and each has an ownership interest in Viamet Pharmaceuticals Holdings, LLC, which is the parent company of Viamet Pharmaceuticals, Inc. H. P. P. and B. H. T.: none to declare.
