Fungal-specific Cyp51 inhibitor VT-1598 demonstrates in vitro activity against Candida and Cryptococcus species, endemic fungi, including Coccidioides species, Aspergillus species and Rhizopus arrhizus

Fungal-specific Cyp51 inhibitor VT-1598 demonstrates in vitro activity against Candida and... Abstract Background 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. Methods 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. Results 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. Conclusions 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). Table 1. 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. a P value <0.05 versus VT-1598. Figure 1. View largeDownload slide 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. Figure 1. View largeDownload slide 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. References 1 Lewis RE. Current concepts in antifungal pharmacology. Mayo Clin Proc  2011; 86: 805– 17. Google Scholar CrossRef Search ADS PubMed  2 Arendrup MC. Update on antifungal resistance in Aspergillus and Candida. Clin Microbiol Infect  2014; 20 Suppl 6: 42– 8. Google Scholar CrossRef Search ADS PubMed  3 Sanglard D. Emerging threats in antifungal-resistant fungal pathogens. Front Med (Lausanne)  2016; 3: 11. Google Scholar PubMed  4 Hargrove TY, Garvey EP, Hoekstra WJ et al.   Crystal structure of the new investigational drug candidate VT-1598 in complex with Aspergillus fumigatus sterol 14α-demethylase provides insights into its broad-spectrum antifungal activity. Antimicrob Agents Chemother  2017; 61: e00570-17. Google Scholar CrossRef Search ADS PubMed  5 Yates CM, Garvey EP, Shaver SR et al.   Design and optimization of highly-selective, broad spectrum fungal CYP51 inhibitors. Bioorg Med Chem Lett  2017; 27: 3243– 8. Google Scholar CrossRef Search ADS PubMed  6 Tintelnot K, De Hoog GS, Antweiler E et al.   Taxonomic and diagnostic markers for identification of Coccidioides immitis and Coccidioides posadasii. Med Mycol  2007; 45: 385– 93. Google Scholar CrossRef Search ADS PubMed  7 Rinaldi MG. Use of potato flakes agar in clinical mycology. J Clin Microbiol  1982; 15: 1159– 60. Google Scholar PubMed  8 Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts—Third Edition: Approved Standard M27-A3 . CLSI, Wayne, PA, USA, 2008. 9 Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi—Second Edition: Approved Standard M38-A2 . CLSI, Wayne, PA, USA, 2008. 10 Wiederhold NP, Gil VG, Gutierrez F et al.   First detection of TR34 L98H and TR46 Y121F T289A Cyp51 mutations in Aspergillus fumigatus isolates in the United States. J Clin Microbiol  2016; 54: 168– 71. Google Scholar CrossRef Search ADS PubMed  11 Abe A, Oda Y, Asano K et al.   Rhizopus delemar is the proper name for Rhizopus oryzae fumaric-malic acid producers. Mycologia  2007; 99: 714– 22. Google Scholar CrossRef Search ADS PubMed  12 Shubitz LF, Trinh HT, Galgiani JN et al.   Evaluation of VT-1161 for treatment of coccidioidomycosis in murine infection models. Antimicrob Agents Chemother  2015; 59: 7249– 54. Google Scholar CrossRef Search ADS PubMed  13 Thompson GR3rd, Barker BM, Wiederhold NP. Large-scale evaluation of in vitro amphotericin B, triazole, and echinocandin activity against Coccidioides species from U.S. institutions. Antimicrob Agents Chemother  2017; 61: e02634–16. Google Scholar CrossRef Search ADS PubMed  14 Gebremariam T, Wiederhold NP, Fothergill AW et al.   VT-1161 protects immunosuppressed mice from Rhizopus arrhizus var. arrhizus infection. Antimicrob Agents Chemother  2015; 59: 7815– 7. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Fungal-specific Cyp51 inhibitor VT-1598 demonstrates in vitro activity against Candida and Cryptococcus species, endemic fungi, including Coccidioides species, Aspergillus species and Rhizopus arrhizus

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© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Abstract

Abstract Background 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. Methods 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. Results 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. Conclusions 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). Table 1. 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. a P value <0.05 versus VT-1598. Figure 1. View largeDownload slide 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. Figure 1. View largeDownload slide 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. References 1 Lewis RE. Current concepts in antifungal pharmacology. Mayo Clin Proc  2011; 86: 805– 17. Google Scholar CrossRef Search ADS PubMed  2 Arendrup MC. Update on antifungal resistance in Aspergillus and Candida. Clin Microbiol Infect  2014; 20 Suppl 6: 42– 8. Google Scholar CrossRef Search ADS PubMed  3 Sanglard D. Emerging threats in antifungal-resistant fungal pathogens. Front Med (Lausanne)  2016; 3: 11. Google Scholar PubMed  4 Hargrove TY, Garvey EP, Hoekstra WJ et al.   Crystal structure of the new investigational drug candidate VT-1598 in complex with Aspergillus fumigatus sterol 14α-demethylase provides insights into its broad-spectrum antifungal activity. Antimicrob Agents Chemother  2017; 61: e00570-17. Google Scholar CrossRef Search ADS PubMed  5 Yates CM, Garvey EP, Shaver SR et al.   Design and optimization of highly-selective, broad spectrum fungal CYP51 inhibitors. Bioorg Med Chem Lett  2017; 27: 3243– 8. Google Scholar CrossRef Search ADS PubMed  6 Tintelnot K, De Hoog GS, Antweiler E et al.   Taxonomic and diagnostic markers for identification of Coccidioides immitis and Coccidioides posadasii. Med Mycol  2007; 45: 385– 93. Google Scholar CrossRef Search ADS PubMed  7 Rinaldi MG. Use of potato flakes agar in clinical mycology. J Clin Microbiol  1982; 15: 1159– 60. Google Scholar PubMed  8 Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts—Third Edition: Approved Standard M27-A3 . CLSI, Wayne, PA, USA, 2008. 9 Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi—Second Edition: Approved Standard M38-A2 . CLSI, Wayne, PA, USA, 2008. 10 Wiederhold NP, Gil VG, Gutierrez F et al.   First detection of TR34 L98H and TR46 Y121F T289A Cyp51 mutations in Aspergillus fumigatus isolates in the United States. J Clin Microbiol  2016; 54: 168– 71. Google Scholar CrossRef Search ADS PubMed  11 Abe A, Oda Y, Asano K et al.   Rhizopus delemar is the proper name for Rhizopus oryzae fumaric-malic acid producers. Mycologia  2007; 99: 714– 22. Google Scholar CrossRef Search ADS PubMed  12 Shubitz LF, Trinh HT, Galgiani JN et al.   Evaluation of VT-1161 for treatment of coccidioidomycosis in murine infection models. Antimicrob Agents Chemother  2015; 59: 7249– 54. Google Scholar CrossRef Search ADS PubMed  13 Thompson GR3rd, Barker BM, Wiederhold NP. Large-scale evaluation of in vitro amphotericin B, triazole, and echinocandin activity against Coccidioides species from U.S. institutions. Antimicrob Agents Chemother  2017; 61: e02634–16. Google Scholar CrossRef Search ADS PubMed  14 Gebremariam T, Wiederhold NP, Fothergill AW et al.   VT-1161 protects immunosuppressed mice from Rhizopus arrhizus var. arrhizus infection. Antimicrob Agents Chemother  2015; 59: 7815– 7. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Feb 1, 2018

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