Abstract
Manogepix (MGX) targets the conserved fungal Gwt1 enzyme required for acylation of inositol early in the glycosylphosphatidylinositol biosynthesis pathway. The prodrug fosmanogepix is currently in clinical development for the treatment of invasive fungal infections. We determined that the median frequencies of spontaneous mutations conferring reduced susceptibility to MGX in Candida albicans, C. glabrata, and C. parapsilosis ranged from 3 × 10-8 to <1.85 × 10-8 Serial passage on agar identified mutants of C. albicans and C. parapsilosis with reduced susceptibility to MGX; however, this methodology did not result in C. glabrata mutants with reduced susceptibility. Similarly, serial passage in broth resulted in ≤2-fold changes in population MIC values for C. tropicalis, C. auris, and C. glabrata A spontaneous V163A mutation in the Gwt1 protein of C. glabrata and a corresponding C. albicans heterozygous V162A mutant were obtained. A C. glabrata V163A Gwt1 mutant generated using CRISPR, along with V162A and V168A mutants expressed in C. albicans and Saccharomyces cerevisiae Gwt1, respectively, all demonstrated reduced susceptibility to MGX versus control strains, suggesting the importance of this valine residue to MGX binding across different species. Cross-resistance to the three major classes of antifungals was evaluated, but no changes in susceptibility to amphotericin B or caspofungin were observed in any mutant. No change was observed in fluconazole susceptibility, with the exception of a single non-Gwt1 mutant, where a 4-fold increase in the fluconazole MIC was observed. MGX demonstrated a relatively low potential for resistance development, consistent with other approved antifungal agents and those in clinical development.
Highlights
C. albicans 90028C. glabrata 2001 C. parapsilosis 22019Derivation WT Spontaneous Serial passage WT SpontaneousCRISPR WT Spontaneous Strain5.1 5.2 5.3 4.15 P20-1 P20-2 P20-35.1 5.2 5.3 5.4 RNP15.2 5.3 5.4 5.5 passage 10 (P10)-1 P10-2 P10-3 P10-4MGX MIC ratio
We evaluated the potential for Candida species to develop resistance to MGX by analyzing both spontaneous mutation frequencies and resistance development by serial passage
Resistance to all classes of antifungal agents has been previously observed, and the underlying mechanisms have been studied in great detail [26]
Summary
Resistance development by spontaneous mutation. (i) Susceptibility. The MIC of MGX was determined for three Candida ATCC strains (C. albicans 90028, C. glabrata 2001, and C. parapsilosis 22019) using the Clinical and Laboratory Standard Institute (CLSI) M27-A3 broth microdilution method and reading of the MIC at 50% growth inhibition relative to the growth control [21]. The broth MIC values were used to determine the ranges of concentrations for agar MIC assays using Sabouraud dextrose agar (SDA) plates. The spontaneous frequency of resistance to MGX was determined in triplicate on agar at 4ϫ MIC for the three Candida strains using 245-by-245-mm square bioassay dishes, as previously described [22, 23]. Spontaneous mutation frequencies were calculated by dividing the number of resistant colonies on a given plate by the plating CFU. Individual colonies were obtained from selected populations and assessed for MIC, followed by sequencing the GWT1 gene from the strain Using this methodology, mutants were selected by plating large numbers of cells [106] across a wide range of drug concentrations. For C. albicans 90028, MIC values increased 8-fold from 0.016 to 0.125 g/ml after 18 serial passages. The total population MIC increased 8- to 16-fold for C
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