De Novo Acquisition of Resistance to SCY-078 in Candida glabrata Involves FKS Mutations That both Overlap and Are Distinct from Those Conferring Echinocandin Resistance

TEXT

The echinocandins are preferred first-line agents for treating severe invasive fungal infections due to Candida species (1). They alter the integrity of the fungal cell wall via inhibition of the biosynthesis of its major component, β-(1,3)-glucan (2). The echinocandins target the catalytic subunit of the enzymatic complex β-(1,3)-d-glucan synthase, which is encoded by the FKS genes. The three FDA-approved echinocandins (caspofungin, anidulafungin, and micafungin) are only available as intravenous formulations, which limits their application, especially as oral step-down agents as often required outside the hospital setting. SCY-078 (formerly MK-3118) is an orally active semisynthetic derivative of the natural product enfumafungin, which is a potent inhibitor of fungal β-(1,3)-d-glucan synthases (3) with a chemical structure that is distinct from that of the echinocandins (Fig. 1). SCY-078 is highly active against clinically important Candida species, including some variants that are resistant to echinocandin drugs (4) (5). There is some cross-resistance between echinocandin-resistant strains and SCY-078, but little is known about the spectrum of de novo reduced susceptibility induced by this new chemical class of antifungal agent. The goal of this study was to understand the nature of de novo reduced susceptibility following in vitro selection with SCY-078 against the important human pathogen Candida glabrata, as it is the predominant Candida species responsible for antifungal drug resistance (azole and echinocandin) observed in the United States and Europe (6).

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FIG 1

Structures of SCY-078, a semisynthetic enfumafungin derivative, and micafungin, an echinocandin.

In order to obtain spontaneous isolates with reduced susceptibility to SCY-078, C. glabrata cells (1 × 10⁶) from different susceptible clinical isolates (Table 1) were plated on SCY-078-containing yeast extract-peptone-dextrose (YPD) plates (1% yeast extract, 2% peptone, 2% glucose) on a range of 1 dilution above to 1 dilution below the MIC (0.25 to 1 μg/ml). SCY-078-resistant (SR) isolates were selected following incubation at 37°C for 5 days, as modified from Balashov (7). The susceptible strains were obtained from epidemiologically distinct patients with bloodstream infection (BSI). Two strains, BAD55 and BAD144, showed a point mutation in the mismatch repair gene MSH2 (P208S) that has been linked to a higher propensity to breakthrough antifungal treatment in vitro (8). Breakthrough colonies were detected only on media plates containing 0.5× the MIC (0.25 μg/ml) of the antifungal drug for all strains except BAD55. After the initial selection, a total of 104 isolates (40 for BAD40, 48 for BAD144, and 16 for BAD47) were screened for growth on YPD plates containing increasing concentrations of SCY-078 (0.5 to 4 μg/ml). Of the 104 isolates, 17 failed to grow when subcultured on YPD plates with 4 μg/ml of SCY-078 and were not further analyzed. Antifungal susceptibility testing was performed in triplicate for the remaining 87 isolates in accordance with the guidelines described in CLSI document M27-A3 (9) for SCY-078 and the comparator agent micafungin (MCF). MCF (Astellas Pharma, Inc.) and SCY-078 (Scynexis, Inc.) were obtained as standard powders from their manufacturers and dissolved in water (MCF) or 100% dimethyl sulfoxide (DMSO) (SCY-078). Geometric MIC distributions ranged from 0.31 to 8 and 0.03 to 2.52 μg/ml for SCY-078 and MCF, respectively (Table 2).

Reduced echinocandin susceptibility leading to therapeutic failure has been linked to mutations in two highly conserved hot spot regions (HS1 and HS2) in the catalytic FKS subunit of β-(1,3)-d-glucan synthase (10, 11). Since SCY-078 also targets β-(1,3)-d-glucan synthase, we determined the spectrum of possible mutations in the SR C. glabrata isolates by sequencing both FKS1 and FKS2 genes. A total of 15 SR isolates harbored mutations in either the FKS1 (n = 2) or FKS2 (n = 13) genes, leading to an amino acid change (Table 2). The majority of mutations were localized to amino acid (aa) Phe659 within the HS1 region of FKS2 (aa 659 to 667). In the clinical setting, fks mutations affecting Fks1 Ser629, Fks2 Ser663, and Phe659 in C. glabrata are some of the most commonly observed mutations. These mutations confer some of the largest MIC (and glucan synthase 50% inhibitory concentration [IC₅₀]) shifts (10, 12); in the present in vitro study, only one echinocandin-resistant locus, Phe659, was identified. Interestingly, three resistant isolates displayed mutations in other regions of FKS2. One isolate contained a mutation upstream of HS1 (BAD40-18 [Glu655Ala]), while another had a mutation downstream of HS2 (aa 1374 to 1381) (BAD40-18 [Ala1390Asp]). Isolate BAD40-36 contained a Trp715Leu mutation, for which the equivalent has been shown to alter echinocandin susceptibility in Saccharomycescerevisiae (13). Isolates bearing FKS mutations exhibited higher MIC values for SCY-078 and MCF relative to their parent strain (Table 2), with fold decreases in susceptibility ranging from 1.58 to 16 and 2 to 84 for SCY-078 and MCF, respectively. Notably, one isolate displayed an elevated MIC for MCF but lacked a mutation in either FKS1 or FKS2 (BAD144-6; MIC, ≥0.25 μg/ml).

To better assess direct inhibition of SCY-078 on glucan synthase, the kinetic inhibition parameter IC₅₀ (half maximal inhibitory concentration) was determined for glucan synthases from wild-type and SCY-078-resistant fks-containing strains. Product-entrapped β-(1,3)-d-glucan synthase complexes were extracted as described previously (10). The inhibition curves for MCF and SCY-078 against the wild-type (WT) C. glabrata isolates showed the typical pattern of β-(1,3)-d-glucan synthase echinocandin susceptibility reported for other sensitive Candida species (12) with mean IC₅₀s of 1.64 to 5.18 ng/ml for MCF and 13.97 to 82.15 ng/ml for SCY-078 (Table 3).

Evaluation of kinetic inhibition of the product-entrapped enzymes isolated from SR strains yielded higher IC₅₀s for SCY-078 and MCF than enzymes from the corresponding parent strains. The Fks2 Phe659del mutant was strongly insensitive to all drugs and exhibited 44- and >121-fold increases in IC₅₀ for MCF and SCY-078, respectively. The Fks1 Phe625del mutant was also highly insensitive, with 11- and 40-fold increases for MCF and SCY-078, respectively (Fig. 2 and Table 3). In contrast, a point mutation (Phe625Leu) at this same locus had little effect on MIC, and the isolated enzyme displayed strong sensitivity to the assayed drugs. Regarding the novel mutations found outside the hot spot (HS) in the FKS2 gene, GS enzymes containing either Ala1390Asp or Trp715Leu proved to be highly insensitive to SCY-078 (20- and 29-fold increases in IC₅₀, respectively) (Table 3). This correlates well with the high MICs these mutants showed for SCY-078 (4 μg/ml for Trp715Leu and 8 μg/ml for Ala1390Asp) but not for MCF (0.3 μg/ml for Trp715Leu and 0.12 μg/ml for Ala1390Asp). The fact that different mutations in the FKS genes had such diverse IC₅₀ profiles suggests that even though SCY-078 and the echinocandins inhibit the same target enzyme, the interaction sites between these two types of antifungal drugs overlap but are not identical.

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FIG 2

Antifungal inhibition profiles of enriched GS complexes from representative susceptible and SCY-078-resistant C. glabrata isolates for micafungin (MCF) and SCY-078.

We observed a similar pattern in IC₅₀s in previous work (5); mutations in Ser645 in Candida albicans within hot spot 1 in the FKS1 gene resulted in an increase in susceptibility to SCY-078 compared to caspofungin (5.5-fold change), but mutations in Phe641 proved to be extremely resistant to both antifungal drugs. In C. glabrata, substitutions at the Phe659 locus within hot spot 1 in the FKS2 gene (equivalent to Phe641 in C. albicans) showed an increase in the susceptibility to SCY-078 compared to that to CSF (3.6-fold change). This behavior is also consistent with the MIC data (5).

In summary, C. glabrata isolates harboring FKS mutations show reduced susceptibility to SCY-078 (1.58-to 16-fold increase in MIC values for SCY-078). The isolated enzymes harboring mutations in or outside the echinocandin-resistant HS regions were more sensitive to MCF, with an IC₅₀ between 3- to 43-fold lower than those for SCY-078. Of the 15 SCY-078-resistant isolates generated from the screen showing FKS mutations, 13 of them contained a mutation in FKS2, correlating with previous findings that glucan synthase expression may be predominantly derived from FKS2 in C. glabrata (11). The absence of mutations found at prominent echinocandin resistance hot spot loci Ser663 (FKS2) and Ser629 (FKS1) is consistent with in vitro studies showing that SCY-078 is active against echinocandin-resistant strains with mutations at these positions (4, 5). This would suggest that FKS2 may be under greater selective pressure relative to FKS1, and the identification of resistant mutations inside and outside FKS hot spot regions indicates a partially shared interaction site among SCY-078 and the echinocandins (Fig. 3). This is also supported by previous in vitro selection studies involving both caspofungin and micafungin in which other prominent loci associated with echinocandin resistance were identified (13).

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FIG 3

Prediction model of C. glabrata Fks2 membrane protein topology (gray bars, transmembrane regions [TMH]; hot spot regions are marked with green circles). Note that amino acids in positions 715 and 1390 (marked with blue dots) are in TMHVI and TMHVIII, respectively. In silico TMH predictions were generated using TMHMM (http://www.cbs.dtu.dk/services/TMHMM ) and PRO-TMHMM (http://topcons.cbr.su.se ).

In conclusion, the molecular target on glucan synthase conferring resistance to SCY-078 displays partial overlap with that for echinocandin drugs, but the binding site appears to be nonidentical.