Chemical Genomics-Based Antifungal Drug Discovery: Targeting Glycosylphosphatidylinositol (GPI) Precursor Biosynthesis

Abstract

Steadily increasing antifungal drug resistance and persistent high rates of fungal-associated mortality highlight the dire need for the development of novel antifungals. Characterization of inhibitors of one enzyme in the GPI anchor pathway, Gwt1, has generated interest in the exploration of targets in this pathway for further study. Utilizing a chemical genomics-based screening platform referred to as the Candida albicans fitness test (CaFT), we have identified novel inhibitors of Gwt1 and a second enzyme in the glycosylphosphatidylinositol (GPI) cell wall anchor pathway, Mcd4. We further validate these targets using the model fungal organism Saccharomyces cerevisiae and demonstrate the utility of using the facile toolbox that has been compiled in this species to further explore target specific biology. Using these compounds as probes, we demonstrate that inhibition of Mcd4 as well as Gwt1 blocks the growth of a broad spectrum of fungal pathogens and exposes key elicitors of pathogen recognition. Interestingly, a strong chemical synergy is also observed by combining Gwt1 and Mcd4 inhibitors, mirroring the demonstrated synthetic lethality of combining conditional mutants of GWT1 and MCD4. We further demonstrate that the Mcd4 inhibitor M720 is efficacious in a murine infection model of systemic candidiasis. Our results establish Mcd4 as a promising antifungal target and confirm the GPI cell wall anchor synthesis pathway as a promising antifungal target area by demonstrating that effects of inhibiting it are more general than previously recognized.

Keywords: Candida albicans fitness test; GPI; GWT1; MCD4; antifungal; chemical biology; glycosylphosphatidylinositol; natural product; next-generation sequencing; yeast cell wall.

Figure 1

Individual C. albicans heterozygous deletion mutants contain two unique barcodes (red and blue boxes) flanking the deleted allele. The CaFT strain pool contains ∼5400 heterozygote deletion mutants, comprising >90% genome coverage. Aliquots of the pool are treated with a sub-MIC of the growth inhibitory compound or mock treatment and grown for 20 generations. The relative abundance of each strain, which reflects their chemical sensitivity to the compound, is subsequently determined by DNA microarray analysis using PCR amplified barcodes of each heterozygote. The response of each heterozygote to the effects of the compound is then appraised by calculating a normalized Z score, with a positive value indicating hypersensitivity and a negative value reflecting resistance (or hyposensitivity) to the tested compound. See Xu et al. for details of Z-score calculation. In this example, the strain highlighted in red is uniquely hypersensitive to the compound (C) treatment and depleted from the pool versus the mock-treated (M) control.

Figure 2

CaFT-based screening identifies GPI inhibitors. (A) CaFT summary of C. albicans heterozygote hypersensitivity to G884 across multiple drug concentrations. Note increasing strain sensitivities to G884 are displayed left to right (second row), based on the sum Z score across each independent CaFT experiment. Individual Z scores for each heterozygote strain are listed (rows 3–6), with Z scores highlighted in a color scale and heat map format. Note the GWT1 heterozygote reproducibly displays greatest sensitivity to G884, and GPI1 and SPT14 heterozygotes display modest resistance to G884. (B) CaFT summary of C. albicans heterozygote hypersensitivity to M743 across multiple drug concentrations. Rank order of strain sensitivities and Z scores are highlighted as in (A). Note that the MCD4 heterozygote displays greatest hypersensitivity to M743, whereas additional sensitive heterozygotes correspond to COPI coatamer subunits. (C) Chemical structures of G884, G365, M743, M720, and gepinacin. (D) Image of the S. cerevisiae GPI precursor and predicted enzymatic steps in precursor biosynthesis targeted by the above inhibitors.

Figure 3

S. cerevisiae drug resistant mutant isolation and characterization of GPI inhibitors. (A) Summary of Gwt1 and Mcd4 drug resistant amino acid substitution mutants and altered susceptibility to GPI inhibitors versus control antifungal agents, amphotericin B (AmB), fluconazole (FLZ), and caspofungin (CSP). Specific amino acid substitutions and causal nucleotide mutations are shown. Note G884^R mutations were isolated using wild-type (wt) strain S288c, whereas M743 ^R and M720 ^R mutants were isolated from a pdr5Δ strain otherwise isogenic to BY4700. Also note cross-resistance is specifically observed between M743 and its semisynthetic analogue, M720, among Mcd4 amino acid drug-resistant isolates as well as between G884 and gepinacin with Gwt1-G132W. (B) G884^R amino acid substitutions (boxed) map to Gwt1. Predicted topology of S. cerevisiae Gwt1 is shown as recently determined with C-terminal sequence residing in cytosol. (C) M743^R and M720^R amino acid substitutions (boxed) map to Mcd4. Predicted topology of S. cerevisiae Mcd4 is shown as previously described, with the KKTQ C-terminal sequence residing in the lumen of the ER.

Figure 4

G884 and G365 inhibit Gwt1 acylation. (A) TLC of products of an in vitro acylation assay using membranes from S. cerevisiae. Controls show that in the absence of ATP and CoA the acylated band is not produced. In the presence of phopholipase C only the acylated band is preserved. Inhibitors of MCD4, M720, and M743 do not inhibit Gwt1-dependent acylation. The acylation reactions were incubated with 2 μg/mL of gepinacin, M720, and M743 and 30 μg/mL of G884 and G365. All GPI inhibitor MIC values for S. cerevisiae are listed in Table 1. (B) Dose response of the acylation inhibition by G884 and G365. The amount of compound in the reaction is given above the blot as μg/mL. (C) G884 preferentially inhibits the fungal enzyme, Gwt1. Growth curves of S. cerevisiae strains contain either the human enzyme Pig-W or the fungal enzyme, Gwt1, as their sole source of inositol acylating activity. The origin of replication for the low-copy plasmids is CEN and for the high-copy plasmids is 2 μm. (D) Induction of the unfolded protein response in cells carrying a GFP reporter construct showing strong induction by inhibitors of Gwt1 and Mcd4. GFP expression was monitored by flow cytometry.

Figure 5

GWT1 and MCD4 display a synthetic lethal genetic interaction and cognate inhibitors possess highly synergistic antifungal activity. (A) Drug synergy of GPI inhibitor combinations G884 and M720 or (B) G884 and M720 determined by standard checkerboard analysis against C. albicans strain 2323. Drug concentrations and fractional inhibitory concentrations (FIC) used to evaluate synergy are indicated. Chemical synergy is achieved provided the sum FIC of the two agents (referred to as the FIC index, or FICI) is ≤0.5. FICI = 0.5 is indicated by the diagonal blue line. (C) Tabular summary of the FICI values of Gwt1 and Mcd4 inhibitors tested against C. albicans and S. cerevisiae. (D) GWT1 and MCD4 exhibit a synthetic lethal genetic interaction in yeast. Spore progeny of a gwt1-20::natMX4/GWT1, mcd4-500::kanMX4/MCD4 double-heterozygous diploid strain dissected onto synthetic complete (SC) medium and incubated at 30 °C for 4 days. Large colonies are identified as wild-type; smaller colonies are either gwt1-20 or mcd4-500 haploids (depending on drug resistance marker; natR, (black square); kanR, (white circle)), and microcolonies maintaining both markers are gwt1-20, mcd4-500 double mutants.

Figure 6

GPI inhibitors unmask cell surface β-glucan and induce TNFα secretion in macrophages. (A) Fluorescence photomicrographs of C. albicans and β-(1–3)-glucan (green) immunoreactivity with anti-β-(1–3)-glucan antibody following treatment with GPI inhibitors (G365, G884, M743, M720, and gepinacin), the control antifungal agent fluconazole (FLZ), or DMSO. All drug treatments were performed at a drug concentration equivalent to IC₄₀ to ensure immunoreactivity is not indirectly due to cell death. Propidium iodide staining confirms minimal cell death for all GPI inhibitors tested at their IC₄₀ concentration versus fluconazole. Images are merged fluorescence and phase contrast. (B) ELISA quantification of secreted TNFα by RAW264.7 macrophage co-incubated with C. albicans strain 2323 treated at MIC₂₀ and MIC₄₀ values for each of the above agents.

Figure 7

In vivo efficacy of M720 in a murine systemic infection model of candidiasis. DBA/2 mice were infected with C. albicans strain MY1055 and treated ip with M720 or caspofungin (CAS) at the indicated doses (mg/kg) either twice daily (bid) or once daily (qd). Kidneys were aseptically collected at 4 days after infectious challenge, and log reduction of colony-forming units (CFU) per gram of kidney tissue was calculated on the basis of kidney burden of vehicle-treated (5% DMSO or H₂O, respectively) control group. Note the limit of detection (LOD) is 1.5 × 10² CFU/g, as indicated by the dashed line. (∗) P < 0.05, (∗∗) P < 0.01, and (∗∗∗) P < 0.001 significance versus vehicle control.