High-Resolution Genetics Identifies the Lipid Transfer Protein Sec14p as Target for Antifungal Ergolines

Abstract

Invasive infections by fungal pathogens cause more deaths than malaria worldwide. We found the ergoline compound NGx04 in an antifungal screen, with selectivity over mammalian cells. High-resolution chemogenomics identified the lipid transfer protein Sec14p as the target of NGx04 and compound-resistant mutations in Sec14p define compound-target interactions in the substrate binding pocket of the protein. Beyond its essential lipid transfer function in a variety of pathogenic fungi, Sec14p is also involved in secretion of virulence determinants essential for the pathogenicity of fungi such as Cryptococcus neoformans, making Sec14p an attractive antifungal target. Consistent with this dual function, we demonstrate that NGx04 inhibits the growth of two clinical isolates of C. neoformans and that NGx04-related compounds have equal and even higher potency against C. neoformans. Furthermore NGx04 analogues showed fungicidal activity against a fluconazole resistant C. neoformans strain. In summary, we present genetic evidence that NGx04 inhibits fungal Sec14p and initial data supporting NGx04 as a novel antifungal starting point.

Fig 1. Chemogenomic profiling.

Structure, haploinsufficiency profiling (HIP) and homozygous profiling (HOP) of NGx04. A) The structure of NGx04 is shown. B) HIP and C) HOP were performed around IC₃₀ (8 μM) in duplicates and the average of the profiles is shown. The MADL score is given on the y-axis and indicates how sensitive a particular deletion strain is to a given compound, compared to its no-drug control. The Z-score is given on the x-axis and is a statistical measure of how frequently a particular deletion strain is affected within the so far profiled compounds [26]. Circles depict non-essential deletions, whereas squares essential deletions. Hyper-sensitive deletions are found bottom left and hyper-resistant deletions top right of the panels. D) Hypersensitivity of a heterozygous SEC14/sec14 strain across 4011 diverse compounds (x-axis) is displayed as Z-score (y-axis). NGx04 (arrow) was found to exert the strongest effect. Validation of the hit candidates from HIP E), HOP F) was performed by single strain growth inhibition. The indicated strains were grown for 24 h in the presence of increasing concentrations of NGx04 and growth measured by turbidity (OD₆₀₀). Duplicate values were determined and used for logistic regression. The calculated IC₅₀ values are displayed.

Fig 2. Resistance mapping.

Functional variomics screen for NGx04-resistant mutants. A) The SEC14 ORFs of 88 NGx04-resistant colonies were amplified by PCR and sequenced. Four mutations mapping to the lipid-binding part of Sec14p were found. The identified amino acid changes with their respective frequencies are given. B) The plasmids coding for various SEC14 proteins were re-transformed into a fresh wild type yeast strain and serial dilutions of the cultures spotted on either 150 μM of NGx04 or DMSO control, incubated for 3 days at 30°C and scored for growth. C) The strains were grown for 24 h in the presence of increasing concentrations of NGx04 and growth measured by turbidity (OD₆₀₀). Duplicate values were determined and the 18 h time point was used for logistic regression. The calculated IC₅₀ values displayed.

Fig 3. Active site antagonism.

Phosphatidylinositol (PI) and phosphatidylcholine (PC) competition. A) The crystal structure of Phosphatidylcholine (PC) bound to the yeast Sfh1protein (PDB ID: 3B7Q), (where R is oleoyl and R’ is palmitol). PC hydrophobic tail forms van der Waals interactions with a cluster of hydrophobic residue, whereas the phosphate moiety is interacting with the side-chain hydroxyl groups of residues S175 and T177, and the ethanolamine nitrogen contacts the phenolic oxygen of Y113. B) The proposed docking pose of NGx04 in the putative binding site of the yeast protein structure is shown (PDB ID:1AUA). In the suggested binding mode, the ester moiety of the ligand forms a hydrogen bond with Y111 while the ergoline amine forms H-bonds with the T175. The ergoline moiety is also forming van der Waals interactions with the following residues: Y122, Y151, S173, M177 and F212. C) Wild type yeast was grown in presence of increasing concentrations of NGx04, supplemented with either 200 μM PI, 200 μM PC or no phospholipid, incubated at 30°C and growth followed over time by measuring turbidity (OD₆₀₀). Duplicate values were determined and the time points at 18 h were used for logistic regression.

Fig 4. Fungal selectivity.

Mammalian cell growth inhibition and cytotoxicity of NGx04. A) Dose-response curves for NGx04 were generated in growth inhibition assay and IC₅₀ values determined in duplicates [39]. IC₅₀ values and growth inhibition (Amax) are displayed. 76% of the tested cell lines were not significantly inhibited by NGx04. B) A dose-response curves for NGx04 was generated in a MTS cytotoxicity assay using HepG2 cells in triplicates and a toxicity curve generated by logistic regression. C) Overlay of the crystal structure of the yeast Sec14p (PDB ID: 1AUA, protein structure highlighted in blue) and its human orthologue SEC14L2 (PDB ID: 1OLM, protein structure highlighted in green) reveals that the amino acid residues belonging to the putative binding site are not conserved between the yeast and the human protein. D) Overlay of the docked NGx04 in the yeast Sec14p (PDB ID: 1AUA) and its human orthologue SEC14L2 (PDB ID: 1OLM). The amino acid residues of the yeast protein structure have been hidden whereas the surface of the putative binding site of the human protein structure has been added to highlight the difference in the overall shape between the active site of the two species.

Fig 5. Antifungal testing.

C. neoformans growth inhibition by NGx04 scaffolds. NGx04 and close analogues were tested against C. neoformans ATCC 36556 and ATCC 90113 in a 72 h proliferation assay and MIC-2 (50% inhibition) and MIC-0 (no visible fungal growth) determined. Triplicate values are given in μg/ml. Amphotericin B was included as positive control.