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. 2024 Jan;9(1):29-34.
doi: 10.1038/s41564-023-01542-4. Epub 2023 Dec 27.

Aspergillus fumigatus strains that evolve resistance to the agrochemical fungicide ipflufenoquin in vitro are also resistant to olorofim

Norman van Rhijn  1 , Isabelle S R Storer  1 , Mike Birch  2 , Jason D Oliver  2 , Michael J Bottery  1 , Michael J Bromley  3
Affiliations

Aspergillus fumigatus strains that evolve resistance to the agrochemical fungicide ipflufenoquin in vitro are also resistant to olorofim

Norman van Rhijn et al. Nat Microbiol. 2024 Jan.

Abstract

Widespread use of azole antifungals in agriculture has been linked to resistance in the pathogenic fungus Aspergillus fumigatus. We show that exposure of A. fumigatus to the agrochemical fungicide, ipflufenoquin, in vitro can select for strains that are resistant to olorofim, a first-in-class clinical antifungal with the same mechanism of action. Resistance is caused by non-synonymous mutations within the target of ipflufenoquin/olorofim activity, dihydroorotate dehydrogenase (DHODH), and these variants have no overt growth defects.

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Conflict of interest statement

M. J. Bromley is a former employee of F2G Ltd (until 2007), the company that developed the antifungal olorofim. He has received historic funding for PhD studentships from F2G Ltd but has no current financial interest in F2G Ltd. M.B. and J.D.O. are employed by F2G Ltd. Experiments carried out at the University of Manchester were done independently and without input or incumbrance from F2G Ltd. F2G Ltd contribution was solely in relation to the IC50 analysis of AfDHODH variants. All remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Ipflufenoquin acts against DHODH.
a, MIC determination of ipflufenoquin (0.175–50 mg l−1) against A. fumigatus MFIG001 in RPMI-1640 (black) using the EUCAST methodology and with addition of 10 mM uridine and uracil (grey). OD600 was measured after 48 h. Three biological replicates were assessed. Data are presented as mean ± s.e.m. b, Molecular docking of FMN and orotate (left in cyan and blue, respectively) and olorofim and ipflufenoquin (right, orange and purple, respectively) to an Alphafold2 model of A. fumigatus DHODH. The pocket of the active site is shown in grey. c, Protein inhibition assay of ipflufenoquin towards wildtype and G119 variants of DHODH. At least five biological replicates were assessed. Data are presented as mean ± s.e.m. (n = 5). d, MIC determination of G119 variants (G119C, G119S and G119A) against ipflufenoquin (right) and olorofim (left) according to EUCAST methodology. OD600 was measured after 48 h. Three biological replicates were assessed. Data are presented as mean ± s.e.m. Source data
Fig. 2
Fig. 2. Ipflufenoquin can select for olorofim-resistant A. fumigatus.
a, MIC determination of H116R, L164P and V200E variant mutants against ipflufenoquin and olorofim according to EUCAST methodology. OD600 was measured after 48 h. Three biological replicates were assessed. Data are presented as mean ± s.e.m. b, Protein inhibition assay of ipflufenoquin towards wildtype, V200E and H116R variants of DHODH. At least five biological replicates were assessed. Data are presented as mean ± s.e.m. (n = 5). c, Frequency of mutants after mixed-inoculum experiments to determine basal fitness of each mutant on solid and liquid MM medium. Three biological replicates were assessed and individual data from replicates are shown. d, Frequency of mutants after low (0.015 mg l−1) and high (0.12 mg l−1) olorofim challenge in a mixed-inoculum competition assay. Three biological replicates were assessed and individual data from replicates are shown. e, Frequency of mutants after low (3.125 mg l−1) and high (25 mg l−1) ipflufenoquin challenge in a mixed-inoculum competition assay. Three biological replicates were assessed and individual data from replicates are shown. Source data
Extended Data Fig. 1
Extended Data Fig. 1. MIC determination of olorofim and ipflufenoquin.
MIC determination of olorofim and ipflufenoquin against G119 variants of A. fumigatus. Three biological replicates were assessed.
Extended Data Fig. 2
Extended Data Fig. 2. MIC determination for natural A. fumigatus isolates of olorofim and ipflufenoquin.
A: Phylogenetic tree highlighting natural A. fumigatus isolates used (n = 30) sampled across a wide genetic diversity of the species, tree generated in Rhodes et al.. B and C: MIC determination of olorofim (B) and ipflufenoquin (C) against thirty natural A. fumigatus isolates. Three biological replicates were assessed per isolate. Data are presented as the mean with SEM. The MIC is shown as a dotted line. D: Correlation of the AUC to ipflufenoquin and olorofim of the 30 natural A. fumigatus isolates. Three biological replicates were assessed per isolate. Data are presented as the mean with SEM. Source data
Extended Data Fig. 3
Extended Data Fig. 3. An Alphafold2 model of A. fumigatus DHODH.
A: superposition of human DHODH (PDB: 2PRH_1, grey) and A. fumigatus DHODH (teal). The AfDHODH modelled region was Asp110-Leu523, which included a region of low per-residue confidence scores (pIDDT), Arg426-Val478, which is shown as a long loop. hDHODH and AfDHODH share 35% identify and 84% coverage across the full-length sequences. B: co-crystal structure of hDHODH (grey) in complex with orotate, shown in grey. The predicted pose of orotate from molecular docking with AfDHODH (teal) is superimposed. C: co-crystal structure of hDHODH (grey) in complex with FMN, shown in grey. The predicted pose of FMN from molecular docking with AfDHODH (teal) is superimposed. D: Predicted plDDT for 5 predicted structures from AlphaFold2. We used the rank 1 model. A score of <50 is considered very low confidence. Models show a region of low per-residue confidence, Arg316-Val368, which is shown as a long loop (A). However, this region was distal to the binding pocket discussed and therefore not of functional relevance to this work.
Extended Data Fig. 4
Extended Data Fig. 4. Rank 9 AfDHODH docking models.
Olorofim and ipflufenoquin were docked to the predicted active site of AfDHODH using VSPipe with the standard parameters. The top 9 poses are shown with olorofim in magenta and ipflufenoquin in orange. Binding energies and residues relevant for decreased susceptibility to olorofim and ipflufenoquin are displayed for each pose.
Extended Data Fig. 5
Extended Data Fig. 5. Sanger sequencing validation of G119 variants.
The pyrE gene was amplified by PCR and Sanger sequencing over the G119 position was performed. No other mutations within the pyrE coding region were observed. Source data
Extended Data Fig. 6
Extended Data Fig. 6. Determination of ipflufenoquin and olorofim against pmsA mutants.
A) workflow of fluctuation assays in A. fumigatus. Non-selective environment generate a genetically diverse population, for which resistant mutants are selected for on ipflufenoquin containing medium. B) MIC determination of olorofim and ipflufenoquin against G119 variants of A. fumigatus according to EUCAST methodology. OD600 was measured after 48 hours. Three biological replicates were assessed. Data are presented as the mean with SEM.
Extended Data Fig. 7
Extended Data Fig. 7. Sanger sequencing validation of H116R, L164P and V200E variants.
The pyrE gene was amplified by PCR and Sanger sequencing over the H116, L164 and V200 position was performed. No other mutations within the pyrE coding region were observed. Source data
Extended Data Fig. 8
Extended Data Fig. 8. AUC analysis of A. fumigatus isolates to olorofim and ipflufenoquin.
AUC of MIC curves to A: olorofim and B: ipflufenoquin presented in Figs. 1, 2 and Extended Data Fig. 2. Each point represents an independent replicate (N = 3 for pyrE mutants, N = 90 for natural isolates, N = 6 for MFIG001). Crossbar shows the mean within groups. Significant differences between groups calculated using two-sided t-tests with Bonferri correction for multiple comparisons, * < 0.05, ** < 0.01, *** < 0.001, (ANOVA A: olorofim F7,109 = 511, p < 0.0001, B: ipflufenoquin F7,109 = 141, p < 0.0001 (pairwise comparisons in Source Data).
Extended Data Fig. 9
Extended Data Fig. 9. Phenotypic characterisation of resistant mutants.
A) Representative images of spot assays on Sabouraud agar medium of MFIG001 and mutant isolates. B) Radial growth was measured after 48 hours on Sabouraud agar medium. Data are presented as the mean ± s.d. of three experiments. P values were determined by two-sided ANOVA with Tukey multiple comparison between means from a sample size of n = 3. C) Sporulation was measured after 3 days by inoculating MM cultures with equal spores. Spores were harvested through Miracloth and counted on a haemocytometer. Data are presented as the mean ± s.d. of three experiments. P values were determined by two-sided ANOVA from a sample size of n = 3. Source data
Extended Data Fig. 10
Extended Data Fig. 10. Competitive fitness in liquid RPMI-1640.
A) Relative abundance of MFIG001 and mutants in RPMI-1640. Abundance was calculated as the amount of reads per isolate normalised to total QC-passed sequencing reads. Data are presented for independent replicates of three experiments. B) Abundance of MFIG001 and mutants in low (0.015 mg/L) and high (0.12 mg/L) olorofim challenge in a mixed inoculum competition assay. Data are presented for independent replicates of three experiments. C) Abundance of MFIG001 and mutants in low (3.125 mg/L) and high (25 mg/L) ipflufenoquin challenge in a mixed inoculum competition assay. Data are presented for independent replicates of three experiments.
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