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. 2022 Dec;11(1):703-714.
doi: 10.1080/22221751.2022.2034485.

Resistance profiling of Aspergillus fumigatus to olorofim indicates absence of intrinsic resistance and unveils the molecular mechanisms of acquired olorofim resistance

Jochem B Buil  1   2 , Jason D Oliver  3 , Derek Law  3 , Tim Baltussen  1 , Jan Zoll  1   2 , Margriet W J Hokken  1 , Marlou Tehupeiory-Kooreman  1   2 , Willem J G Melchers  1   2 , Mike Birch  3 , Paul E Verweij  1   2
Affiliations

Resistance profiling of Aspergillus fumigatus to olorofim indicates absence of intrinsic resistance and unveils the molecular mechanisms of acquired olorofim resistance

Jochem B Buil et al. Emerg Microbes Infect. 2022 Dec.

Abstract

Olorofim (F901318) is a new antifungal currently under clinical development that shows both in vitro and in vivo activity against a number of filamentous fungi including Aspergillus fumigatus. In this study, we screened A. fumigatus isolates for intrinsic olorofim-resistant A. fumigatus and evaluated the ability of A. fumigatus to acquire an olorofim-resistant phenotype. No intrinsic resistance was found in 975 clinical A. fumigatus isolates. However, we found that isolates with increased olorofim MICs (> 8 mg/L) could be selected using a high number of conidia and olorofim exposure under laboratory conditions. Assessment of the frequency of acquired olorofim resistance development of A. fumigatus was shown to be higher than for voriconazole but lower than for itraconazole. Sequencing the PyrE gene of isogenic isolates with olorofim MICs of >8 mg/L identified various amino acid substitutions with a hotspot at locus G119. Olorofim was shown to have reduced affinity to mutated target protein dihydroorotate dehydrogenase (DHODH) and the effect of these mutations was proven by introducing the mutations directly in A. fumigatus. We then investigated whether G119 mutations were associated with a fitness cost in A. fumigatus. These experiments showed a small but significant reduction in growth rate for strains with a G119V substitution, while strains with a G119C substitution did not exhibit a reduction in growth rate. These in vitro findings were confirmed in an in vivo pathogenicity model.

Keywords: Aspergillosis; F901318; antifungal resistance; fungal; virulence.

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Conflict of interest statement

J.B reports grants from F2G Ltd and Gilead Sciences. J.O, D.L and M.B. are employees and shareholders of F2G Ltd. P.E. reports grants from Mundipharma, F2G Ltd, Pfizer, Gilead Sciences, and Cidara and nonfinancial support from IMMY for work outside the submitted study.

Figures

Figure 1.
Figure 1.
Olorofim resistance frequency. Frequency of resistance observed of six A. fumigatus isolates when 106–109 spores were incubated on RPMI agar plates containing either 0.5 mg/L olorofim (OLO), 4 mg/L voriconazole (VOR) or 8 mg/L itraconazole (ITC). A. fumigatus ATCC 204305 b. A. fumigatus AZN 8196 c. A. fumigatus V052-35 (TR34/L98H, azole resistant) d. A. fumigatus V139-36 e. A. fumigatus V180-37 and f. A. fumigatus V254-51. *P ≤ 0.05 **P ≤ 0.01, ns Not significant.
Figure 2.
Figure 2.
IC50s of wildtype and mutant DHODH. The inhibition of DHODH activity by a range of olorofim concentrations was measured for the recombinant wild type Af293 enzyme and the Gly119 mutants indicated. Lines were fitted using log (inhibitor) vs response – Variable slope (four parameters) in Graphpad Prism. R squares were 0.998 for Af_DHODH (WT), 0.556 for Af_DHODH (G119V), 0.924 for Af_DHODH (G119A), 1.000 for Af_DHODH (G119S) and 0.9680 for Af_DHODH (G119C).
Figure 3.
Figure 3.
Radial growth rate of isolate AZN8196 and Af293 and olorofim-resistant progeny. Colony diameters are displayed for (a) isolate AZN8196 and 2 olorofim-resistant progeny isolates AZN8196_OLR1 (G119V) and AZN8196_OLR2 (G119C) with and (b) Af293, Af293_OLR5 (G119S), Af293_OLR7 (G119C) Af293 OLR9 (G119V).
Figure 4.
Figure 4.
In vivo virulence model. Survival of mice inoculated with (a) olorofim wildtype strain AZN8196 and olorofim-resistant progeny AZN8196_OLR1 and AZN8196_OLR2, and (b) olorofim wildtype strain Af293 and olorofim-resistant progeny Af293_OLR5, Af293_OLR7 and Af293_OLR9. Eight mice were inoculated with each strain.
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References

    1. Bongomin F, Gago S, Oladele RO, et al. . Global and multi-national prevalence of fungal diseases-estimate precision. J Fungi. 2017 Oct 18;3(4):E57. - PMC - PubMed
    1. van de Veerdonk FL, Kolwijck E, Lestrade PP, et al. . Influenza-associated aspergillosis in critically ill patients. Am J Respir Crit Care Med. 2017 Aug 15;196(4):524–527. - PubMed
    1. Schauwvlieghe A, Rijnders BJA, Philips N, et al. . Invasive aspergillosis in patients admitted to the intensive care unit with severe influenza: a retrospective cohort study. Lancet Respir Med. 2018 Oct;6(10):782–792. - PubMed
    1. White PL, Dhillon R, Cordey A, et al. . A national strategy to diagnose COVID-19 associated invasive fungal disease in the ICU. Clin Infect Dis. 2020 Aug 29;73(7):e1634–e1644. - PMC - PubMed
    1. Lamoth F, Glampedakis E, Boillat-Blanco N, et al. . Incidence of invasive pulmonary aspergillosis among critically ill COVID-19 patients. Clin Microbiol Infect. 2020 Jul 10;26(12):1706–1708. - PMC - PubMed