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

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.

The importance of antimicrobial resistance in medical mycology

“…Thus, both olorofim and manogepix may offer hope against resistant pathogens. Nonetheless, it must be noted that resistance has been observed in vitro with exposure to each of these therapeutic candidates 105 – 107 .…”

The novel agrochemical fungicide ipflufenoquin drives cross-resistance to olorofim in the human pathogen Aspergillus fumigatus.

“…Using an Alphafold2 generated model of the A. fumigatus (Af)DHODH (Extended Data 2) we predict that both ipflufenoquin and olorofim bind within the active site of the enzyme (binding energies: orotate -7.0 kcalm/mol, FMN: -10.8 kcal/mol, olorofim: -8.2 kcal/mol, ipflufenoquin: -9.2 kcal/mol) (Figure 1B) with the larger olorofim molecule but not ipflufenoquin extending from the active site to a region proximal to G119. Furthermore, we clearly demonstrate that ipflufenoquin directly inhibits the enzymatic activity of AfDHODH, although the IC50 is more than 15 times higher than that previously observed for olorofim (IC50: 0.77 µM ipflufenoquin vs 0.044 µM olorofim, Figure 1C) [14,17].…”

Antagonism of the Azoles to Olorofim and Cross-Resistance Are Governed by Linked Transcriptional Networks in Aspergillus fumigatus

“…The changes in sensitivity in these isolates were less extreme than seen for the azoles, indicating that the frequency of olorofim resistance may be lower than that seen for itraconazole. Indeed, this hypothesis was supported by a recent study that revealed the frequency of olorofim resistance was variable between strains ranging from 1.3 × 10 −7 to 6.9 × 10 −9 , while for itraconazole resistance occurred at an order of magnitude higher (1.2 × 10 −6 and 3.3 × 10 −8 ) ( 23 ). It is unsurprising, given the mechanism of action of olorofim, that the transcription factors that we identified in this screen either have well-defined roles in regulating nitrogen utilization or have been linked to this function in our study.…”

“…A recent study suggests that levels of resistance to olorofim in a collection of clinical isolates of A. fumigatus are low. Only 1 of 976 clinical isolates exhibited preexisting olorofim resistance caused by a single nucleotide polymorphism (SNP) in the pyrE gene ( 23 ).…”