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

Aspergillosis, in its various manifestations, is a major cause of morbidity and mortality. Very few classes of antifungal drugs have been approved for clinical use to treat these diseases and resistance to the first-line therapeutic class, the triazoles are increasing. A new class of antifungals that target pyrimidine biosynthesis, the orotomides, are currently in development with the first compound in this class, olorofim in late-stage clinical trials. In this study, we identified an antagonistic action of the triazoles on the action of olorofim. We showed that this antagonism was the result of an azole-induced upregulation of the pyrimidine biosynthesis pathway. Intriguingly, we showed that loss of function in the higher order transcription factor, HapB a member of the heterotrimeric HapB/C/E (CBC) complex or the regulator of nitrogen metabolic genes AreA, led to cross-resistance to both the azoles and olorofim, indicating that factors that govern resistance were under common regulatory control. However, the loss of azole-induced antagonism required decoupling of the pyrimidine biosynthetic pathway in a manner independent of the action of a single transcription factor. Our study provided evidence for complex transcriptional crosstalk between the pyrimidine and ergosterol biosynthetic pathways.