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. IMPORTANCE: Aspergillosis is a spectrum of diseases and a major cause of morbidity and mortality. To treat these diseases, there are a few classes of antifungal drugs approved for clinical use. Resistance to the first line treatment, the azoles, is increasing. The first antifungal, olorofim, which is in the novel class of orotomides, is currently in development. Here, we showed an antagonistic effect between the azoles and olorofim, which was a result of dysregulation of the pyrimidine pathway, the target of olorofim, and the ergosterol biosynthesis pathway, the target of the azoles.This work was supported by the Wellcome Trust grant number 219551/Z/19/Z and 208396/Z/17/Z to M.J.B. C.V. was funded by a postdoctoral fellowship from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP-BEPE 2020/01131-5).S

Functional analysis of the Aspergillus fumigatus kinome reveals a DYRK kinase involved in septal plugging is a novel antifungal drug target

More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. The azole class of antifungals represent first line therapeutics for most of these infections however resistance is rising. Identification of novel antifungal targets that, when inhibited, synergise with the azoles will aid the development of agents that can improve therapeutic outcomes and supress the emergence of resistance. As part of the A. fumigatus genome-wide knockout program (COFUN), we have completed the generation of a library that consists of 120 genetically barcoded null mutants in genes that encode the protein kinase cohort of A. fumigatus. We have employed a competitive fitness profiling approach (Bar-Seq), to identify targets which when deleted result in hypersensitivity to the azoles and fitness defects in a murine host. The most promising candidate from our screen is a previously uncharacterised DYRK kinase orthologous to Yak1 of Candida albicans, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. Here we show that the orthologue YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress via phosphorylation of the Woronin body tethering protein Lah. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and impacts growth in murine lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit Yak1 in C. albicans prevents stress mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.</p