Screening of chemical libraries for new antifungal drugs against Aspergillus fumigatus reveals the potential mechanism of action of miltefosine

Abstract

Aspergillus fumigatus is an important fungal pathogen and the main etiological agent of aspergillosis, a disease characterized by a noninvasive process that can evolve to a more severe clinical manifestation called invasive pulmonary aspergillosis (IPA) in immunocompromised patients. The antifungal arsenal to threat aspergillosis is very restricted. Azoles are the main therapeutic approach to control IPA, but the emergence of azole-resistant A. fumigatus isolates has significantly increased over the last decades. Therefore, new strategies are necessary to combat aspergillosis and drug repurposing has emerged as an efficient and alternative approach for identifying new antifungal drugs. Here, we used a screening approach to analyze A. fumigatus in vitro susceptibility to 1,127 compounds. A. fumigatus was more susceptible to 10 compounds, including miltefosine, a drug that displayed fungicidal activity against A. fumigatus. By screening an A. fumigatus transcription factor null library, we identified a single mutant, which has the rmiA (resistant to miltefosine) gene deleted, conferring a phenotype of susceptibility to miltefosine. The transcriptional profiling (RNA-seq) of the wild-type and the {Delta}rmiA strains and the Chromatin Immunoprecipitation coupled to next generation sequencing (ChIP-Seq) of a RmiA-tagged strain exposed to miltefosine revealed genes of the sphingolipids pathway that are directly or indirectly regulated by RmiA. Sphingolipids analysis demonstrated that the mutant has overall decreased levels of sphingolipids when growing in the presence of miltefosine. The identification of RmiA represents the first genetic element described and characterized which plays a direct role in miltefosine response in fungi. Author summaryThe filamentous fungus Aspergillus fumigatus causes a group of diseases named aspergillosis and their development occurs after the inhalation of conidia dispersed in the environment. Very few classes of antifungal drugs are available for aspergillosis treatment, e.g., azoles, but the emergence of global resistance to azoles in A. fumigatus clinical isolates has increased over the last decades. Repositioning or repurposing drugs already available on the market is an interesting and faster opportunity for the identification of novel antifungals agents. By using a repurposing strategy, we identified 10 different compounds that impact A. fumigatus survival. One of these compounds, miltefosine, demonstrated fungicidal activity against A. fumigatus. The mechanism of action of miltefosine is unknown and aiming to get more insights about it, we identified a transcription factor RmiA (Resistant to miltefosine) important for miltefosine resistance. Our results suggest that miltefosine plays antifungal activity against A. fumigatus interfering in the sphingolipids biosynthesis.