The Dynamic Influence of Olorofim (F901318) on the Cell Morphology and Organization of Living Cells of Aspergillus fumigatus

Abstract

The first characterized antifungal in the orotomide class is olorofim. It targets the de novo pyrimidine biosynthesis pathway by inhibiting dihydroorotate dehydrogenase (DHODH). The pyrimidines uracil, thymine and cytosine are the building blocks of DNA and RNA; thus, inhibition of their synthesis is likely to have multiple effects, including affecting cell cycle regulation and protein synthesis. Additionally, uridine-5′-triphosphate (UTP) is required for the formation of uridine-diphosphate glucose (UDP-glucose), which is an important precursor for several cell wall components. In this study, the dynamic effects of olorofim treatment on the morphology and organization of Aspergillus fumigatus hyphae were analyzed microscopically using confocal live-cell imaging. Treatment with olorofim led to increased chitin content in the cell wall, increased septation, enlargement of vacuoles and inhibition of mitosis. Furthermore, vesicle-like structures, which could not be stained or visualized with a range of membrane- or vacuole-selective dyes, were found in treated hyphae. A colocalization study of DHODH and MitoTracker Red FM confirmed for the first time that A. fumigatus DHODH is localized in the mitochondria. Overall, olorofim treatment was found to significantly influence the dynamic structure and organization of A. fumigatus hyphae.

Review of the Novel Investigational Antifungal Olorofim

“…UTP is also important for the formation of the DNA/RNA pyrimidine derivatives cytosine, thymine, and uracil, as well as cell cycle regulation. Following 24 h of olorofim exposure, mitosis within A. fumigatus was halted, which is consistent with cell cycle arrest, and this was postulated to be due to a decrease in cellular levels of pyrimidine, leading to an inability to fully replicate DNA [ 28 ]. In addition to de novo synthesis, fungi are also able to acquire pyrimidine by scavenging from the environment.…”

“…Thus, inhibition of pyrimidine synthesis by olorofim may affect the fungal cell wall and result in cell lysis [ 27 ]. An in vitro study reported that exposure of A. fumigatus hyphae to olorofim (0.1 μg/mL for 24 h) led to significant reductions in 1,3-β- d -glucan at the hyphal tips and at the periphery of the mycelium [ 28 ]. Interestingly, chitin content was significantly increased, which may be due to a compensatory mechanism that is known to occur with reduced 1,3-β- d -glucan levels following echinocandin exposure in different fungal species [ 29 , 30 ].…”

“…In addition to its effect on hyphal growth and viability, A. fumigatus exposure to olorofim has been shown to lead to increased hyphal septation, reduces the size of hyphal compartments, and increases in vacuolar volume [ 28 ]. The enlargement of vacuoles may be related to cell cycle arrest, as cytoplasmic volume may be an important trigger for the G1 cell cycle phase in which mRNA and proteins are synthesized in preparation for mitosis.…”

“…Highly vacuolated compartments are believed to be arrested in the G1 cell cycle phase. It has been postulated that large vacuole formation may be a sign of the activation of autophagy in response to olorofim exposure [ 28 ]. It is currently unknown if the wide range of effects observed against A. fumigatus also occur against different fungal species when exposed to olorofim.…”

DHODH and cancer: promising prospects to be explored

“…Moreover, DHODH inhibitors exhibited efficacy against virus, including respiratory syncytial virus [ 109 ], rotavirus [ 110 ], foot-and-mouth disease virus infection [ 111 ], flavivirus which contains dengue virus, Zika virus, and Japanese encephalitis virus [ 112 ], and notably, against RNA viruses including newly emerged coronavirus SARS-CoV-2 [ 113 ]. DHODH inhibitors are also found to be effective against oomycete phytophthora infestans [ 114 ], schistosomiasis [ 115 , 116 ], invasive fungal infections [ 117 ], eumycetoma [ 118 ], and malaria [ 119 – 122 ].…”