Characterisation of putative class 1A DHODH-like proteins from Mucorales and dematiaceous mould species

Abstract

Olorofim is a new antifungal in clinical development which has a novel mechanism of action against dihydroorotate dehydrogenase (DHODH). DHODH form a ubiquitous family of enzymes in the de novo pyrimidine biosynthetic pathway and are split into class 1A, class 1B and class 2. Olorofim specifically targets the fungal class 2 DHODH present in a range of pathogenic moulds. The nature and number of DHODH present in many fungal species have not been addressed for large clades of this kingdom. Mucorales species do not respond to olorofim; previous work suggests they have only class 1A DHODH and so lack the class 2 target that olorofim inhibits. The dematiaceous moulds have mixed susceptibility to olorofim, yet previous analyses imply that they have class 2 DHODH. As this is at odds with their intermediate susceptibility to olorofim, we hypothesised that these pathogens may maintain a second class of DHODH, facilitating pyrimidine biosynthesis in the presence of olorofim. The aim of this study was to investigate the DHODH repertoire of clinically relevant species of Mucorales and dematiaceous moulds to further characterise these pathogens and understand variations in olorofim susceptibility. Using bioinformatic analysis, S. cerevisiae complementation and biochemical assays of recombinant protein, we provide the first evidence that two representative members of the Mucorales have only class 1A DHODH, substantiating a lack of olorofim susceptibility. In contrast, bioinformatic analyses initially suggested that seven dematiaceous species appeared to harbour both class 1A-like and class 2-like DHODH genes. However, further experimental investigation of the putative class 1A-like genes through yeast complementation and biochemical assays characterised them as dihydrouracil oxidases rather than DHODHs. These data demonstrate variation in dematiaceous mould olorofim susceptibility is not due to a secondary DHODH and builds on the growing picture of fungal dihydrouracil oxidases as an example of horizontal gene transfer.

Comprehensive Review on the Virulence Factors and Therapeutic Strategies with the Aid of Artificial Intelligence against Mucormycosis

“…The absence of antifungal efficacy in Olorofim, class 2 DHODH inhibitor, against Mucorales species was attributed to the presence of class 1 DHODH in Mucorales, which is significantly different from the class 2 DHODH found in susceptible fungal species, hence making it a novel therapeutic target. Loss of this enzyme was connected to cell wall integrity and increased sensitivity to cell wall-damaging chemicals including amphotericin B by Banerjee et al, and Pinder et al further confirmed that the Mucorales species M. circinelloides and R. arrhizus contain active class 1A DHODH. − …”

The Role of Olorofim in the Treatment of Filamentous Fungal Infections: A Review of In Vitro and In Vivo Studies

“…and Cryptococcus spp., remain unaffected due to nucleotide modifications throughout the years affecting the enzyme's active site [3]. Additionally, the inability to affect class 1A DHODH in some Mucorales may explain olorofim's natural inefficacy against this fungal group [30].…”