Wider properties of olorofim, a novel orotomide compound

  • Ressa De Radigues

Student thesis: Unknown

Abstract

There is an unmet need for novel antifungal drugs, with resistance to the available drugs on the rise. Olorofim is a novel, first-in-class antifungal agent targeting the fourth enzyme, the class 2 DHODH, in the de novo pyrimidine biosynthesis pathway. It has good potency against Aspergillus spp, Scedosporium spp, Lomentospora spp, some dimorphic fungi such as Talaromyces and some species of the genus Fusarium. However, some clinically relevant Fusarium spp present with variable MICs: Neocosmospora solani (previously F. solani) is not susceptible to olorofim and presents with MICs of >8 ug/mL, while F. oxysporum has slightly higher MICs than Aspergillus spp at 0.5 ug/mL. Through bioinformatical means a second DHODH-like gene, a putative class 1A, was identified within the Fusarium spp genomes. Characterization of the DHODH repertoire of these species was accomplished through a variety of methods. These include Fusarium spp DHODH gene annotation, RNAseq in the presence of olorofim, DHODH protein modelling and the docking of olorofim. Recombinant constructs of both DHODH-like genes were expressed bacterially, purified and their activity tested. The recombinant Fusarium spp class 2 DHODH were also tested for olorofim inhibition. Lastly, a complementation study was undertaken utilizing A. fumigatus TetOFF:DHODH strain as background. The Fusarium spp putative class 1A or 2 DHODH genes were coupled to two different promoters and phenotypes of the constructs were studied in the presence and absence of pyrimidines. It was found that F. oxysporum has a much higher transcriptional response to olorofim than that of N. solani. Olorofim seemed to impact the nitrogen assimilation pathway and affect chitin-associated gene expression. There was no differential expression of either putative class 1A or class 2 DHODH upon exposure to olorofim in either strain of Fusarium spp. The expressed putative class 1A DHODH enzyme from N. solani or F. oxysporum were not active under these conditions but class 2 DHODH from both strains were active. The class 2 DHODH enzyme of F. oxysporum was inhibited by the drug but the N. solani enzyme was not susceptible to olorofim in these conditions. The complementation study showed that N. solani and F. oxysporum putative class 1A DHODH cannot rescue the growth of A. fumigatus TetOFF:DHODH but class 2 can. In conclusion, it appears that the class 1A DHODH gene is not responsible for raised olorofim MICs in N. solani or F. oxysporum. This means that the Fusarium studied do not utilize a second DHODH when class 2 DHODH is inhibited by olorofim. It also shows that the N. solani class 2 DHODH is not inhibited by olorofim, but the F. oxysporum enzyme remains sensitive to the drug. This work provides further data recognizing olorofim as a valid treatment for some Fusarium infections but that it should be treated on a species-specific basis due to differences in the target class 2 DHODH and not the presence of a functional class 1A DHODH.
Date of Award1 Aug 2023
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorMichael Bromley (Supervisor) & Paul Bowyer (Supervisor)

Keywords

  • CRISPR/cas9
  • heterologus protein expression
  • olorofim
  • antifungal drug discovery
  • fungal infection

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