Invasive fungal infections are responsible for an estimated 1.5 million deaths per year. At the moment, azole antifungal therapy is the first line treatment option, but issues such as drug-drug interactions, toxicity, variable PK and emerging resistance make the discovery of new antifungal agents acting via new mechanisms an important task. F901318 is the lead candidate of the novel class of orotomide antifungals and acts via inhibition of DHODH, a key enzyme in pyrimidine biosynthesis, a mechanism not shared with current antifungals. F901318 has a mould-only spectrum and has excellent in vitro activity against all the clinically significant Aspergillus spp. with MICs below 0.03 μg/ml.
In this study we synthesised several fluorescent analogues of F901318 and investigated their antifungal activity, uptake and cellular localisation and distribution
A total of five fluorescent variants of F901318 have been synthesised by conjugating an F901318 analogue to a fluorophore. These analogues have been tested for spectrum and antifungal activity using fluorescence measurements, MIC tests and DHODH inhibition assays. Uptake, localisation and distribution of the most active compound, F901848, by Aspergilli was further analysed using confocal fluorescence microscopy.
Although displaying a good fluorescent signal, most analogues lacked potent antifungal activity compared to F901318. F901848 showed the best activity with MICs of 0.8 μg/ml against A. niger and A. flavus and inhibited A. niger DHODH with an IC50 of 37 nM. This compound is conjugated to fluorophore 4-chloro-7-nitrobenzofurazan (NBD-Cl) and fluorescence was observed at an excitation/emission of 490/550 nm. Confocal microscopy with A. flavus showed that F901848 was rapidly taken up by A. flavus within minutes and was spread throughout the hyphae.
Together our results show that fluorescent compound analogues can be used to study uptake, localisation and distribution of compounds by Aspergillus spp. The successful design of a fluorescent version of F901318 that retains some DHODH inhibition and antifungal activity has provided a useful tool for further investigation of orotomide action.