Background: There is a need for new antifungal drugs acting via novel mechanisms to combat an increasing incidence of serious invasive fungal disease. The limited existing systemic therapies have liabilities such as toxicities, drug-drug interactions, variable kinetics, and increasing resistance. F901318 is the most advanced member of a new class of antifungal agents, the orotomides, which have a novel mechanism of action distinct from those of currently available clinical antifungal agents.
Methods & Results: The orotomide class was discovered by whole cell screening of a small molecule chemical library against clinical fungal isolates. Initial hits were developed through iterative rounds of molecular design and whole cell susceptibility testing. F901318 is the most advanced analog from this program. The target of F901318 was identified as dihydroorotate dehydrogenase (DHODH) through a combination of microbiological, biochemical and molecular techniques. DHODH, an important enzyme in pyrimidine biosynthesis, was identified as the target of F901318 from a screen employing an Aspergillus nidulansgenomic library carried on an AMA plasmid. AMA transformants overexpressing DHODH were shown to be resistant to F901318. Knockout of the DHODH gene on the AMA plasmid restored sensitivity to F901318. Attempts to obtain mutants resistant to F901318 by repeated passage in the presence of drug failed. The antifungal action of F901318 in vitro is reversed by the addition of high concentrations of pyrimidines (circa 5mM, compared to a human serum concentration of 15 µM) confirming that the pyrimidine biosynthesis pathway is targeted in the whole cell. Inhibition of DHODH causes rapid cessation of fungal growth, and F901318 is a potent, competitive, reversible inhibitor of the recombinant Aspergillus fumigatus protein in vitro: IC50 = 44 nM +/- 10 nM (+/- standard deviation; n=11). The enzyme is also present in mammalian cells but F901318 is a very poor inhibitor of the human form of the enzyme (IC50 > 90 µM) giving a >2000 fold degree of selectivity.
Conclusion: F901318 belongs to a novel class of antifungal agents, the orotomides, which arrest pyrimidine biosynthesis via inhibition of DHODH. The discovery of the orotomides and F901318 represent an important step in the development of novel antifungal drugs to target systemic fungal infections in man.
- ICAAC 2015 (55th)