The novelty of our studies starts first with the structures of active compounds, heretofore not reported (18
). Further, from our current data, we have established that compounds with a BZT core structure have antifungal activity against a variety of human pathogen isolates, including dermatophytic fungi ( and ). The compounds are for the most part fungicidal, have kill-time ratios that compare favorably to those of amphotericin B and micafungin, and are inhibitory against a variety of clinical isolates of Candida
spp. that are resistant to triazoles, micafungin, and amphotericin B.
We realize that the BZT compounds have only modest activity (μg/ml) against our panel of fungal pathogens, but their fungicidal activity compelled us to understand the MOA of one (DFD-VI-15). Initial experiments on MOA employed macromolecular synthesis determinations in treated and untreated cultures. However, inhibition of protein, RNA, or ergosterol synthesis by DFD-VI-15 was not observed after 1 to 3 h of treatment (data not included). Also, while from the same scaffold, unpublished data indicate that BD-I-186 may target cell separation, suggesting a different MOA.
In this paper, we placed considerable importance on chemogenomics to understand the MOA of DFD-VI-15. Chemogenomics is the application of genomics with chemistry (5
) in the pursuit of target discovery. As DFD-VI-15 was significantly active against S. cerevisiae
= 6 μg/ml, YPD broth), the availability of mutant libraries of yeast provided an attractive approach to MOA studies. We chose initially to use the homozygous null, diploid library of ~4,700 nonessential gene mutants. The prevailing paradigm is that nontarget gene products are believed to buffer or protect (or even compensate for) an inhibited target (52
). Mutants lacking these genes have a hypersensitivity phenotype. Oppositely, resistant mutants can represent the target since the lack of a target confers resistance. Susceptibility assays identified hypersensitive and resistant mutants. Cluster analysis using FunSpec and phenotype experiments have helped us construct a hypothetical MOA model.
Our screens yielded 31 mutants that were hypersensitive to DFD-VI-15, and of these mutants, 11 lacked mitochondrial or mitochondrium-related encoding genes. The next largest category of hypersensitive mutants (five) lacked genes involved in amino acid biosynthesis. Other categories include regulators of hyphal growth, protein sorting, iron utilization, chromatin remodeling, and stress adaptation. Annotation data for 27 mutants are shown in . The alignment of 11 genes associated directly or indirectly with mitochondrial functions follows that of other investigators. For example, Altmann and Westermann (2
) utilized a library of 768 S. cerevisiae
mutants lacking essential genes to identify those genes that were required for normal mitochondrial morphogenesis. Of these, 119 essential genes that clustered in essential cell pathways that included ergosterol biosynthesis, mitochondrial import and assembly, vesicular trafficking and secretion, actin cytoskeleton-dependent transport, and ubiqutin/26S protesome-dependent protein degradation were described. Our list of genes of hypersensitive mutants includes those functional annotations that are required for normal mitochondrial morphology as described previously (2
We also identified three mutants that were resistant to DFD-VI-15, including one lacking ACE2
. In C. albicans
mutant has been also implicated in a number of functions, and it is important to our current observations, as it is a positive regulator of glycolysis (31
). Genes encoding glycolytic metabolism were downregulated while respiratory genes associated with the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and ATP synthesis were upregulated in the C. albicans ace2
null mutant (41
). The resistance of the ACE2
mutant to DFD-VI-15 may be explained by a compensatory change that elevates mitochondrial functions and, consequently, survival of treated cells. However, our phenotype data suggest that DFD-VI-15 inhibits mitochondrial respiration, as parental cells have mitochondrial defects, including increased ROS and membrane depolarization. Further, anaerobically grown cells (especially S. cerevisiae
) are more resistant to the compound, pointing to a role for aerobic respiration as inhibited by DFD-VI-15.
We advocate mitochondria as rich sources of antifungal targets for several reasons. First, there are significant fungus- and even Candida
-specific proteins and pathways of mitochondrial respiration. For example, most fungi, but not S. cerevisiae
, have an alternative oxidase (AOX) pathway and a parallel pathway (PAR) not found in mammalian cells (1
). Most, but not all, of the complex I proteins of the electron transport chain (ETC) are highly conserved. Second, mitochondria are essential for energy production and, consequently, many cell functions. Therefore, inhibition of fungus-specific proteins should affect numerous cell activities. Third, published data indicate that inhibition of respiratory complexes and the AOX pathway by antimycin and benzohydroxamate (BHAM), respectively, convert caspofungin-resistant strains of C. parapsilosis
to a hypersensitivity phenotype (13
). Fourth, inhibitors of mitochondrial targets are even suggested for the treatment of human diseases such as type 2 diabetes, certain cancers, and neurodegenerative diseases (28
). Exploitation of mitochondrial targets is compelling, especially given the lack of new antifungal targets.