Amino Acid-Derived 1,2-Benzisothiazolinone Derivatives as Novel Small-Molecule Antifungal Inhibitors: Identification of Potential Genetic Targets

Abstract

We have identified four synthetic compounds (DFD-VI-15, BD-I-186, DFD-V-49, and DFD-V-66) from an amino acid-derived 1,2-benzisothiazolinone (BZT) scaffold that have reasonable MIC 50 values against a panel of fungal pathogens. These compounds have no structural similarity to existing antifungal drugs. Three of the four compounds have fungicidal activity against Candida spp., Cryptococcus neoformans, and several dermatophytes, while one is fungicidal to Aspergillus fumigatus. The kill rates of our compounds are equal to those in clinical usage. The BZT compounds remain active against azole-, polyene-, and micafungin-resistant strains of Candida spp. A genetics-based approach, along with phenotype analysis, was used to begin mode of action (MOA) studies of one of these compounds, DFD-VI-15. The genetics-based screen utilized a homozygous deletion collection of approximately 4,700 Saccharomyces cerevisiae mutants. We identified mutants that are both hypersensitive and resistant. Using FunSpec, the hypersensitive mutants and a resistant ace2 mutant clustered within a category of genes related directly or indirectly to mitochondrial functions. In Candida albicans, the functions of the Ace2p transcription factor include the regulation of glycolysis. Our model is that DFD-VI-15 targets a respiratory pathway that limits energy production. Supporting this hypothesis are phenotypic data indicating that DFD-VI-15 causes increased cell-reactive oxidants (ROS) and a decrease in mitochondrial membrane potential. Also, the same compound has activity when cells are grown in a medium containing glycerol (mitochondrial substrate) but is much less active when cells are grown anaerobically.

Antifungal Drug Discovery: The Process and Outcomes

“…An advantage here is that a minimal level of toxicity of ‘repurposed’ compounds may have already been established. A second type of library is compounds that are derived from newly synthesized synthetic scaffolds (Table 1) [16]. The activity of a specific scaffold is determined followed by lead optimization to identify the ‘most’ active synthetic derivative(s) of that scaffold.…”

“…In addition to the use of the DTP library as a source of compounds, we have screened a group of synthetic, low-molecular-weight compounds that were provided to the laboratory through an ongoing collaboration [16,31]. Scaffolds such as the amino acid-derived 1,2-benzisothiazolinone (BZT; BD-I-186) scaffold were screened against pathogens that included Candida species Cryptococcus neoformans, Aspergillus fumigatus and several dermatophytic fungi [16,31].…”

“…Scaffolds such as the amino acid-derived 1,2-benzisothiazolinone (BZT; BD-I-186) scaffold were screened against pathogens that included Candida species Cryptococcus neoformans, Aspergillus fumigatus and several dermatophytic fungi [16,31]. Lead optimization of the BZT scaffold identified structure–activity relationships (SARs) that established the importance of a heterocyclic ring, a methyl group and a phenyl ring for optimal antifungal activity.…”

“…These compounds have no structural similarity to existing antifungal drugs [31]. All compounds caused synergy with fluconazole, had fungicidal concentrations that were similar to micafungin and nearly that of AmpB, had effective time-kill (fungicidal concentrations) and minimal in vitro toxicity against human cell lines (3 of 4 compounds) [16]. …”

“…One of the compounds (BD-I-186) displayed no toxicity in vitro and had very good activity against several fungal pathogens mentioned above (Table 2) [16] [Gay-Andrieu F, Groutas W, Li D, Alpha M, Calderone R, Unpublished Data]. Using the S. cerevisiae 6000+ heterozygous mutant library, which allows for the identification of haploinsufficiency or reduced fitness mutants, mentioned below in detail, we have demonstrated that this compound appears to target the kinetochore complex of S. cerevisiae [Gay-Andrieu F, Groutas W, Li D, Alpha M, Calderone R, Unpublished Data].…”