Overcoming Fungal Echinocandin Resistance through Inhibition of the Non-essential Stress Kinase Yck2.

New strategies are urgently needed to counter the threat to human health posed by drug-resistant fungi. To explore an as-yet unexploited target space for antifungals, we screened a library of protein kinase inhibitors for the ability to reverse resistance of the most common human fungal pathogen, Candida albicans, to caspofungin, a widely used antifungal. This screen identified multiple 2,3-aryl-pyrazolopyridine scaffold compounds capable of restoring caspofungin sensitivity. Using chemical genomic, biochemical, and structural approaches, we established the target for our most potent compound as Yck2, a casein kinase 1 family member. Combination of this compound with caspofungin eradicated drug-resistant C. albicans infection while sparing co-cultured human cells. In mice, genetic depletion of YCK2 caused an ∼3-log10 decline in fungal burden in a model of systemic caspofungin-resistant C. albicans infection. Structural insights and our tool compound's profile in culture support targeting the Yck2 kinase function as a broadly active antifungal strategy.

Functional divergence of a global regulatory complex governing fungal filamentation.

Morphogenetic transitions are prevalent in the fungal kingdom. For a leading human fungal pathogen, Candida albicans, the capacity to transition between yeast and filaments is key for virulence. For the model yeast Saccharomyces cerevisiae, filamentation enables nutrient acquisition. A recent functional genomic screen in S. cerevisiae identified Mfg1 as a regulator of morphogenesis that acts in complex with Flo8 and Mss11 to mediate transcriptional responses crucial for filamentation. In C. albicans, Mfg1 also interacts physically with Flo8 and Mss11 and is critical for filamentation in response to diverse cues, but the mechanisms through which it regulates morphogenesis remained elusive. Here, we explored the consequences of perturbation of Mfg1, Flo8, and Mss11 on C. albicans morphogenesis, and identified functional divergence of complex members. We observed that C. albicans Mss11 was dispensable for filamentation, and that overexpression of FLO8 caused constitutive filamentation even in the absence of Mfg1. Harnessing transcriptional profiling and chromatin immunoprecipitation coupled to microarray analysis, we identified divergence between transcriptional targets of Flo8 and Mfg1 in C. albicans. We also established that Flo8 and Mfg1 cooperatively bind to promoters of key regulators of filamentation, including TEC1, for which overexpression was sufficient to restore filamentation in the absence of Flo8 or Mfg1. To further explore the circuitry through which Mfg1 regulates morphogenesis, we employed a novel strategy to select for mutations that restore filamentation in the absence of Mfg1. Whole genome sequencing of filamentation-competent mutants revealed chromosome 6 amplification as a conserved adaptive mechanism. A key determinant of the chromosome 6 amplification is FLO8, as deletion of one allele blocked morphogenesis, and chromosome 6 was not amplified in evolved lineages for which FLO8 was re-located to a different chromosome. Thus, this work highlights rewiring of key morphogenetic regulators over evolutionary time and aneuploidy as an adaptive mechanism driving fungal morphogenesis.