laminaran and cercosporamide

The Pkc1-mediated cell wall integrity-signaling pathway is highly conserved in fungi and is essential for fungal growth. We thus explored the potential of targeting the Pkc1 protein kinase for developing broad-spectrum fungicidal antifungal drugs through a Candida albicans Pkc1-based high-throughput screening. We discovered that cercosporamide, a broad-spectrum natural antifungal compound, but previously with an unknown mode of action, is actually a selective and highly potent fungal Pkc1 kinase inhibitor. This finding provides a molecular explanation for previous observations in which Saccharomyces cerevisiae cell wall mutants were found to be highly sensitive to cercosporamide. Indeed, S. cerevisiae mutant cells with reduced Pkc1 kinase activity become hypersensitive to cercosporamide, and this sensitivity can be suppressed under high-osmotic growth conditions. Together, the results demonstrate that cercosporamide acts selectively on Pkc1 kinase and, thus, they provide a molecular mechanism for its antifungal activity. Furthermore, cercosporamide and a beta-1,3-glucan synthase inhibitor echinocandin analog, by targeting two different key components of the cell wall biosynthesis pathway, are highly synergistic in their antifungal activities. The synergistic antifungal activity between Pkc1 kinase and beta-1,3-glucan synthase inhibitors points to a potential highly effective combination therapy to treat fungal infections.

Topics: Amphotericin B; Animals; Antifungal Agents; Benzofurans; beta-Glucans; Biological Assay; Candida albicans; Drug Synergism; Enzyme Activation; Fungal Proteins; Gene Expression Regulation, Fungal; Glucosyltransferases; Humans; Isoenzymes; Microbial Sensitivity Tests; Molecular Structure; Phosphatidylserines; Protein Kinase C; Protein Kinase Inhibitors

k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.

Topics: Amino Acid Sequence; Base Sequence; Benzofurans; beta-Glucans; Blotting, Western; Chromosomes, Fungal; Cloning, Molecular; DNA, Fungal; Drug Resistance, Microbial; Fungal Proteins; Genes, Fungal; Glucans; Glucosyltransferases; Hydrolases; Killer Factors, Yeast; Kinetics; Membrane Proteins; Molecular Sequence Data; Molecular Weight; Open Reading Frames; Pichia; Protein Biosynthesis; Proteins; Recombinant Proteins; Restriction Mapping; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Schizosaccharomyces pombe Proteins; Sequence Homology, Amino Acid; Transcription Factors