Azole-class compounds are widely employed as antifungals in the treatment of nosicomial and life-threatening fungal infections. The effectiveness of azole-class antibiotics is limited by their fungistatic rather than fungicidal mechanism of action resulting in the emergence of azole-resistant strains of many pathogenic species of fungi. Both of these problems can be averted by disabling the calcium signaling network of fungal cells, which becomes activated in response to antibiotics and acts as a defense mechanism that promotes survival of fungal cells during long-term exposure to the antibiotics. Remarkably, inhibitors of the calcium signaling network convert azole-class antibiotics from fungistats to fungicides. Two natural compounds that inhibit the fungal calcium signaling network (Cyclosporine and FK506) are known to be highly effective fungicidal co-drugs but they cannot be used to combat fungal infections because of their potent immunosuppressive effects in humans. Compounds that inhibit other fungi-specific components of the calcium signaling network would be ideal for improvement of antifungal therapies. We have recently identified a non-selective cation channel (NSCC) that functions at the apex of the calcium signaling network and we demonstrated that NSCCs are essential in yeasts for survival when exposed to azoles and other antibiotics. Being located at the cell surface and expressed only in fungi, the NSCC represents an ideal target for development of novel fungicidal co-drugs. Here we will develop cell-based assays that can be used for high-throughput screening of chemical libraries for compounds that specifically target fungal NSCCs. Specifically;we will express Nsc1 proteins from various species of pathogenic fungi in Nsc1-deficient mutants of Saccharomyces cerevisiae non-pathogenic model yeast) and determine the optimal environmental conditions in which the NSCCs inhibit yeast cell growth. Preliminary data show that NSCCs confer sensitivity to high sodium ions in the medium. Thus, NSCC inhibitors should restore yeast growth in simple culture media containing high salt. We will also develop optimal secondary assays using fluorescent stains and flow cytometry that directly measure cell death caused by NSCC-deficiency. The combination of cell-based growth and death assays will provide a powerful platform for the discovery of NSCC inhibitors.
A commonly prescribed class of antibiotics used to combat fungal infections can slow the growth of fungal pathogens but cannot kill them. By disabling fungal defenses with a secondary drug, these same antibiotics become potent fungicides. The research proposed here will develop primary and secondary assay methods that can be used for the identification of novel compounds that disable fungal defenses to common antibiotics. The assays focus on a highly drugable target enzyme - the NSCC - which occurs only in fungi. Therefore, the compounds identified by such an approach would have few side-effects in humans and massively increase the effectiveness of current antifungal antibiotics.
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