Evaluation of Gyp7 Protein Ability to Coordinate and Regulate Mitochondrial Genomes Stability

Cellular creation of adenosine triphosphate, ATP, is essential for eukaryotic cells to function properly. The ATP molecule drives most of the biochemical and metabolic pathways of the cell. The cell\u27s ATP is produced in the mitochondria. Mutations within the genome of the mitochondria will alter the cell\u27s ability to generate A TP. Preliminary work has shown that loss of the Gyp 7p in Saccharomyces cerevisiae blocks the ability of mitochondria to properly function. The Gyp 7 gene was isolated using a technique called two-hybrid analysis with a known mitochondrial protein called llvSp, which was used as \u27bait\u27. We have shown that a deletion of the Gyp7 gene is not essential for cellular viability in S. cerevisiae. We observed that loss of Gyp 7 decreases both the occurrence of point mutations at microsatellite sequences as well as decreasing the rate at which recombination between direct-repeat DNA sequences occurs. This contributes to the effective that cellular respiration mutation rate increase when Gyp7p is removed. Gyp7 encodes for the production of the GTPase-activating protein (GAP) Gyp7p within the Ypt/Rab transport GTPase pathway. This pathway is involved in protein trafficking within the cell

Cryptococcus neoformans phosphoinositide-dependent kinase 1 (PDK1) ortholog is required for stress tolerance and survival in murine phagocytes

Cryptococcus neoformans PKH2-01 and PKH2-02 are orthologous to mammalian PDK1 kinase genes. Although orthologs of these kinases have been extensively studied in S. cerevisiae, little is known about their function in pathogenic fungi. In this study, we show that PKH2-02 but not PKH2-01 is required for C. neoformans to tolerate cell wall, oxidative, nitrosative, and antifungal drug stress. Deletion of PKH2-02 leads to decreased basal levels of Pkc1 activity and, consequently, reduced activation of the cell wall integrity mitogen-activated protein kinase (MAPK) pathway in response to cell wall, oxidative, and nitrosative stress. PKH2-02 function also is required for tolerance of fluconazole and amphotericin B, two important drugs for the treatment of cryptococcosis. Furthermore, OSU-03012, an inhibitor of human PDK1, is synergistic and fungicidal in combination with fluconazole. Using a Galleria mellonella model of low-temperature cryptococcosis, we found that PKH2-02 is also required for virulence in a temperature-independent manner. Consistent with the hypersensitivity of the pkh2-02Δ mutant to oxidative and nitrosative stress, this mutant shows decreased survival in murine phagocytes compared to that of wild-type (WT) cells. In addition, we show that deletion of PKH2-02 affects the interaction between C. neoformans and phagocytes by decreasing its ability to suppress production of tumor necrosis factor alpha (TNF-α) and reactive oxygen species. Taken together, our studies demonstrate that Pkh2-02-mediated signaling in C. neoformans is crucial for stress tolerance, host-pathogen interactions, and both temperature-dependent and -independent virulence

Antifungal Activity of Tamoxifen: In Vitro and In Vivo Activities and Mechanistic Characterization▿ †

Tamoxifen (TAM), an estrogen receptor antagonist used primarily to treat breast cancer, has well-recognized antifungal properties, but the activity of TAM has not been fully characterized using standardized (i.e., CLSI) in vitro susceptibility testing, nor has it been demonstrated in an in vivo model of fungal infection. In addition, its mechanism of action remains to be clearly defined at the molecular level. Here, we report that TAM displays in vitro activity (MIC, 8 to 64 μg/ml) against pathogenic yeasts (Candida albicans, other Candida spp., and Cryptococcus neoformans). In vivo, 200 mg/kg of body weight per day TAM reduced kidney fungal burden (−1.5 log10 CFU per g tissue; P = 0.008) in a murine model of disseminated candidiasis. TAM is a known inhibitor of mammalian calmodulin, and TAM-treated yeast show phenotypes consistent with decreased calmodulin function, including lysis, decreased new bud formation, disrupted actin polarization, and decreased germ tube formation. The overexpression of calmodulin suppresses TAM toxicity, hypofunctional calmodulin mutants are hypersensitive to TAM, and TAM interferes with the interaction between Myo2p and calmodulin, suggesting that TAM targets calmodulin as part of its mechanism of action. Taken together, these experiments indicate that the further study of compounds related to TAM as antifungal agents is warranted

Structure-activity relationships for the antifungal activity of selective estrogen receptor antagonists related to tamoxifen.

Cryptococcosis is one of the most important invasive fungal infections and is a significant contributor to the mortality associated with HIV/AIDS. As part of our program to repurpose molecules related to the selective estrogen receptor modulator (SERM) tamoxifen as anti-cryptococcal agents, we have explored the structure-activity relationships of a set of structurally diverse SERMs and tamoxifen derivatives. Our data provide the first insights into the structural requirements for the antifungal activity of this scaffold. Three key molecular characteristics affecting anti-cryptococcal activity emerged from our studies: 1) the presence of an alkylamino group tethered to one of the aromatic rings of the triphenylethylene core; 2) an appropriately sized aliphatic substituent at the 2 position of the ethylene moiety; and 3) electronegative substituents on the aromatic rings modestly improved activity. Using a cell-based assay of calmodulin antagonism, we found that the anti-cryptococcal activity of the scaffold correlates with calmodulin inhibition. Finally, we developed a homology model of C. neoformans calmodulin and used it to rationalize the structural basis for the activity of these molecules. Taken together, these data and models provide a basis for the further optimization of this promising anti-cryptococcal scaffold

High-Throughput Screen in Cryptococcus neoformans Identifies a Novel Molecular Scaffold That Inhibits Cell Wall Integrity Pathway Signaling

Cryptococcus neoformans is one of the most important human fungal pathogens; however, no new therapies have been developed in over 50 years. Fungicidal activity is crucially important for an effective anticryptococal agent and, therefore, we screened 361,675 molecules against C. neoformans using an adenylate kinase release assay that specifically detects fungicidal activity. A set of secondary assays narrowed the set of hits to molecules that interfere with fungal cell wall integrity and identified three benzothioureas with low in vitro mammalian toxicity and good in vitro anticryptococcal (minimum inhibitory concentration = 4 μg/mL). This scaffold inhibits signaling through the cell wall integrity MAP kinase cascade. Structure–activity studies indicate that the thiocarbonyl moiety is crucial for activity. Genetic and biochemical data suggest that benzothioureas inhibit signaling upstream of the kinase cascade. Thus, the benzothioureas appear to be a promising new scaffold for further exploration in the search for new anticryptococcal agents

Antifungal Phenothiazines: Optimization, Characterization of Mechanism, and Modulation of Neuroreceptor Activity

New classes of antifungal drugs are an urgent unmet clinical need. One approach to the challenge of developing new antifungal drugs is to optimize the antifungal properties of currently used drugs with favorable pharmacologic properties, so-called drug or scaffold repurposing. New therapies for cryptococcal meningitis are particularly important given its worldwide burden of disease and limited therapeutic options. We report the first systematic structure–activity study of the anticryptococcal properties of the phenothiazines. We also show that the antifungal activity of the phenothiazine scaffold correlates well with its calmodulin antagonism properties and, thereby, provides the first insights into the mechanism of its antifungal properties. Guided by this mechanism, we have generated improved trifluoperazine derivatives with increased anticryptococcal activity and, importantly, reduced affinity for receptors that modulate undesired neurological effects. Taken together, these data suggest that phenothiazines represent a potentially useful scaffold for further optimization in the search for new antifungal drugs

Antifungal activity of 2-ethenyl-substituted tamoxifen derivatives.

<p>The structure, molecule number used in the text, minimum inhibitory concentration (MIC, μg/mL) against <i>C</i>.<i>neoformans</i> (first number) and MIC against <i>C</i>. <i>albicans</i> are provided.</p