1522. Fungal Cytological Profiling of Candida albicans Exposed to Diverse Antifungal Agents Including the Novel Gwt1 inhibitor APX001A
Session: Poster Abstract Session: Preclinical Study with New Antibiotics and Antifungals
Friday, October 6, 2017
Room: Poster Hall CD
Posters
  • Poster_1522_Sharp_et_al.pdf (872.2 kB)
  • Background:  Bacterial cytological profiling accelerates drug discovery efforts by determining the mechanism of action (MOA) of newly developed antibacterial agents. Our goal was to adapt this technology to the identification and study of the MOA of antifungal compounds. Here we explore the utility of Fungal Cytological Profiling (FCP) of C. albicans in revealing changes in morphology over time using for 6 antifungal agents with unique MOA using fluorescently labeled compounds that specifically stain a variety of subcellular structures including DNA and membranes. Included in the analysis was the novel broad spectrum Gwt1 inhibitor APX001A, the active moiety of the prodrug APX001 which is currently in clinical trials for invasive fungal infections.

    Methods: The MICs of 6 antifungals vs C. albicans were determined by CLSI methodology. For FCP, antifungals were added to cultures (1x105cells/mL) in RPMI 1640 (buffered with MOPS) at concentrations near MIC: APX001A (0.064 µg/mL); caspofungin (1µg/mL); fluconazole (2 µg/mL); flucytosine (2 µg/mL); amphotericin B (1 µg/mL) and nikkomycin (3.33 µg/mL) incubated at 35°C with shaking. At 4 hr and 24 hr, treated cultures were stained with various dyes (for 15 min), and staining examined under the fluorescence microscope. Dyes included FM 4-64 (membranes), DAPI (DNA), and Sytox Green (cell viability). High resolution fluorescence microscopy, image analysis and quantitation of cytological parameters (cell length, width, shape, DNA content) were used to create a cytological profile for each growth condition.

    Results:  Unique cytological signatures strongly correlated with antifungal MOA: FCZ resulted in rounded cells that lacked hyphal forms; APX001A resulted in abundant intracellular membrane labeling at 4 hr, consistent with an endoplasmic reticulum stress response, with cell death (Sytox Green Staining) at 24 hr.

    Conclusion:  FCP is a rapid and accurate method to establish MOA and distinguish between antifungals that inhibit specific biosynthetic pathways (e.g., cell wall) and sub-pathways (glucan vs chitin synthesis). In addition, this technology can be useful in drug discovery program to determine on-target vs off-target activity of newly synthesized molecules.

    Marc Sharp, PhD1, Quinlyn Soltow, PhD2, Karen Joy Shaw, PhD2 and Joseph Pogliano, PhD1, (1)Linnaeus Bioscience, San Diego, CA, (2)Amplyx Pharmaceuticals Inc., San Diego, CA

    Disclosures:

    M. Sharp, Linnaeus: Employee , Salary

    Q. Soltow, Amplyx Pharmaceuticals Inc.: Employee , Salary

    K. J. Shaw, Amplyx Pharmaceuticals Inc.: Employee , Salary
    Linnaeus: Consultant , Consulting fee

    J. Pogliano, Linnaeus: Employee , Salary

    Findings in the abstracts are embargoed until 12:01 a.m. PDT, Wednesday Oct. 4th with the exception of research findings presented at the IDWeek press conferences.