Identifying Protein Targets of Novel Compounds with Broad-Spectrum Antifungal Activity
LisaMichelle Pecaro
Co-Presenters: Individual Presentation
College: The Dorothy and George Hennings College of Science, Mathematics and Technology
Major: Biotechnology/Molecular Biology - STEM 5 Year B.S./M.S.
Faculty Research Mentor: Stephen Parent
Abstract:
Annually, an estimated 6.5 million cases of invasive fungal infection are associated with 3.8 million deaths worldwide (Fig. 1). However, the effectiveness of current antifungal therapies is limited by factors such as patient tolerability and the increasing emergence of drug-resistant fungal isolates. This highlights the need for antifungal therapeutics with alternative mechanisms of action. Kathera Bioscience, Inc. discovered a proprietary series of novel compounds with broad-spectrum antifungal activity (Fig. 2), but whose mechanism of action is unknown.The goal of this study is to identify the specific fungal protein targets of compound KP-100289, thus enabling its further optimization into a broad-spectrum therapeutic capable of treating life-threatening fungal infections. To identify this target(s), we performed a pulldown assay, incubating lysate of the well-characterized model organism Saccharomyces cerevisiae with a biotinylated derivative of the antifungal compound (immobilized on streptavidin beads) as bait (Fig. 3 - 4). Biotin-blocked streptavidin beads served as a control for non-specific protein binding. Bound proteins were eluted with acidic buffer, then separated via SDS-PAGE (Fig. 5). Lastly, excised protein bands were digested with trypsin and analyzed through the use of liquid chromatography and mass spectrometry (Fig. 6 - 7).