Date of Award

12-2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Todd B. Reynolds

Committee Members

Jeffrey M. Becker, Shawn R. Campagna, Elizabeth M. Fozo, Richard E. Lee

Abstract

Treatments for Candida albicans systemic infections are limited to three classes of antifungals, some of which have harmful side effects or increasing instances of antifungal resistance. These shortcomings illustrate a need for new antifungals. The phosphatidylserine (PS) synthase, Cho1p, represents a potential drug target that can address this need. Cho1p is involved in a major phospholipid biosynthesis pathway and represents a novel drug target for three key reasons: 1) It is required for virulence, indicating that inhibitors of Cho1p would render the organism incapable of causing infection; 2) it is absent in mammals, so inhibitors potentially have no toxic side effects; and 3) the enzyme appears to be well conserved across fungi, indicating that inhibitors could be broadly effective against many fungal pathogens. In order to take full advantage of Cho1p as a drug target, we performed several screens of small molecules for ones that inhibit Cho1p. In one screen we identified SB-224289 that seems to act by inducing large scale endocytosis. A second screen identified the compound Lee-3664 which demonstrates selective toxicity against C. albicans. Further, we performed a detailed biochemical characterization of Cho1p where we identified substrate binding sites as well as the Km and apparent Vmax for each substrate. Computational modeling has produced a useful model of what Cho1p might look like and has further increased interest in crystallizing the protein in future work. The results of this dissertation will lead to more direct approaches for finding Cho1p inhibitors, leading ultimately to more effective antifungals, as well as a greater understanding of enzymes involved in phospholipid biosynthesis, which is necessary for virulence in this pathogen.

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