Doctoral Dissertations

Orcid ID

0000-0001-8760-877X

Date of Award

12-2023

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Todd B. Reynolds

Committee Members

Barry D. Bruce, Elizabeth M. Fozo, Erik R. Zinser

Abstract

Candida albicans phosphatidylserine (PS) synthase, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic candidiasis due to its importance in virulence, absence in the host and conservation among fungal pathogens. This dissertation is focused on the identification of inhibitors for this membrane enzyme. Cho1 has two substrates: cytidyldiphosphate-diacylglycerol (CDP-DAG) and serine. Previous studies identified a conserved CDP-alcohol phosphotransferase (CAPT) binding motif present within Cho1, and here we revealed that mutations in all but one conserved amino acid within the CAPT motif resulted in decreased Cho1. For serine, we have predicted a serine-binding site based on sequence alignment and found that some of the residues in this putative serine-binding site are required for Cho1 function. One residue, R189, is particularly interesting because it was suggested to be involved in serine binding.

Then, we attempted to perform a small molecule screening on C .albicans Cho1, which will be facilitated by purified Cho1 protein. Due to the transmembrane nature, several solubilizing reagents were used to solubilize Cho1 protein. Digitonin was determined to be the best detergent as it retained the most PS synthase activity. Pull-downs of HA-tagged Cho1 in the digitonin-solubilized fraction reveal an apparent MW of Cho1 consistent with a hexamer. Biochemical and electron microscopy analysis suggest that the hexamer is composed of a trimer of dimers. Cho1 protein was then purified to near-homogeneity as a hexamer and was optimized for high activity to be used in the small drug screening.

For the screening, we developed a nucleotidase-coupled malachite green-based screen against purified Cho1. Over 7,300 molecules curated from repurposing chemical libraries were interrogated in primary and dose-responsivity assays using this platform, and seven compounds were identified to inhibit purified Cho1. Among all, compound CBR-5884 disrupted in vivo Cho1 function by inducing phenotypes consistent with the cho1∆∆ mutant, including a reduction of cellular PS levels. Kinetic curves and computational docking suggest that CBR-5884 competes with serine for binding of Cho1 with a Ki of 1,550 ± 245.6 nM, thus this compound has the potential for further drug design.

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