Abstract
Systemic infections by Candida spp. are associated with high mortality rates, partly due to limitations in current antifungals, highlighting the need for novel drugs and drug targets. The fungal phosphatidylserine synthase, Cho1, from Candida albicans is a logical antifungal drug target due to its importance in virulence, absence in the host, and conservation among fungal pathogens. Inhibitors of Cho1 could serve as lead compounds for drug development, so we developed a target-based screen for inhibitors of purified Cho1. This enzyme condenses serine and cytidyldiphosphate-diacylglycerol (CDP-DAG) into phosphatidylserine (PS) and releases cytidylmonophosphate (CMP). Accordingly, we developed an in vitro nucleotidase-coupled malachite-green-based high throughput assay for purified C. albicans Cho1 that monitors CMP production as a proxy for PS synthesis. Over 7,300 molecules curated from repurposing chemical libraries were interrogated in primary and dose-responsivity assays using this platform. The screen had a promising average Z’ score of ~0.8, and seven compounds were identified that inhibit Cho1. Three of these, ebselen, LOC14, and CBR-5884, exhibited antifungal effects against C. albicans cells, with fungicidal inhibition by ebselen and fungistatic inhibition by LOC14 and CBR-5884. Only CBR-5884 showed evidence of disrupting in vivo Cho1 function by inducing phenotypes consistent with the cho1∆∆ mutant, including a reduction of cellular PS levels. Kinetics curves and computational docking indicate that CBR-5884 competes with serine for binding to Cho1 with a Ki of 1,550 ± 245.6 nM. Thus, this compound has the potential for development into an antifungal compound.
| Original language | English |
|---|---|
| Journal | mBio |
| Volume | 15 |
| Issue number | 5 |
| DOIs | |
| State | Published - May 2024 |
Funding
The authors would like to thank Dr. Glen E. Palmer and Dr. Brian M. Peters from the University of Tennessee Health Science Center for their pKE4 and CaHygB-flipper plasmids. Reese Saho, a summer undergraduate student from Ohio Northern University, also helped with setting up the PS synthase assay. We also thank him for his work. Conceived and designed the experiments: Y.Z., G.A.P., J.M.R., R.E.L., T.B.R. Performed the experiments: Y.Z., G.A.P., M.M.M., J.M., J.M.R., E.K.P., J.L., C.F.A. Analyzed the data: Y.Z., G.A.P., T.B.R. Contributed reagents/materials/analysis tools: J.M.R., R.E.L., M.D.B., T.B.R. Wrote the original draft: Y.Z. Writing & Editing: Y.Z., G.A.P., P.M.O., C.F.A., T.B.R. All authors contributed to the article and approved the submitted version.
Keywords
- CBR-5884
- Candida
- Cho1
- phosphatidylserine
- small-molecule screening