Harnessing Clickable Acylated Glycerol Probes as Chemical Tools for Tracking Glycerolipid Metabolism

Ancajas, Christelle F.; Carr, Adam J.; Lou, Jinchao; Sagar, Ruhani; Zhou, Yue; Reynolds, Todd B.; Best, Michael D.

doi:10.1002/chem.202300417

“…This probe consists of a serine analog carrying an azide tag for click chemistry, C- l -SerN 3 . This probe has been demonstrated to be incorporated into live cell membranes by infiltrating lipid metabolism to produce azide-tagged PS analogs that can be post-labeled by click-tagging with fluorophores to localized/quantify PS in cells ( 59 , 60 ). Wild-type C. albicans was grown in YPD to early log phase before C- l -SerN 3 was added, along with CBR-5884 or DMSO ( Fig.…”

Section: Resultsmentioning

confidence: 99%

The small molecule CBR-5884 inhibits the Candida albicans phosphatidylserine synthase

Zhou,

Phelps,

Mangrum

et al. 2024

mBio

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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 K i of 1,550 ± 245.6 nM. Thus, this compound has the potential for development into an antifungal compound. IMPORTANCE Fungal phosphatidylserine synthase (Cho1) is a logical antifungal target due to its crucial role in the virulence and viability of various fungal pathogens, and since it is absent in humans, drugs targeted at Cho1 are less likely to cause toxicity in patients. Using fungal Cho1 as a model, there have been two unsuccessful attempts to discover inhibitors for Cho1 homologs in whole-cell screens prior to this study. The compounds identified in these attempts do not act directly on the protein, resulting in the absence of known Cho1 inhibitors. The significance of our research is that we developed a high-throughput target-based assay and identified the first Cho1 inhibitor, CBR-5884, which acts both on the purified protein and its function in the cell. This molecule acts as a competitive inhibitor with a K i value of 1,550 ± 245.6 nM and, thus, has the potential for development into a new class of antifungals targeting PS synthase.

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“…This probe consists of a serine analogue carrying an azide tag for click chemistry, C-L-SerN 3 . This probe has been demonstrated to be incorporated into live cell membranes by infiltrating lipid metabolism to produce azide-tagged PS analogues that can be post-labeled by click-tagging with fluorophores to localized/quantify PS in cells (57, 58). Wildtype C. albicans was grown in YPD to early log phase before C-L-SerN 3 was added, along with CBR-5884 or DMSO (Figure 5C).…”

Section: Resultsmentioning

confidence: 99%

The small molecule CBR-5884 inhibits theCandida albicansphosphatidylserine synthase

Zhou,

Phelps,

Mangrum

et al. 2023

Preprint

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Systemic infections byCandida 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, fromCandida albicansis 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 anin vitronucleotidase-coupled malachite green-based high throughput assay for purifiedC. albicansCho1 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 inhibitory effects againstC. albicanscells, with fungicidal inhibition by ebselen and fungistatic inhibition by LOC14 and CBR-5884. Only CBR-5884 showed evidence of disruptingin vivoCho1 function by inducing phenotypes consistent with thecho1ΔΔ 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 aKiof 1,550 ± 245.6 nM. Thus, this compound has the potential for development into an antifungal compound.

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Probing Glycerolipid Metabolism using a Caged Clickable Glycerol‐3‐Phosphate Probe

Lou,

Ancajas,

Zhou

et al. 2024

ChemBioChem

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In this study, we present the probe SATE‐G3P‐N3 as a novel tool for metabolic labeling of glycerolipids (GLs) to investigate lipid metabolism in yeast cells. By introducing a clickable azide handle onto the glycerol backbone, this probe enables general labeling of glycerolipids. Additionally, this probe contains a caged phosphate moiety at the glycerol sn‐3 position to not only facilitate probe uptake by masking negative charge but also to bypass the phosphorylation step crucial for initiating phospholipid synthesis, thereby enhancing phospholipid labeling. The metabolic labeling activity of the probe was thoroughly assessed through cellular fluorescence microscopy, mass spectrometry (MS), and thin‐layer chromatography (TLC) experiments. Fluorescence microscopy analysis demonstrated successful incorporation of the probe into yeast cells, with labeling predominantly localized at the plasma membrane. LCMS analysis confirmed metabolic labeling of various phospholipid species (PC, PS, PA, PI, and PG) and neutral lipids (MAG, DAG, and TAG), and GL labeling was corroborated by TLC. These results showcased the potential of the SATE‐G3P‐N3 probe in studying GL metabolism, offering a versatile and valuable approach to explore the intricate dynamics of lipids in yeast cells.

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Harnessing Clickable Acylated Glycerol Probes as Chemical Tools for Tracking Glycerolipid Metabolism

Cited by 3 publications

References 55 publications

The small molecule CBR-5884 inhibits the Candida albicans phosphatidylserine synthase

The small molecule CBR-5884 inhibits the Candida albicans phosphatidylserine synthase

The small molecule CBR-5884 inhibits theCandida albicansphosphatidylserine synthase

Probing Glycerolipid Metabolism using a Caged Clickable Glycerol‐3‐Phosphate Probe

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