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Published on: April 19, 2017
Fluorescence Polarization Binding Assay for Aspergillus fumigatus Virulence Factor UDP-Galactopyranose Mutase
Jun Qi1, Michelle Oppenheimer, Pablo Sobrado
1Department of Biochemistry, Virginia Tech, Blacksburg, VA 26061, USA.
Abstract:
Aspergillus fumigatus is an opportunistic human pathogenic fungus responsible for deadly lung infections in immunocompromised individuals. Galactofuranose (Galf) residues are essential components of the cell wall and play an important role in A. fumigatus virulence. The flavoenzyme UDP-galactopyranose mutase (UGM) catalyzes the isomerization of UDP-galactopyranose to UDP-galactofuranose, the biosynthetic precursor of Galf. Thus, inhibitors of UGM that block the biosynthesis of Galf can lead to novel chemotherapeutics for treating A. fumigatus-related diseases. Here, we describe the synthesis of fluorescently labeled UDP analogs and the development of a fluorescence polarization (FP) binding assay for A. fumigatus UGM (AfUGM). High-affinity binding to AfUGM was only obtained with the chromophore TAMRA, linked to UDP by either 2 or 6 carbons with K(d) values of 2.6 ± 0.2 μM and 3.0 ± 0.7 μM, respectively. These values were ~6 times lower than when UDP was linked to fluorescein. The FP assay was validated against several known ligands and displayed an excellent Z' factor (0.79 ± 0.02) and good tolerance to dimethyl sulfoxide.
Insights
Researchers developed a fluorescence polarization assay to find new drugs targeting Aspergillus fumigatus. The assay uses fluorescently labeled UDP analogs to inhibit UDP-galactopyranose mutase, crucial for fungal cell wall integrity and virulence.
Area of Science:
- Biochemistry
- Mycology
- Medicinal Chemistry
Background:
- Aspergillus fumigatus causes life-threatening lung infections in immunocompromised individuals.
- Galactofuranose (Galf) in the fungal cell wall is vital for A. fumigatus virulence.
- UDP-galactopyranose mutase (UGM) is essential for synthesizing Galf.
Purpose of the Study:
- To develop a fluorescence polarization (FP) binding assay for A. fumigatus UGM (AfUGM).
- To identify high-affinity fluorescently labeled UDP analogs for AfUGM inhibition.
- To establish a screening method for novel anti-fungal therapeutics.
Main Methods:
- Synthesis of fluorescently labeled UDP analogs.
- Development and validation of an FP binding assay for AfUGM.
- Determination of binding affinities (K(d) values) using the FP assay.
Main Results:
- TAMRA-labeled UDP analogs showed high-affinity binding to AfUGM (K(d) values of 2.6-3.0 μM).
- Binding affinity was significantly higher compared to fluorescein-labeled analogs.
- The FP assay demonstrated excellent performance with a Z' factor of 0.79 and good DMSO tolerance.
Conclusions:
- A validated FP assay for AfUGM was established.
- Fluorescently labeled UDP analogs, particularly TAMRA conjugates, are effective tools for studying AfUGM.
- This assay provides a foundation for discovering new drugs targeting A. fumigatus infections.
