Fluorescence Polarization Binding Assay forAspergillus fumigatusVirulence Factor UDP-Galactopyranose Mutase

Jun Qi,Pablo Sobrado,Michelle Oppenheimer

doi:10.4061/2011/513905

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 Kd 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.

Highlights

Aspergillus fumigatus is an opportunistic human pathogen responsible for diseases such as allergic reactions and lung infections, including bronchopulmonary aspergillosis (ABPA) and invasive pulmonary aspergillosis (IPA) [1, 2]
We report the development of an fluorescence polarization (FP) assay that can be used in a highthroughput format for the identification of inhibitors of Af UDP-galactopyranose mutase (UGM), which we believe will lead to the development of new therapeutics against A. fumigatus-related diseases
If the UDP fluorescent probe binds to A. fumigatus UGM (Af UGM) and is excited with plane-polarized light, the resulting enzyme-ligand complex tumbles slowly in solution, and the fluorescence emission remains polarized (Figure 3(a))

Summary

Introduction

Aspergillus fumigatus is an opportunistic human pathogen responsible for diseases such as allergic reactions and lung infections, including bronchopulmonary aspergillosis (ABPA) and invasive pulmonary aspergillosis (IPA) [1, 2]. This fungus is a significant health threat to immunocompromised patients, such as organ transplant recipients and people with AIDS or leukemia [3, 4]. Four fluorescently labeled UDP derivatives including two known UDP-fluorescein analogs (1 and 2, Figure 2) and two novel UDP-TAMRA analogs (3 and 4, Figure 2) were synthesized to be used as fluorescent probes in the FP assay Their concentrations were optimized to obtain a stable FP signal with minimal standard deviation, and their Kd values were determined by measuring the anisotropy changes as a function of Af UGM concentration. This fast convenient one-step FP assay is suitable for a high-throughput screening to identify Af UGM inhibitors

Materials and Methods

Results and Discussion

Conclusion