Modifying the Siderophore Triacetylfusarinine C for Molecular Imaging of Fungal Infection

Abstract

Purpose: Aspergillus fumigatus produces the siderophore triacetylfusarinine C (TAFC) for iron acquisition which is essential for its virulence. Therefore, TAFC is a specific marker for invasive aspergillosis. We have shown previously that positron emission tomography (PET) imaging with [ 68 Ga]TAFC exhibited excellent targeting properties in an A. fumigatus rat infection model. In this study, we aimed to prepare TAFC analogs modifying fusarinine C (FSC) by acylation with different carbon chain lengths as well as with charged substituents and investigated the influence of introduced substituents on preservation of TAFC characteristics in vitro and in vivo. Procedures: Fifteen TAFC derivatives were prepared and labeled with gallium-68. In vitro uptake assays were carried out in A. fumigatus under iron-replete as well as iron-depleted conditions and distribution coefficient was determined. Based on these assays, three compounds, [ 68 Ga]tripropanoyl(FSC) ([ 68 Ga]TPFC), [ 68 Ga]diacetylbutanoyl(FSC) ([ 68 Ga]DABuFC), and [ 68 Ga]trisuccinyl(FSC) ([ 68 Ga]FSC(suc) 3 ), with high, medium, and low in vitro uptake in fungal cultures, were selected for further evaluation. Stability and protein binding were evaluated and in vivo imaging performed in the A. fumigatus rat infection model. Results: In vitro uptake studies using A. fumigatus revealed specific uptake of mono-and trisubstituted TAFC derivatives at RT. Lipophilicities as expressed by logD were 0.34 to − 3.80. The selected compounds displayed low protein binding and were stable in PBS and serum. Biodistribution and image contrast in PET/X-ray computed tomography of [ 68 Ga]TPFC and [ 68 Ga]DABuFC were comparable to [ 68 Ga]TAFC, whereas no uptake in the infected region was observed with [ 68 Ga]FSC(suc) 3 . Conclusions: Our studies show the possibility to modify TAFC without losing its properties and specific recognition by A. fumigatus. This opens also new ways for multimodality imaging or theranostics of fungal infection by introducing functionalities such as fluorescent dyes or antifungal moieties.

Hybrid Imaging of Aspergillus fumigatus Pulmonary Infection with Fluorescent, 68Ga-Labelled Siderophores

“…For [Fe]DAFC-Cy5, rapid uptake into hyphae of A. fumigatus with pronounced visualisation of subcellular organelles was observed 5 min after compound application, which remained visible for more than two hours. In contrast, no uptake of [Fe]DAFC-Cy5 was found by A. terreus, which lacks uptake of [Fe]TAFC (14). Remarkably, the Cy5 carboxylic acid dye alone showed rapid hyphal uptake in both A. fumigatus and A. terreus.…”

“…Uptake assays were performed as previously published [14]. Briefly, 180 µL of A. fumigatus culture in iron-depleted and iron-replete media were added in 96-well MultiScreen Filter Plates HTS (1 µm glass fiber filter, Merck Millipore, Darmstadt, Germany) and pre-incubated for 15 min with either PBS or [Fe]TAFC (blocking solution).…”

“…To investigate the ability of A. fumigatus to use iron containing siderophores for its metabolism, a mutant strain (∆sidA/∆ftrA) lacking two genes, sidA and ftrA was point inoculated (10 4 conidia) in 24-well plates, containing 0.5 mL of aspergillus minimal media agar with [Fe]siderophore concentrations ranging from 0.1-50 µM. Plates were incubated for 48 h at 37 • C in a humidity chamber and visually assessed afterwards [14].…”

“…To measure utilization of the synthesized siderophore conjugates, a previously described bioassay was employed (14). Therefore, the A. fumigatus ∆sidA/∆ftrA mutant strain was used, which lacks both siderophore biosynthesis and reductive iron assimilation.…”

“…TAFC and FsC consist of three N5-cis-anhydromevalonyl-N5-hydroxy-L-ornithine units which are linked by ester bonds to a cyclic molecule ( Figure 1). The three amino groups are either acetylated (TAFC) or present as free amines (FsC), that allow chemical modification [14]. A high iron binding affinity of pM = 31.8 enables TAFC trans-chelation of ferric irons from transferrin (pM = 23.6) in the human host [15].…”