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Over the last years Gallium-68 ((68)Ga) has received tremendous attention for labeling of radiopharmaceuticals for positron emission tomography (PET). (68)Ga labeling of biomolecules is currently based on bifunctional chelators containing aminocarboxylates (mainly DOTA and NOTA). We have recently shown that cyclic peptide siderophores have very good complexing properties for (68)Ga resulting in high specific activities and excellent metabolic stabilities, in particular triacetylfusarinine-C (TAFC). We postulated, that, starting from its deacetylated form (Fusarinine-C (FSC)) trimeric bioconjugates are directly accessible to develop novel targeting peptide based (68)Ga labeled radiopharmaceuticals. As proof of principle we report on the synthesis and (68)Ga-radiolabeling of a trimeric FSC-RGD conjugate, [(68)Ga]FSC-(RGD)3, targeting αvβ3 integrin, which is highly expressed during tumor-induced angiogenesis. Synthesis of the RGD peptide was carried out applying solid phase peptide synthesis (SPPS), followed by the coupling to the siderophore [Fe]FSC via in situ activation using HATU/HOAt and DIPEA. Subsequent demetalation allowed radiolabeling of FSC-(RGD)3 with (68)Ga. The radiolabeling procedure was optimized regarding peptide amount, reaction time, temperature as well buffer systems. For in vitro evaluation partition coefficient, protein binding, serum stability, αvβ3 integrin binding affinity, and tumor cell uptake were determined. For in vitro tests as well as for the biodistribution studies αvβ3 positive human melanoma M21 and αvβ3 negative M21-L cells were used. [(68)Ga]FSC-(RGD)3 was prepared with high radiochemical yield (>98%). Distribution coefficient was -3.6 revealing a hydrophilic character, and an IC50 value of 1.8±0.6 nM was determined indicating a high binding affinity for αvβ3 integrin. [(68)Ga]FSC-(RGD)3 was stable in PBS (pH7.4), FeCl3- and DTPA-solution as well as in fresh human serum at 37°C for 2hours. Biodistribution assay confirmed the receptor specific uptake found in vitro. Uptake in the αvβ3 positive tumor was 4.3% ID/g 60min p.i. which was 3-fold higher than the monomeric [(68)Ga]NODAGA-RGD. Tumor to blood ratio of approx. 8 and tumor to muscle ratio of approx. 7 were observed. [(68)Ga]FSC-(RGD)3 serves as an example for the feasibility of a novel class of bifunctional chelators based on cyclic peptide siderophores and shows excellent targeting properties for αvβ3 integrin in vivo for imaging tumor-induced neovascularization., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Introduction: Siderophores are low-molecular-mass iron chelators serving as iron transporters for almost all bacteria, fungi and some plants. Iron is an essential element for majority of organisms and plays an important role in virulence of pathogenic organisms. (68)Ga is a positron emitter with complexing properties comparable to those of Fe(III) and readily available from a generator. Initial studies with (68)Ga-triacetylfusarinine C (TAFC) showed excellent targeting properties in a rat infection model. We report here on the in vitro and in vivo evaluation of other siderophores radiolabelled with (68)Ga as potential radiopharmaceuticals for infection imaging., Methods: (68)Ga labelling was performed using acetate buffer. Stability, log P and protein binding values were determined. In vitro uptake was tested using iron-deficient and iron-sufficient Aspergillus fumigatus (A.f.) cultures. Biodistribution of (68)Ga-siderophores was studied in Balb/c mice., Results: Significant differences among studied siderophores were observed in labelling efficiency, stability and protein binding. Uptake in A.f. cultures was highly dependent on iron load and type of the siderophore. In mice, (68)Ga-TAFC and (68)Ga-ferrioxamine E (FOXE) showed rapid renal excretion and low blood values even at a short period after injection; in contrast, (68)Ga-ferricrocin and (68)Ga-ferrichrome revealed high retention in blood and (68)Ga-fusarinine C showed very high kidney retention., Conclusions: Some of the studied siderophores bind (68)Ga with high affinity and stability, especially (68)Ga-TAFC and (68)Ga-FOXE. Low values of protein binding, high and specific uptake in A.f., and excellent in vivo biodistribution make them favourable agents for Aspergillus infection imaging., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aspergillus fumigatus is the most common airborne fungal pathogen for humans. In this mold, iron starvation induces production of the siderophore triacetylfusarinine C (TAFC). Here we demonstrate a link between TAFC and ergosterol biosynthetic pathways, which are both critical for virulence and treatment of fungal infections. Consistent with mevalonate being a limiting prerequisite for TAFC biosynthesis, we observed increased expression of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase (Hmg1) under iron starvation, reduced TAFC biosynthesis following lovastatin-mediated Hmg1 inhibition, and increased TAFC biosynthesis following Hmg1 overexpression. We identified enzymes, the acyl-CoA ligase SidI and the enoyl-CoA hydratase SidH, linking biosynthesis of mevalonate and TAFC, deficiency of which under iron starvation impaired TAFC biosynthesis, growth, oxidative stress resistance, and murine virulence. Moreover, inactivation of these enzymes alleviated TAFC-derived biosynthetic demand for mevalonate, as evidenced by increased resistance to lovastatin. Concordant with bilateral demand for mevalonate, iron starvation decreased the ergosterol content and composition, a phenotype that is mitigated in TAFC-lacking mutants.

The opportunistic fungal pathogen Aspergillus fumigatus produces four types of siderophores, low-molecular-mass iron chelators: it excretes fusarinine C (FsC) and triacetylfusarinine C (TAFC) for iron uptake and accumulates ferricrocin (FC) for hyphal and hydroxyferricrocin (HFC) for conidial iron distribution and storage. Siderophore biosynthesis has recently been shown to be crucial for fungal virulence. Here we identified a new component of the fungal siderophore biosynthetic machinery: AFUA_1G04450, termed SidL. SidL is conserved only in siderophore-producing ascomycetes and shows similarity to transacylases involved in bacterial siderophore biosynthesis and the N(5)-hydroxyornithine:anhydromevalonyl coenzyme A-N(5)-transacylase SidF, which is essential for TAFC biosynthesis. Inactivation of SidL in A. fumigatus decreased FC biosynthesis during iron starvation and completely blocked FC biosynthesis during iron-replete growth. In agreement with these findings, SidL deficiency blocked conidial accumulation of FC-derived HFC under iron-replete conditions, which delayed germination and decreased the size of conidia and their resistance to oxidative stress. Remarkably, the sidL gene is not clustered with other siderophore-biosynthetic genes, and its expression is not affected by iron availability. Tagging of SidL with enhanced green fluorescent protein suggested a cytosolic localization of the FC-biosynthetic machinery. Taken together, these data suggest that SidL is a constitutively active N(5)-hydroxyornithine-acetylase required for FC biosynthesis, in particular under iron-replete conditions. Moreover, this study revealed the unexpected complexity of siderophore biosynthesis, indicating the existence of an additional, iron-repressed N(5)-hydroxyornithine-acetylase.

Unlabelled: The diagnosis of invasive pulmonary aspergillosis (IPA) is difficult and lacks specificity and sensitivity. In the pathophysiology of Aspergillus fumigatus, iron plays an essential role as a nutrient during infection. A. fumigatus uses a specific and highly efficient iron uptake mechanism based on iron-complexing ferric ion Fe(III) siderophores, which are a requirement for A. fumigatus virulence. We aimed to evaluate the potential of siderophores radiolabeled with (68)Ga, a positron emitter with complexing properties comparable to those of Fe(III), as a radiopharmaceutical for imaging IPA., Methods: (68)Ga radiolabeling of the A. fumigatus siderophores desferri-triacetylfusarinine C (TAFC) and desferri-ferricrocin (FC) was performed at high specific activity. Stability, protein binding, and log P values were determined. In vitro uptake in A. fumigatus cultures was tested under varying conditions. Biodistribution was studied in healthy noninfected BALB/c mice, and uptake was studied in a model of A. fumigatus infection using immunosuppressed Lewis rats., Results: High-specific-activity (68)Ga labeling could be achieved, and resulting complexes were stable in serum, toward diethylenetriaminepentaacetic acid and Fe(III) challenge. Both siderophores showed hydrophilic properties ((68)Ga-TAFC, log P = -2.59; (68)Ga-FC, log P = -3.17) with low values of protein binding for (68)Ga-TAFC (<2%). Uptake of both siderophores was highly dependent on the mycelial iron load and could be blocked with an excess (10 microM) of siderophore or NaN(3), indicating specific, energy-dependent uptake. In noninfected mice, (68)Ga-TAFC showed rapid renal excretion and low blood values (1.6 +/- 0.37 percentage injected dose per gram [%ID/g] at 30 min); in urine only intact (68)Ga-TAFC was detected. In contrast, (68)Ga-FC revealed high retention in blood (16.1 +/- 1.07 %ID/g at 90 min) and rapid metabolism. In the rat IPA model, lung uptake of (68)Ga-TAFC was dependent on the severity of infection, with less than 0.04 %ID/g in control rats (n = 5) and 0.29 +/- 0.11 %ID/g in mildly infected (n = 3) and 0.95 +/- 0.37 %ID/g in severely infected (n = 4) rats. PET showed focal accumulation in infected lung tissue., Conclusion: Both siderophores bound (68)Ga with high affinity, and (68)Ga-TAFC, especially, showed high stability. (68)Ga-TAFC displayed highly selective accumulation by A. fumigatus subspecies in vitro and in vivo. The high and specific uptake by A. fumigatus proves the potential of (68)Ga-labeled siderophores for the specific detection of A. fumigatus during infection. They hold promise as new PET agents for IPA.

Iron is an essential metal for virtually all organisms. Iron acquisition is well characterized for various organisms, whereas intracellular iron distribution is poorly understood. In contrast to bacteria, plants, and animals, most fungi lack ferritin-mediated iron storage but possess an intracellular siderophore shown to be involved in iron storage. Here we demonstrate that deficiency in the intracellular siderophore ferricrocin causes iron starvation in conidia of Aspergillus fumigatus, demonstrating that ferricrocin is also involved in intra- and transcellular iron distribution. Thus, ferricrocin represents the first intracellular iron transporter identified in any organism.