Antifungal Siderophore Conjugates for Theranostic Applications in Invasive Pulmonary Aspergillosis Using Low-Molecular TAFC Scaffolds

Abstract

Invasive pulmonary aspergillosis (IPA) is a life-threatening form of fungal infection, primarily in immunocompromised patients and associated with significant mortality. Diagnostic procedures are often invasive and/or time consuming and existing antifungals can be constrained by dose-limiting toxicity and drug interaction. In this study, we modified triacetylfusarinine C (TAFC), the main siderophore produced by the opportunistic pathogen Aspergillus fumigatus (A. fumigatus), with antifungal molecules to perform antifungal susceptibility tests and molecular imaging. A variation of small organic molecules (eflornithine, fludioxonil, thiomersal, fluoroorotic acid (FOA), cyanine 5 (Cy5) with antifungal activity were coupled to diacetylfusarinine C (DAFC), resulting in a “Trojan horse” to deliver antifungal compounds specifically into A. fumigatus hyphae by the major facilitator transporter MirB. Radioactive labeling with gallium-68 allowed us to perform in vitro characterization (distribution coefficient, stability, uptake assay) as well as biodistribution experiments and PET/CT imaging in an IPA rat infection model. Compounds chelated with stable gallium were used for antifungal susceptibility tests. [Ga]DAFC-fludioxonil, -FOA, and -Cy5 revealed a MirB-dependent active uptake with fungal growth inhibition at 16 µg/mL after 24 h. Visualization of an A. fumigatus infection in lungs of a rat was possible with gallium-68-labeled compounds using PET/CT. Heterogeneous biodistribution patterns revealed the immense influence of the antifungal moiety conjugated to DAFC. Overall, novel antifungal siderophore conjugates with promising fungal growth inhibition and the possibility to perform PET imaging combine both therapeutic and diagnostic potential in a theranostic compound for IPA caused by A. fumigatus.

The Siderophore Transporters Sit1 and Sit2 Are Essential for Utilization of Ferrichrome-, Ferrioxamine- and Coprogen-Type Siderophores in Aspergillus fumigatus

“…In conclusion, recognition of ferrioxamines by Sit1 does not involve the termini of linear ferrioxamines, and modification of the termini does not block their uptake. These findings are possibly helpful for generating conjugates of ferrioxamines with toxic molecules to design novel antifungal drugs with microbial uptake specificity in a Trojan horse approach [ 26 , 53 ]. In this respect, it is noteworthy that SITs are fungal-specific transporters [ 10 ]; ferrioxamines are also used by bacteria but the transporters are of different types [ 54 , 55 ] and might have different characteristics for conjugates.…”

Uptake of the Siderophore Triacetylfusarinine C, but Not Fusarinine C, Is Crucial for Virulence of Aspergillus fumigatus

“…Previous studies indicated that A. fumigatus internalizes several fluorescent and nonfluorescent TAFC derivatives, including fluorescent [Fe]DAFC-FITC ( 16 – 18 ). Epifluorescence microscopy demonstrated that the internalization of [Fe]DAFC-FITC depends on MirB but not MirD ( Fig.…”

“…In the present study, epifluorescence microscopy revealed that MirB-mediated transport tolerates the conjugation of fluorescent FITC to TAFC (DAFC-FITC). Moreover, previous studies indicated that A. fumigatus internalizes several other fluorescent and nonfluorescent TAFC derivatives ( 16 – 18 ). These results emphasize the translational potential of SITs for the development of a novel antifungal drug mechanism based on the conjugation of toxic compounds to siderophores.…”

“…Consequently, there is a need for alternatives. One possibility for the development of novel antifungal drugs is a Trojan horse approach ( 16 – 18 ), in which toxic compounds are conjugated to siderophores for selective import into the pathogen, a concept that has been realized in a recently FDA-approved drug against bacterial infections ( 19 ). Noteworthy, in this respect, the A. fumigatus siderophore transporter Sit1 was found to mediate the uptake and, consequently, the antifungal activity of VL-2397, which resembles a ferrichrome-type siderophore ( 20 ).…”

Desferrioxamine B-Mediated Pre-Clinical In Vivo Imaging of Infection by the Mold Fungus Aspergillus fumigatus

“…As the efficient uptake of a radiolabeled siderophore by a pathogen is crucial for effective imaging, these findings might help to improve the properties of siderophores used for imaging. In a proof-of-concept study, Pfister et al previously demonstrated that certain siderophores could be used in a theranostic approach, in which antimicrobial therapy is combined with a diagnostic approach [ 38 ]. In this study, 68 Ga-imaging was combined with the fusion of antimicrobials to the siderophore to exploit the so-called Trojan horse approach.…”