Optimization of Artificial Siderophores as 68Ga-Complexed PET Tracers for In Vivo Imaging of Bacterial Infections.

The diagnosis of bacterial infections at deep body sites benefits from noninvasive imaging of molecular probes that can be traced by positron emission tomography (PET). We specifically labeled bacteria by targeting their iron transport system with artificial siderophores. The cyclen-based probes contain different binding sites for iron and the PET nuclide gallium-68. A panel of 11 siderophores with different iron coordination numbers and geometries was synthesized in up to 8 steps, and candidates with the best siderophore potential were selected by a growth recovery assay. The probes [68Ga]7 and [68Ga]15 were found to be suitable for PET imaging based on their radiochemical yield, radiochemical purity, and complex stability in vitro and in vivo. Both showed significant uptake in mice infected with Escherichia coli and were able to discern infection from lipopolysaccharide-triggered, sterile inflammation. The study qualifies cyclen-based artificial siderophores as readily accessible scaffolds for the in vivo imaging of bacteria.

Efficient Iron and ROS Nanoscavengers for Brain Protection after Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) will be accompanied by the overload of iron and reactive oxygen species (ROS) following hematoma clearance. Although deferoxamine (DFO) has been widely utilized as a clinical first-line siderophore to remove the iron overload, the ROS-inducing damage still greatly limits the therapeutic effect of DFO. To address this issue, we designed and fabricated a series of dual-functional macromolecular nanoscavengers featuring high-density DFO units and catechol moieties. Note that the former units could effectively remove the iron overload, while the latter ones could efficiently deplete the ROS. The resulting nanoscavengers efficiently down-regulate the iron and ROS levels as well as significantly reduce the cell death in both iron-overloaded RAW 264.7 cells and the ICH mice model. This work suggests a novel clue for the ICH-ameliorated iron-depleting interventional therapeutic regimen.

Hydroxamate siderophores: Natural occurrence, chemical synthesis, iron binding affinity and use as Trojan horses against pathogens.

Hydroxamic acids are an important class of molecules, in particular because of their metal-chelating ability. Microorganisms, including pathogenic bacteria, use hydroxamate-based entities (siderophores), among others, to acquire Fe (III). The "Trojan horse" strategy exploits the need of bacteria for this metal by using Fe (III) active transporters to carry antibacterial or bactericidal moieties into the bacterial cell. Many natural Trojan horses (sideromycins) are derived from hydroxamic acids, thereby reflecting their potency. Various artificial sideromycins and their antibacterial activities have been reported. This review discusses the structural aspects of the hydroxamate-siderophores isolated in the last two decades, the chemical synthesis of their building blocks, their binding affinity towards Fe (III), and their application as Trojan horses (weaknesses and strengths).

Siderophore-Antibiotic Conjugate Design: New Drugs for Bad Bugs?

Antibiotic resistance is a global health concern and a current threat to modern medicine and society. New strategies for antibiotic drug design and delivery offer a glimmer of hope in a currently limited pipeline of new antibiotics. One strategy involves conjugating iron-chelating microbial siderophores to an antibiotic or antimicrobial agent to enhance uptake and antibacterial potency. Cefiderocol (S-649266) is a promising cephalosporin-catechol conjugate currently in phase III clinical trials that utilizes iron-mediated active transport and demonstrates enhanced potency against multi-drug resistant (MDR) Gram-negative pathogens. Such molecules demonstrate that siderophore-antibiotic conjugates could be important future medicines to add to our antibiotic arsenal. This review is written in the context of the chemical design of siderophore-antibiotic conjugates focusing on the differing siderophore, linker, and antibiotic components that make up conjugates. We selected chemically distinct siderophore-antibiotic conjugates as exemplary conjugates, rather than multiple analogues, to highlight findings to date. The review should offer a general guide to the uninitiated in the molecular design of siderophore-antibiotic conjugates.

Design, solid-phase synthesis and evaluation of enterobactin analogs for iron delivery into the human pathogen Campylobacter jejuni.

The human enteropathogen Campylobacter jejuni, like many bacteria, employs siderophores such as enterobactin for cellular uptake of ferric iron. This transport process has been shown to be essential for virulence and presents an attractive opportunity for further study of the permissiveness of this pathway to small-molecule intervention and as inspiration for the development of synthetic carriers that may effectively transport cargo into Gram-negative bacteria. In this work, we have developed a facile and robust microscale assay to measure growth recovery of C. jejuni NCTC 11168 in liquid culture as a result of ferric iron uptake. In parallel, we have established the solid-phase synthesis of catecholamide compounds modeled on enterobactin fragments. Applying these methodological developments, we show that small synthetic iron chelators of minimal dimensions provide ferric iron to C. jejuni with equal or greater efficiency than enterobactin.

Chemical Synthesis of Staphyloferrin B Affords Insight into the Molecular Structure, Iron Chelation, and Biological Activity of a Polycarboxylate Siderophore Deployed by the Human Pathogen Staphylococcus aureus.

Staphyloferrin B (SB) is a citrate-based polycarboxylate siderophore produced and utilized by the human pathogen Staphylococcus aureus for acquiring iron when colonizing the vertebrate host. The first chemical synthesis of SB is reported, which enables further molecular and biological characterization and provides access to structural analogues of the siderophore. Under conditions of iron limitation, addition of synthetic SB to bacterial growth medium recovered the growth of the antibiotic resistant community isolate S. aureus USA300 JE2. Two structural analogues of SB, epiSB and SBimide, were also synthesized and employed to investigate how epimerization of the citric acid moiety or imide formation influence its function as a siderophore. Epimerization of the citric acid stereocenter perturbed the iron-binding properties and siderophore function of SB as evidenced by experimental and computational modeling studies. Although epiSB provided growth recovery to S. aureus USA300 JE2 cultured in iron-deficient medium, the effect was attenuated relative to that of SB. Moreover, SB more effectively sequestered the Fe(III) bound to human holo-transferrin, an iron source of S. aureus, than epiSB. SBimide is an imide analogous to the imide forms of other citric acid siderophores that are often observed when these molecules are isolated from natural sources. Here, SBimide is shown to be unstable, converting to native SB at physiological pH. SB is considered to be a virulence factor of S. aureus, a pathogen that poses a particular threat to public health because of the number of drug-resistant strains emerging in hospital and community settings. Iron acquisition by S. aureus is important for its ability to colonize the human host and cause disease, and new chemical insights into the structure and function of SB will inform the search for new therapeutic strategies for combating S. aureus infections.

Iron(III)-templated macrolactonization of trihydroxamate siderophores.

A method was developed to synthesize macrocyclic trihydroxamate siderophores using optimized Yamaguchi macrolactonization conditions. The natural ability of siderophores to bind iron(III) was exploited to template the reactions and allowed for rapid reaction rates, high product conversions, and the formation of large macrolactone rings up to 35 atoms. An X-ray structure of a 33-membered macrolactone siderophore-Fe(III) complex is presented.

Iron transport-mediated drug delivery: practical syntheses and in vitro antibacterial studies of tris-catecholate siderophore-aminopenicillin conjugates reveals selectively potent antipseudomonal activity.

An artificial tris-catecolate siderophore with a tripodal backbone and its conjugates with ampicillin and amoxicillin were synthesized. Both conjugates exhibited significantly enhanced in vitro antibacterial activities against Gram-negative species compared to the parent drugs, especially against Pseudomonas aeruginosa . The conjugates appeared to be assimilated by an induced bacterial iron transport process as their activities were inversely related to iron concentration. The easily synthesized tris-catecolate siderophore has great potential for future development of various drug conjugates to target antibiotic-resistant Gram-negative bacteria.

Carbohydrate functionalized iron(III) complexes as biomimetic siderophores.

Catechol functionalized iron(III) glycodendrimers have been prepared using a self-assembly process for targeting a specific strain of E. coli bacteria and have been shown to exhibit carbohydrate-lectin mediated iron delivery in growth promotion assay.

Chemical synthesis and biological evaluation of gallidermin-siderophore conjugates.

The lantibiotic gallidermin was modified at lysine residues by regioselective attachment of derivatives of pyochelin, agrobactin and desferrioxamine B with the objective of having siderophore receptors of Gram-negative bacteria transport the antibiotic-iron chelator conjugate through the outer membrane. All of the conjugates retained activity against the Gram-positive indicator strain, Lactococcus lactis subsp. cremoris HP. However, testing of the conjugates against several Gram-negative strains yielded unexpected results. Bacteria treated with 100 µM of the conjugates complexed with Fe(3+) grew better than bacteria grown in iron-free media but worse than bacteria grown in the same media supplemented with 10 µM FeCl(3). Although these findings indicate that the conjugates are unable to inhibit the growth of Gram-negative bacteria, they indicate penetration of the outer membrane and provide structure-activity information for design of other lantibiotic conjugates. The synthetic strategy is applicable for linking biomarkers or fluorescence probes to gallidermin for studies on its localization and mode of action. As there are many lantibiotics that operate with unknown mechanisms of action, this chemical approach provides a means to modify such peptides with biomarkers for biological investigations.

Synthetic and natural iron chelators: therapeutic potential and clinical use.

Iron-chelation therapy has its origins in the treatment of iron-overload syndromes. For many years, the standard for this purpose has been deferoxamine. Recently, considerable progress has been made in identifying synthetic chelators with improved pharmacologic properties relative to deferoxamine. Most notable are deferasirox (Exjade(®)) and deferiprone (Ferriprox(®)), which are now available clinically. In addition to treatment of iron overload, there is an emerging role for iron chelators in the treatment of diseases characterized by oxidative stress, including cardiovascular disease, atherosclerosis, neurodegenerative diseases and cancer. While iron is not regarded as the underlying cause of these diseases, it does play an important role in disease progression, either through promotion of cellular growth and proliferation or through participation in redox reactions that catalyze the formation of reactive oxygen species and increase oxidative stress. Thus, iron chelators may be of therapeutic benefit in many of these conditions. Phytochemicals, many of which bind iron, may also owe some of their beneficial properties to iron chelation. This review will focus on the advances in iron-chelation therapy for the treatment of iron-overload disease and cancer, as well as neurodegenerative and chronic inflammatory diseases. Established and novel iron chelators will be discussed, as well as the emerging role of dietary plant polyphenols that effectively modulate iron biochemistry.

Pseudomonas fluorescens CHA0 produces enantio-pyochelin, the optical antipode of the Pseudomonas aeruginosa siderophore pyochelin.

The siderophore pyochelin is made by a thiotemplate mechanism from salicylate and two molecules of cysteine. In Pseudomonas aeruginosa, the first cysteine residue is converted to its D-isoform during thiazoline ring formation whereas the second cysteine remains in its L-configuration, thus determining the stereochemistry of the two interconvertible pyochelin diastereoisomers as 4'R, 2''R, 4''R (pyochelin I) and 4'R, 2''S, 4''R (pyochelin II). Pseudomonas fluorescens CHA0 was found to make a different stereoisomeric mixture, which promoted growth under iron limitation in strain CHA0 and induced the expression of its biosynthetic genes, but was not recognized as a siderophore and signaling molecule by P. aeruginosa. Reciprocally, pyochelin promoted growth and induced pyochelin gene expression in P. aeruginosa, but was not functional in P. fluorescens. The structure of the CHA0 siderophore was determined by mass spectrometry, thin-layer chromatography, NMR, polarimetry, and chiral HPLC as enantio-pyochelin, the optical antipode of the P. aeruginosa siderophore pyochelin. Enantio-pyochelin was chemically synthesized and confirmed to be active in CHA0. Its potential biosynthetic pathway in CHA0 is discussed.

An artificial siderophore for the detection of iron(III).

An artificial siderophore in the form of a squaraine dye (1) has been synthesized. The bidentate ligand chelates to Fe(III) between the deprotonated hydroxyl group on the ortho position of the ring adjacent to the carbonyl group of the cyclobutadiene ring. The optical response is due to a subtle geometry change of 1 on chelation to Fe(III). This artificial siderophore forms a 2 ratio 1 ligand ratio metal complex, as indicated by a sigmoidal isotherm (K(a)= 10(7) M(-1)). The optical response on the addition of Fe(III) is observed at low concentrations in comparison to other metal salts. The X-ray crystal structure and calculated structures of dye (1) are also included, and will be discussed.

Desketoneoenactin-siderophore conjugates for Candida: evidence of iron transport-dependent species selectivity.

We investigated the inhibitory activity of synthetic isocyanurate-based as well as linear mono- and trihydroxamate siderophore-drug conjugates against Candida spp. The conjugated drug was 13C-desketoneoenactin (DE). The MICs of siderophore-drug conjugates were determined in the absence and presence of 2,2'-dipyridyl to restrict iron availability. The ability of various siderophore types to promote growth in an iron-restricted medium was also assayed. Addition of a siderophore portion to the drug strongly impaired the inhibitory activity of DE. However, the activity of the drug conjugates was increased by up to 16-fold in iron-depleted medium for species having their growth strongly promoted by most hydroxamate-type siderophores (C. albicans, C. stellatoidea, and C. pseudotropicalis). The uptake of (55)Fe from ferrichrome and from two siderophore-drug conjugates was improved when C. albicans cells were grown in a low-iron medium. In the same assay, unlabeled ferrichrome was able to compete with the uptake of (55)Fe from both conjugates, indicating a common mechanism of uptake. A C. albicans strain lacking the siderophore transporter CaSit1/CaArn1 was not able to use ferrichrome or the synthetic ornithine-based trihydroxamate siderophore for growth promotion and was much less susceptible to the siderophore-drug conjugates than its isogenic parent strain. In summary, the ability of some Candida spp. to use ferrichrome-like siderophores for growth promotion explains the selective activity of hydroxamate-drug conjugates, and this activity seems to be related to the presence, in C. albicans, of the siderophore transporter CaSit1/CaArn1. New conjugate designs are necessary to fully restore or improve the initial DE activity.

Total synthesis of petrobactin and its homologues as potential growth stimuli for Marinobacter hydrocarbonoclasticus, an oil-degrading bacteria.

A modular synthesis was developed to access petrobactin, a catechol-containing siderophore isolated from Marinobacter hydrocarbonoclasticus. A range of petrobactin homologues with differing dihydroxybenzamide motifs and in one case an increased number of carbons in the polyamine backbone were also synthesized. As such, these systems represent new isomeric probes to study iron transport properties in M. hydrocarbonoclasticus. The synthesis of petrobactin and its homologues and the first biological study of how these agents influence the growth of Mycobacterhydrocarbonoclasticus are reported. New synthetic methods were developed to overcome issues (imide formation) encountered in earlier syntheses. Both the (1)H and (13)C NMR of petrobactin were consistent with the recently revised structure showing that petrobactin in fact contains a 3,4-dihydroxybenzene motif rather than a 2,3-dihydroxybenzene motif. The preliminary biological studies suggested that using the native petrobactin 1b for M. hydrocarbonoclasticus-specific growth stimulation may be a poor strategy for oil-spill cleanup.

Formation of the chromophore of the pyoverdine siderophores by an oxidative cascade.

The pyoverdine chromophore is formed in a reaction involving a two-electron oxidation, a conjugate addition, and a second two-electron oxidation. This oxidative cascade can be carried out with polyphenol oxidase (PPO), MnO(2), and cell-free extracts from Pseudomonas aeruginosa. [reaction: see text]

Synthesis and biological evaluation of new citrate-based siderophores as potential probes for the mechanism of iron uptake in mycobacteria.

Several iron chelators containing alpha,beta-unsaturated hydroxamic acid motifs appended to a citric acid platform were synthesized. Mycobacterium paratuberculosis was then challenged to grow in the presence of a panel of siderophores (mycobactin J, deferrioxamine B, acinetoferrin, and nannochelin A) and the new synthetic agents. Of the structures tested, those containing the trans 2-octenoyl motif were preferred over those with trans cinnamoyl groups. In addition, derivatives containing longer tether lengths between the iron binding ligands (C5) were more efficacious and led to higher growth index values. Perhaps most remarkable was the finding that at 2.4 microM a trans 2-octenoylated, citrate-containing imide 6 was nearly 5-fold more effective in stimulating growth than the native chelator, mycobactin J. In this regard, new structural elements were identified (e.g., an imide motif or 2-octenoyl side chain), whose presence stimulated mycobacterial growth.

Studies and syntheses of siderophores, microbial iron chelators, and analogs as potential drug delivery agents.

Siderophores (microbial iron chelators) play an extremely important role in microbial pathogenicity. Microbial uptake of siderophore-iron complexes through active transport systems allow microbes to survive and proliferate even under iron deficient environments during invasion of a host. Due to their structural complexity, unique iron (III) chelation, acquisition properties, and their therapeutic potential, siderophores have attracted much attention in a broad range of disciplines. Tremendous progress has been made in siderophore syntheses, in determination of the structures and functions of outer membrane receptors (e.g. FhuA and FepA), and in the mechanistic insight into siderophore-iron-mediated active transport processes. One of the important practical applications of this active transport system is development of species-selective active drug transport (the Trojan Horse approach) to potentially treat infections due to drug resistant strains of microbes. Siderophore-drug conjugates have shown great potential in active drug delivery to target pathogenic microbes.