In Silico Analysis of the Ga3+/Fe3+ Competition for Binding the Iron-Scavenging Siderophores of P. aeruginosa-Implementation of Three Gallium-Based Complexes in the "Trojan Horse" Antibacterial Strategy.

The emergence of multidrug-resistant (MDR) microorganisms combined with the ever-draining antibiotic pipeline poses a disturbing and immensely growing public health challenge that requires a multidisciplinary approach and the application of novel therapies aimed at unconventional targets and/or applying innovative drug formulations. Hence, bacterial iron acquisition systems and bacterial Fe2+/3+-containing enzymes have been identified as a plausible target of great potential. The intriguing "Trojan horse" approach deprives microorganisms from the essential iron. Recently, gallium's potential in medicine as an iron mimicry species has attracted vast attention. Different Ga3+ formulations exhibit diverse effects upon entering the cell and thus supposedly have multiple targets. The aim of the current study is to specifically distinguish characteristics of great significance in regard to the initial gallium-based complex, allowing the alien cation to effectively compete with the native ferric ion for binding the siderophores pyochelin and pyoverdine secreted by the bacterium P. aeruginosa. Therefore, three gallium-based formulations were taken into consideration: the first-generation gallium nitrate, Ga(NO3)3, metabolized to Ga3+-hydrated forms, the second-generation gallium maltolate (tris(3-hydroxy-2-methyl-4-pyronato)gallium), and the experimentally proven Ga carrier in the bloodstream-the protein transferrin. We employed a reliable in silico approach based on DFT computations in order to understand the underlying biochemical processes that govern the Ga3+/Fe3+ rivalry for binding the two bacterial siderophores.

"Off-Label Use" of the Siderophore Enterobactin Enables Targeted Imaging of Cancer with Radioactive Ti(IV).

The development of inert, biocompatible chelation methods is required to harness the emerging positron emitting radionuclide 45Ti for radiopharmaceutical applications. Herein, we evaluate the Ti(IV)-coordination chemistry of four catechol-based, hexacoordinate chelators using synthetic, structural, computational, and radiochemical approaches. The siderophore enterobactin (Ent) and its synthetic mimic TREN-CAM readily form mononuclear Ti(IV) species in aqueous solution at neutral pH. Radiolabeling studies reveal that Ent and TREN-CAM form mononuclear complexes with the short-lived, positron-emitting radionuclide 45Ti(IV), and do not transchelate to plasma proteins in vitro and exhibit rapid renal clearance in naïve mice. These features guide efforts to target the 45Ti isotope to prostate cancer tissue through the design, synthesis, and evaluation of Ent-DUPA, a small molecule conjugate composed of a prostate specific membrane antigen (PSMA) targeting peptide and a monofunctionalized Ent scaffold. The [45Ti][Ti(Ent-DUPA)]2- complex forms readily at room temperature. In a tumor xenograft model in mice, selective tumor tissue accumulation (8±5 %, n=5), and low off-target uptake in other organs is observed. Overall, this work demonstrates targeted imaging with 45Ti(IV), provides a foundation for advancing the application of 45Ti in nuclear medicine, and reveals that Ent can be repurposed as a 45Ti-complexing cargo for targeted nuclear imaging applications.

Exploring the Antibacterial Activity and Cellular Fates of Enterobactin-Drug Conjugates That Target Gram-Negative Bacterial Pathogens.

ConspectusSiderophores are secondary metabolites utilized by bacteria to acquire iron (Fe), an essential transition metal nutrient. Fe levels in the host environment are tightly regulated and can be further restricted to starve invading bacterial pathogens in a host-defense process known as nutritional immunity. To survive and colonize the Fe-limited host environment, bacteria produce siderophores and express cognate siderophore transport machinery. These active transport pathways present an opportunity for selective and efficient drug delivery into bacterial cells, motivating decades of research on synthetic siderophore-antibiotic conjugates (SACs) as a Trojan-horse strategy for the development of targeted antibiotics.Enterobactin (Ent) is a triscatecholate siderophore produced and utilized by many Gram-negative bacteria, including all Escherichia coli and Salmonella species. Within these species, pathogenic strains cause a variety of human diseases including urinary tract infections, gastroenteritis, and sepsis. Infections caused by these Gram-negative pathogens can be difficult to treat because of the impermeability of the outer membrane (OM). This impermeability can be overcome by utilizing siderophores as drug delivery vectors for targeting Gram-negative pathogens. Ent is a promising delivery vector because it undergoes active transport across the OM mediated by the Ent uptake machinery after scavenging Fe(III) from the extracellular environment. Despite the well-elucidated chemistry and biology of Ent, its use for SAC development was hampered by the lack of an appropriate functional group for cargo attachment. Our laboratory addressed this need by designing and synthesizing monofunctionalized Ent scaffolds. Over the past decade, we have used these scaffolds to explore Ent-based SACs with a variety of drug warheads, including ß-lactam and fluoroquinolone antibiotics, and Pt(IV) prodrugs. Investigations of the antibacterial activities of these conjugates and their cellular fates have informed our design principles and revealed approaches to achieving enhanced antibacterial potency and pathogen-targeted activity. Collectively, our studies of Ent-drug conjugates have provided discoveries, understanding, and invaluable insights for future design and evaluation of SACs.In this Account, we present the story of our work on Ent-drug conjugates that began about ten years ago with the development of monofunctionalized Ent scaffolds and the design and synthesis of various conjugates based on these scaffolds. We describe the antibacterial activity profiles and uptake pathways of Ent-drug conjugates harboring traditional antibiotics and repurposed platinum anticancer agents as well as studies that address cellular targets and fates. Finally, we discuss other applications of monofunctionalized Ent scaffolds, including a siderophore-based immunization strategy. We intend for this Account to inspire further investigations into the fundamental understanding and translational applications of siderophores and siderophore-drug conjugates.

Iron status and root cell morphology of Arabidopsis thaliana as modified by a bacterial ferri-siderophore.

We previously provided evidence for the contribution of pyoverdine to the iron nutrition of Arabidopsis. In the present article, we further analyze the mechanisms and physiology of the adaptations underlying plant iron nutrition through Fe(III)-pyoverdine (Fe(III)-pvd). An integrated approach combining microscopy and nanoscale secondary ion mass spectrometry (NanoSIMS) on plant samples was adopted to localize pyoverdine in planta and assess the impact of this siderophore on the plant iron status and root cellular morphology. The results support a possible plant uptake mechanism of the Fe(III)-pvd complex by epidermal root cells via a non-reductive process associated with the presence of more vesicles. Pyoverdine was transported to the central cylinder via the symplastic and/or trans-cellular pathway(s), suggesting a possible root-to-shoot translocation. All these processes led to enhanced plant iron nutrition, as previously shown. Overall, these findings suggest that bacterial siderophores contribute to plant iron uptake and homeostasis.

Vectorization via Siderophores Increases Antibacterial Activity of K(RW)3 Peptides against Pseudomonas aeruginosa.

A series of new conjugates comprised from a small synthetic antimicrobial peptide (AMP) and a siderophore-type vector component was designed and tested for activity on P. aeruginosa PAO1 and several genetically modified strains. As AMP, the well-established arginine-tryptophane combination K(RW)3 (P1) was chosen with an added lysine for siderophore attachment. This peptide is easy to prepare, modify, and possesses good anti-bacterial activity. On the vector part, we examined several moieties: (i) the natural siderophore deferoxamine (DFO); (ii) bidentate iron chelators based on the hydroxamate building block (4 a-c) ; (iii) the non-siderophore chelators deferasirox (DFX) and deferiprone-carboxylate (DFP-COOH). All conjugates were prepared by solid phase synthesis techniques and fully characterized by HPLC and mass spectrometry (including HR-MS). 55 Fe uptake assays indicate a receptor-mediated uptake for 4 a-c, DFP-COOH and DFO, which is dependent on the outer membrane transporter FoxA in the case of DFO. All conjugates showed increased antibacterial activity against P. aeruginosa compared to the parent peptide P1 alone when investigated in iron-depleted medium. MIC values were as low as 2 µM (for P1-DFP) on wild type P. aeruginosa. The activity of P1-DFO and P1-DFP was even better on genetically mutated strains unable to produce siderophores (down to 0.5 µM). Although the DFX vector on its own was not able to transport iron inside the bacterial cell as shown by 55 Fe uptake studies, the P1-DFX conjugate had excellent antibacterial activity compared to P1 (2 µM, and as low as 0.25 µM on a receptor-deficient strain unable to produce siderophores), suggesting that the conjugates were indeed recognized and internalized by an (unknown) transporter. Control experiments with an equimolar mixture of P1 and DFX confirm that the observed activity is intrinsic to vectorization. This work thus demonstrates the power of linking small AMPs covalently to siderophores for a new class of Trojan Horse antibiotics, with P1-DFP and P1-DFX being the most potent conjugates.

Discovery of New Siderophores from a Marine Streptomycetes sp. via Combined Metabolomics and Analysis of Iron-Chelating Activity.

The marine-derived Streptomyces sp. FIMYZ-003 strain was found to produce novel siderophores with yields negatively correlated with the iron concentration in the medium. Mass spectrometry (MS)-based metabolomics coupled with metallophore assays identified two novel α-hydroxycarboxylate-type siderophores, fradiamines C and D (3 and 4), together with two related known siderophores, fradiamines A and B (1 and 2). Their chemical structures were elucidated by nuclear magnetic resonance (NMR) and MS experiments. The annotation of a putative fra biosynthetic gene cluster enabled us to propose the biosynthetic pathway of fradiamines A-D. Furthermore, the solution-phase iron-binding activity of fradiamines was evaluated using metabolomics, confirming them as general iron scavengers. Fradiamines A-D exhibited Fe(III) binding activity equivalent to that of deferoxamine B mesylate. Growth analysis of pathogenic microbes demonstrated that fradiamine C promoted the growth of Escherichia coli and Staphylococcus aureus, but fradiamines A, B, and D did not. The results indicate that fradiamine C may serve as a novel iron carrier applicable to antibiotic delivery strategies to treat and prevent foodborne pathogens.

Metabolically versatile psychrotolerant Antarctic bacterium Pseudomonas sp. ANT_H12B is an efficient producer of siderophores and accompanying metabolites (SAM) useful for agricultural purposes.

BACKGROUND: Bacterial siderophores are chelating compounds with the potential of application in agriculture, due to their plant growth-promoting (PGP) properties, however, high production and purification costs are limiting factors for their wider application. Cost-efficiency of the production could be increased by omitting purification processes, especially since siderophores accompanying metabolites (SAM) often also possess PGP traits. In this study, the metabolism versatility of Pseudomonas sp. ANT_H12B was used for the optimization of siderophores production and the potential of these metabolites and SAM was characterized in the context of PGP properties. RESULTS: The metabolic diversity of ANT_H12B was examined through genomic analysis and phenotype microarrays. The strain was found to be able to use numerous C, N, P, and S sources, which allowed for the design of novel media suitable for efficient production of siderophores in the form of pyoverdine (223.50-512.60 µM). Moreover, depending on the culture medium, the pH of the siderophores and SAM solutions varied from acidic (pH < 5) to alkaline (pH > 8). In a germination test, siderophores and SAM were shown to have a positive effect on plants, with a significant increase in germination percentage observed in beetroot, pea, and tobacco. The PGP potential of SAM was further elucidated through GC/MS analysis, which revealed other compounds with PGP potential, such as indolic acetic acids, organic acids, fatty acids, sugars and alcohols. These compounds not only improved seed germination but could also potentially be beneficial for plant fitness and soil quality. CONCLUSIONS: Pseudomonas sp. ANT_H12B was presented as an efficient producer of siderophores and SAM which exhibit PGP potential. It was also shown that omitting downstream processes could not only limit the costs of siderophores production but also improve their agricultural potential.

Cryo-EM structures for the Mycobacterium tuberculosis iron-loaded siderophore transporter IrtAB.

The adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter, IrtAB, plays a vital role in the replication and viability of Mycobacterium tuberculosis (Mtb), where its function is to import iron-loaded siderophores. Unusually, it adopts the canonical type IV exporter fold. Herein, we report the structure of unliganded Mtb IrtAB and its structure in complex with ATP, ADP, or ATP analogue (AMP-PNP) at resolutions ranging from 2.8 to 3.5 Å. The structure of IrtAB bound ATP-Mg2+ shows a "head-to-tail" dimer of nucleotide-binding domains (NBDs), a closed amphipathic cavity within the transmembrane domains (TMDs), and a metal ion liganded to three histidine residues of IrtA in the cavity. Cryo-electron microscopy (Cryo-EM) structures and ATP hydrolysis assays show that the NBD of IrtA has a higher affinity for nucleotides and increased ATPase activity compared with IrtB. Moreover, the metal ion located in the TM region of IrtA is critical for the stabilization of the conformation of IrtAB during the transport cycle. This study provides a structural basis to explain the ATP-driven conformational changes that occur in IrtAB.

Megalochelin, a Tridecapeptide Siderophore from a Talented Streptomycete.

Streptomycetes are bacteria known for their extraordinary biosynthetic capabilities. Herein, we describe the genome and metabolome of a particularly talented strain, Streptomyces ID71268. Its 8.4-Mbp genome harbors 32 bioinformatically predicted biosynthetic gene clusters (BGCs), out of which 10 are expressed under a single experimental condition. In addition to five families of known metabolites with previously assigned BGCs (nigericin, azalomycin F, ectoine, SF2766, and piericidin), we were able to predict BGCs for three additional metabolites: streptochlorin, serpetene, and marinomycin. The strain also produced two families of presumably novel metabolites, one of which was associated with growth inhibitory activity against the human opportunistic pathogen Acinetobacter baumannii in an iron-dependent manner. Bioassay-guided fractionation, followed by extensive liquid chromatography-mass spectrometry (LC-MS) and NMR analyses, established that the molecule responsible for the observed antibacterial activity is an unusual tridecapeptide siderophore with a ring-and-tail structure: the heptapeptide ring is formed through a C-C bond between a 2,3-dihydroxybenzoate (DHB) cap on Gly1 and the imidazole moiety of His7, while the hexapeptide tail is sufficient for binding iron. This molecule, named megalochelin, is the largest known siderophore. The megalochelin BGC encodes a 13-module nonribosomal peptide synthetase for the synthesis of the tridecapeptide, and a copper-dependent oxidase, likely responsible for the DHB-imidazole cross-link, whereas the genes for synthesis of the DHB starter unit are apparently specified in trans by a different BGC. Our results suggest that prolific producers of specialized metabolites may conceal hidden treasures within a background of known compounds.

Synthesis of temporin L hydroxamate-based peptides and evaluation of their coordination properties with iron(III ).

Ferric iron is an essential nutrient for bacterial growth. Pathogenic bacteria synthesize iron-chelating entities known as siderophores to sequestrate ferric iron from host organisms in order to colonize and replicate. The development of antimicrobial peptides (AMPs) conjugated to iron chelators represents a promising strategy for reducing the iron availability, inducing bacterial death, and enhancing simultaneously the efficacy of AMPs. Here we designed, synthesized, and characterized three hydroxamate-based peptides Pep-cyc1, Pep-cyc2, and Pep-cyc3, derived from a cyclic temporin L peptide (Pep-cyc) developed previously by some of us. The Fe3+ complex formation of each ligand was characterized by UV-visible spectroscopy, mass spectrometry, and IR and NMR spectroscopies. In addition, the effect of Fe3+ on the stabilization of the α-helix conformation of hydroxamate-based peptides and the cotton effect were examined by CD spectroscopy. Moreover, the antimicrobial results obtained in vitro on some Gram-negative strains (K. pneumoniae and E. coli) showed the ability of each peptide to chelate efficaciously Fe3+ obtaining a reduction of MIC values in comparison to their parent peptide Pep-cyc. Our results demonstrated that siderophore conjugation could increase the efficacy and selectivity of AMPs used for the treatment of infectious diseases caused by Gram-negative pathogens.

LCN2-Fungal siderophore-iron binding and uptake leads to oxidative stress and cell death in hepatocellular carcinoma cell line HepG2.

Microorganisms produce non-ribosomal peptides called siderophores for the purpose of iron acquisition. Mammalian immune system is well-known for producing small secretory proteins called lipocalins upon bacterial infection. These proteins sequester siderophores produced by invading bacterial pathogens rendering them unable to acquire iron from the host. However, this is not their sole function. In addition to transferrin and lactoferrin, lipocalins are also known to transport siderophore-bound iron to the host cells. While binding of bacterial siderophores with human lipocalin is well studied, binding of the fungal counterpart is still not confirmed and fully understood. Apart from pathogen-affected cells, developing cancerous cells also show varying expression level of different proteins including those involved in iron transport. The possibility of exogenous fungal siderophore-mediated iron transport via lipocalin and its receptor in mammalian cells has not yet been explored much. In present investigation we have checked differential expression of human lipocalin, LCN2 in hepatocellular carcinoma cell lines HepG2 as well as its normal counterpart WRL-68 and computationally determined the feasibility of LCN2 binding with fungal siderophore. Further in case of a stable complex being formed, whether this complex has the ability to transport iron through its specific receptor was assessed. Also, we have tried to explore possible mechanism of fungal-siderophore mediated oxidative stress leading to significant cell death in cancerous cells. This study will thus be useful towards finding a new way of treating hepatocellular carcinoma via inducing siderophore-mediated cell death in cancerous cells.Communicated by Ramaswamy H. Sarma.

Nonribosomal peptide synthetase gene clusters and characteristics of predicted NRPS-dependent siderophore synthetases in Armillaria and other species in the Physalacriaceae.

Fungal secondary metabolites are often pathogenicity or virulence factors synthesized by genes contained in secondary metabolite gene clusters (SMGCs). Nonribosomal polypeptide synthetase (NRPS) clusters are SMGCs which produce peptides such as siderophores, the high affinity ferric iron chelating compounds required for iron uptake under aerobic conditions. Armillaria spp. are mostly facultative necrotrophs of woody plants. NRPS-dependent siderophore synthetase (NDSS) clusters of Armillaria spp. and selected Physalacriaceae were investigated using a comparative genomics approach. Siderophore biosynthesis by strains of selected Armillaria spp. was evaluated using CAS and split-CAS assays. At least one NRPS cluster and other clusters were detected in the genomes studied. No correlation was observed between the number and types of SMGCs and reported pathogenicity of the species studied. The genomes contained one NDSS cluster each. All NDSSs were multi-modular with the domain architecture (ATC)3(TC)2. NDSS clusters of the Armillaria spp. showed a high degree of microsynteny. In the genomes of Desarmillaria spp. and Guyanagaster necrorhizus, NDSS clusters were more syntenic with NDSS clusters of Armillaria spp. than to those of the other Physalacriaceae species studied. Three A-domain orthologous groups were identified in the NDSSs, and atypical Stachelhaus codes were predicted for the A3 orthologous group. In vitro biosynthesis of mainly hydroxamate and some catecholate siderophores was observed. Hence, Armillaria spp. generally contain one highly conserved, NDSS cluster although some interspecific variations in the products of these clusters is expected. Results from this study lays the groundwork for future studies to elucidate the molecular biology of fungal phyto-pathogenicity.

Proteomic Investigation of the Antibacterial Mechanism of Cefiderocol against Escherichia coli.

This study aimed to investigate the antibacterial mechanism of cefiderocol (CFDC) using data-independent acquisition quantitative proteomics combined with cellular and molecular biological assays. Numerous differentially expressed proteins related to the production of NADH, reduced cofactor flavin adenine dinucleotide (FADH2), NADPH and reactive oxygen species (ROS), iron-sulfur cluster binding, and iron ion homeostasis were found to be upregulated by CFDC. Furthermore, parallel reaction monitoring analysis validated these results. Meanwhile, we confirmed that the levels of NADH, ROS, H2O2, and iron ions were induced by CFDC, and the sensitivity of Escherichia coli to CFDC was inhibited by the antioxidant vitamin C, N-acetyl-l-cysteine, and deferoxamine. Moreover, deferoxamine also suppressed the H2O2 stress induced by CFDC. In addition, knockout of the NADH-quinone oxidoreductase genes (nuoA, nuoC, nuoE, nuoF, nuoG, nuoJ, nuoL, nuoM) in the respiratory chain attenuated the sensitivity of E. coli to CFDC far beyond the effects of cefepime and ceftazidime; in particular, the E. coli BW25113 ΔnuoJ strain produced 60-fold increases in MIC to CFDC compared to that of the wild-type E. coli BW25113 strain. The present study revealed that CFDC exerts its antibacterial effects by inducing ROS stress by elevating the levels of NADH and iron ions in E. coli. IMPORTANCE CFDC was the first FDA-approved siderophore cephalosporin antibiotic in 2019 and is known for its Trojan horse tactics and broad antimicrobial activity against Gram-negative bacteria. However, its antibacterial mechanism is not fully understood, and whether it has an impact on in vivo iron ion homeostasis remains unknown. To comprehensively reveal the antibacterial mechanisms of CFDC, data-independent acquisition quantitative proteomics combined with cellular and molecular biological assays were performed in this study. The findings will further facilitate our understanding of the antibacterial mechanism of CFDC and may provide a theoretical foundation for controlling CFDC resistance in the future.

Rational inhibitor design for Pseudomonas aeruginosa salicylate adenylation enzyme PchD.

Pseudomonas aeruginosa is an increasingly antibiotic-resistant pathogen that causes severe lung infections, burn wound infections, and diabetic foot infections. P. aeruginosa produces the siderophore pyochelin through the use of a non-ribosomal peptide synthetase (NRPS) biosynthetic pathway. Targeting members of siderophore NRPS proteins is one avenue currently under investigation for the development of new antibiotics against antibiotic-resistant organisms. Here, the crystal structure of the pyochelin adenylation domain PchD is reported. The structure was solved to 2.11 Å when co-crystallized with the adenylation inhibitor 5'-O-(N-salicylsulfamoyl)adenosine (salicyl-AMS) and to 1.69 Å with a modified version of salicyl-AMS designed to target an active site cysteine (4-cyano-salicyl-AMS). In the structures, PchD adopts the adenylation conformation, similar to that reported for AB3403 from Acinetobacter baumannii.

Slow Kinetics of Iron Binding to Marine Ligands in Seawater Measured by Isotope Exchange Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry.

Current understanding of dissolved iron (Fe) speciation in the ocean is based on two fundamentally different approaches: electrochemical methods that measure bulk properties of a heterogeneous ligand pool and liquid chromatography mass spectrometry methods that characterize ligands at a molecular level. Here, we describe a method for simultaneously determining Fe-ligand dissociation rate constants (kd) of suites of naturally occurring ligands in seawater by monitoring the exchange of ligand-bound 56Fe with 57Fe using liquid chromatography-inductively coupled mass spectrometry. Values of kd were determined for solutions of ferrichrome and ferrioxamine E. In seawater, the dissociation rate constant of ferrichrome (kd = 10 × 10-8 s-1) was greater than that of ferrioxamine E (kd = 3.6 × 10-8 s-1). The rates for both compounds were over twice as fast in seawater compared with pure water, suggesting that seawater salts accelerate dissociation. Isotope exchange experiments on organic extracts of natural seawater indicated that ligand-binding sites associated with chromatographically unresolved dissolved organic matter exchanged Fe more quickly (kd = 1.8 × 10-5 s-1) than amphibactin siderophores (kd = 2.15 × 10-6 s-1) and an unidentified siderophore with m/z 709 (kd = 9.6 × 10-6 s-1). These findings demonstrate that our approach can bridge molecular-level ligand identification with kinetic and thermodynamic metal-binding properties.

Iron coordination to pyochelin siderophore influences dynamics of FptA receptor from Pseudomonas aeruginosa: a molecular dynamics simulation study.

FptA is a TonB-dependent transporter that permits the high affinity binding and transport of Fe(III)-pyochelin complex across the outer membrane of Pseudomonas aeruginosa. Molecular dynamics simulations were employed to FptA receptor and its complexes with pyochelin, and co-crystallized Fe(III)-pyochelin-ethanediol and Fe(III)-pyochelin-water embedded in dilauroyl phosphatidyl choline bilayer for the evaluation of their structural and dynamical properties. The evaluation of properties of the receptor bound to pyochelin molecule and Fe(III)-pyochelin complexes helped to figure out the iron coordination effect on the receptor properties. Moreover, comparison of these four simulation systems revealed further information on the dynamical changes occurred in extracellular loops, in particular loop-7 corresponding to the missing amino acid residues including the close-by loop-8 that was largely affected by the metal coordination to pyochelin. The binding of iron to pyochelin molecule affected the overall structure of the receptor therefore, evaluation fo the gyration radii and hydrogen bonding were evaluated as well as analysis of the pore size were also carried out to understand the effect of metal coordination on the dynamics of the helices which form a kind of translocation channel to transport the siderophore across the FptA protein into the periplasmic space. The properties of each component of the molecular systems were therefore observed to be perturbed by the incorporation of iron to the pyochelin molecule thus demonstrating that the bacteria use its receptor to abstract and transport iron from extracellular environment for its survival and that was made possible to understand at the molecular level through successful implementation of molecular dynamics simulations.

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.

Saccharochelins A-H, Cytotoxic Amphiphilic Siderophores from the Rare Marine Actinomycete Saccharothrix sp. D09.

Siderophores are secreted by microorganisms to survive in iron-depleted conditions, and they also possess tremendous therapeutic potential. Genomic-inspired isolation facilitated the identification of eight amphiphilic siderophores, saccharochelins A-H (1-8), from a rare marine-derived Saccharothrix species. Saccharochelins feature a series of fatty acyl groups appended to the same tetrapeptide skeleton. With the help of gene disruption and heterologous expression, we identified the saccharochelin biosynthetic pathway. The diversity of saccharochelins originates from the flexible specificity of the starter condensation (CS) domain at the beginning of the nonribosomal peptide synthetase (NRPS) toward various fatty acyl substrates. Saccharochelins showed cytotoxicity against several human tumor cell lines, with IC50 values ranging from 2.3 to 17 µM. Additionally, the fatty acid side chains of the saccharochelins remarkably affected the cytotoxicity, suggesting changing the N-terminal acyl groups of lipopeptides may be a promising approach to produce more potent derivatives.

Targeted SPION siderophore conjugate loaded with doxorubicin as a theranostic agent for imaging and treatment of colon carcinoma.

Recently, the siderophores have opened new horizons in nanomedicine. The current study aimed to design a theranostic platform based on superparamagnetic iron oxide nanoparticles-pyoverdine (SPION/PVD) conjugates bound to MUC1 aptamer (MUC1Apt) and loaded with doxorubicin (DOX) as an anti-cancer agent. The SPION/PVD complex was covalently conjugated to MUC1Apt and loaded with DOX to prepare a targeted drug delivery system (SPION/PVD/MUC1Apt/DOX). The investigation of cellular cytotoxicity and uptake of formulations by MTT and flow cytometry in both MUC1 positive (C26) and MUC1 negative (CHO) cell lines revealed that MUC1Apt could improve both cellular uptake and toxicity in the C26 cell line. The evaluation of tumor-targeting activity by in vivo bio-distribution showed that the targeted formulation could enhance tumor inhibitory growth effect and survival rate in C26 tumor-bearing mice. Furthermore, the potential of synthesized SPION/PVD/MUC1Apt/DOX complex as diagnostic agents was investigated by magnetic resonance imaging (MRI) which improved the contrast of tumor site in MRI. Our findings confirm that aptamer-targeted PVD chelated the SPION as a diagnostic agent and loaded with DOX as a chemotherapeutic drug, would be beneficial as a novel theranostic platform.

Structures of a non-ribosomal peptide synthetase condensation domain suggest the basis of substrate selectivity.

Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.