Thermostable homologues of the periplasmic siderophore-binding protein CeuE from Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius.

Siderophore-binding proteins from two thermophilic bacteria, Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius, were identified from a search of sequence databases, cloned and overexpressed. They are homologues of the well characterized protein CjCeuE from Campylobacter jejuni. The iron-binding histidine and tyrosine residues are conserved in both thermophiles. Crystal structures were determined of the apo proteins and of their complexes with iron(III)-azotochelin and its analogue iron(III)-5-LICAM. The thermostability of both homologues was shown to be about 20°C higher than that of CjCeuE. Similarly, the tolerance of the homologues to the organic solvent dimethylformamide (DMF) was enhanced, as reflected by the respective binding constants for these ligands measured in aqueous buffer at pH 7.5 in the absence and presence of 10% and 20% DMF. Consequently, these thermophilic homologues offer advantages in the development of artificial metalloenzymes using the CeuE family.

Siderophore-Linked Ruthenium Catalysts for Targeted Allyl Ester Prodrug Activation within Bacterial Cells.

Due to rising resistance, new antibacterial strategies are needed, including methods for targeted antibiotic release. As targeting vectors, chelating molecules called siderophores that are released by bacteria to acquire iron have been investigated for conjugation to antibacterials, leading to the clinically approved drug cefiderocol. The use of small-molecule catalysts for prodrug activation within cells has shown promise in recent years, and here we investigate siderophore-linked ruthenium catalysts for the activation of antibacterial prodrugs within cells. Moxifloxacin-based prodrugs were synthesised, and their catalyst-mediated activation was demonstrated under anaerobic, biologically relevant conditions. In the absence of catalyst, decreased antibacterial activities were observed compared to moxifloxacin versus Escherichia coli K12 (BW25113). A series of siderophore-linked ruthenium catalysts were investigated for prodrug activation, all of which displayed a combinative antibacterial effect with the prodrug, whereas a representative example displayed little toxicity against mammalian cell lines. By employing complementary bacterial growth assays, conjugates containing siderophore units based on catechol and azotochelin were found to be most promising for intracellular prodrug activation.

Antibiotic-functionalized gold nanoparticles for the detection of active ß-lactamases.

Antimicrobial resistance (AMR) continues to threaten the effective treatment and prevention of bacterial infections. The spread of resistant infections is accelerated by the lack of fast and cost-effective tests for the detection of AMR at the point-of-care. We aimed to address this challenge by developing a diagnostic tool to detect one of the major forms of AMR, the ß-lactamase enzymes. Antibiotic-functionalized gold nanoparticles (AuNPs) have been successfully developed for the detection of ß-lactamases in challenging biological media, namely undiluted urine. Furthermore, these tools are compatible with samples containing a urine sample preservative (boric acid) or hematuria (blood). The functionalized AuNPs interact with the active ß-lactamases, resulting in the hydrolysis of the surface-bound antibiotics, which then inhibits binding of the AuNPs to a capture protein (a penicillin-binding protein) to indicate the presence of active ß-lactamases. We successfully integrated the antibiotic-functionalized AuNPs into a new lateral flow assay (LFA), which can be used to detect active ß-lactamases down to the detection limit of 11 nM. While we demonstrate the use of antibiotic-functionalized AuNPs in an LFA format to provide a novel method of detecting active ß-lactamases, these functionalized AuNPs are amenable to a range of alternative diagnostic technologies and could lead to vital point-of-care diagnostics for the early detection of multi-drug resistant infections.

Synthesis and antimicrobial activity of an SO2-releasing siderophore conjugate.

A novel Trojan Horse conjugate consisting of an SO2-releasing 2,4-dinitrobenzenesulfonamide group attached to the monocatecholate siderophore aminochelin was synthesized to examine whether a bidentate catecholate siderophore unit could help potentiate the antimicrobial activity of SO2-releasing prodrugs. The conjugate obtained displays rapid SO2 release on reaction with glutathione, and proved more active against Staphylococcus aureus than a comparable SO2-releasing prodrug lacking the siderophore unit, although activity required micromolar concentrations. The conjugate was inactive against wild-type Escherichia coli, but activity was observed against an entA mutant strain that is unable to produce its major siderophores. Hence, the poor activity of the conjugate in wild-type E. coli may be due to the production of native siderophores that can compete with the conjugate for iron binding and uptake.

Unveiling the origin of photo-induced enhancement of oxidation catalysis at Mo(VI) centres of Ru(II)-Mo(VI) dyads.

Photo-induced oxidation-enhancement in biomimetic bridged Ru(ii)-Mo(vi) photo-catalyst is unexpectedly photo-activated in ps timescales. One-photon absorption generates an excited state where both photo-oxidized and photo-reduced catalytic centres are activated simultaneously and independently.

Experimental Methods for Evaluating the Bacterial Uptake of Trojan Horse Antibacterials.

The field of antibacterial siderophore conjugates, referred to as Trojan Horse antibacterials, has received increasing attention in recent years, driven by the rise of antimicrobial resistance. Trojan Horse antibacterials offer an opportunity to exploit the specific pathways present in bacteria for active iron uptake, potentially allowing the drugs to bypass membrane-associated resistance mechanisms. Hence, the Trojan Horse approach might enable the redesigning of old antibiotics and the development of antibacterials that target specific pathogens. Critical parts of evaluating such Trojan Horse antibacterials and improving their design are the quantification of their bacterial uptake and the identification of the pathways by which this occurs. In this minireview, we highlight a selection of the biological and chemical methods used to study the uptake of Trojan Horse antibacterials, exemplified with case studies, some of which have led to drug candidates in clinical development or approved antibiotics.

A Salmochelin S4-Inspired Ciprofloxacin Trojan Horse Conjugate.

A novel ciprofloxacin-siderophore Trojan Horse antimicrobial was prepared by incorporating key design features of salmochelin, a stealth siderophore that evades mammalian siderocalin capture via its glycosylated catechol units. Assessment of the antimicrobial activity of the conjugate revealed that attachment of the salmochelin mimic resulted in decreased potency, compared to ciprofloxacin, against two Escherichia coli strains, K12 and Nissle 1917, in both iron replete and deplete conditions. This observation could be attributed to a combination of reduced DNA gyrase inhibition, as confirmed by in vitro DNA gyrase assays, and reduced bacterial uptake. Uptake was monitored using radiolabeling with iron-mimetic 67Ga3+, which revealed limited cellular uptake in E. coli K12. In contrast, previously reported staphyloferrin-based conjugates displayed a measurable uptake in analogous 67Ga3+ labeling studies. These results suggest that, in the design of Trojan Horse antimicrobials, the choice of siderophore and the nature and length of the linker remain a significant challenge.

Synthesis and biochemical evaluation of cephalosporin analogues equipped with chemical tethers.

Molecular probes typically require structural modifications to allow for the immobilisation or bioconjugation with a desired substrate but the effects of these changes are often not evaluated. Here, we set out to determine the effects of attaching functional handles to a first-generation cephalosporin. A series of cephalexin derivatives was prepared, equipped with chemical tethers suitable for the site-selective conjugation of antibiotics to functionalised surfaces. The tethers were positioned remotely from the ß-lactam ring to ensure minimal effect to the antibiotic's pharmacophore. Herein, the activity of the modified antibiotics was evaluated for binding to the therapeutic target, the penicillin binding proteins, and shown to maintain binding interactions. In addition, the deactivation of the modified drugs by four ß-lactamases (TEM-1, CTX-M-15, AmpC, NDM-1) was investigated and the effect of the tethers on the catalytic efficiencies determined. CTX-M-15 was found to favour hydrolysis of the parent antibiotic without a tether, whereas AmpC and NDM-1 were found to favour the modified analogues. Furthermore, the antimicrobial activity of the derivatives was evaluated to investigate the effect of the structural modifications on the antimicrobial activity of the parent drug, cephalexin.

Artificial imine reductases: developments and future directions.

Biocatalytic imine reduction has been a topic of intense research by the artificial metalloenzyme community in recent years. Artificial constructs, together with natural enzymes, have been engineered to produce chiral amines with high enantioselectivity. This review examines the design of the main classes of artificial imine reductases reported thus far and summarises approaches to enhancing their catalytic performance using complementary methods. Examples of utilising these biocatalysts in vivo or in multi-enzyme cascades have demonstrated the potential that artIREDs can offer, however, at this time their use in biocatalysis remains limited. This review explores the current scope of artIREDs and the strategies used for catalyst improvement, and examines the potential for artIREDs in the future.

Surface-Bound Antibiotic for the Detection of ß-Lactamases.

Antimicrobial resistance (AMR) has been identified as a major threat to public health worldwide. To ensure appropriate use of existing antibiotics, rapid and reliable tests of AMR are necessary. One of the most common and clinically important forms of bacterial resistance is to ß-lactam antibiotics (e.g., penicillin). This resistance is often caused by ß-lactamases, which hydrolyze ß-lactam drugs, rendering them ineffective. Current methods for detecting these enzymes require either time-consuming growth assays or antibiotic mimics such as nitrocefin. Here, we report the development of a surface-bound, clinically relevant ß-lactam drug that can be used to detect ß-lactamases and that is compatible with a range of high-sensitivity, low-cost, and label-free analytical techniques currently being developed for point-of-care-diagnostics. Furthermore, we demonstrate the use of these functionalized surfaces to selectively detect ß-lactamases in complex biological media, such as urine.

Mimicking salmochelin S1 and the interactions of its Fe(III) complex with periplasmic iron siderophore binding proteins CeuE and VctP.

A mimic of the tetradentate stealth siderophore salmochelin S1, was synthesised, characterised and shown to form Fe(III) complexes with ligand-to-metal ratios of 1:1 and 3:2. Circular dichroism spectroscopy confirmed that the periplasmic binding proteins CeuE and VctP of Campylobacter jejuni and Vibrio cholerae, respectively, bind the Fe(III) complex of the salmochelin mimic by preferentially selecting Λ-configured Fe(III) complexes. Intrinsic fluorescence quenching studies revealed that VctP binds Fe(III) complexes of the mimic and structurally-related catecholate ligands, such as enterobactin, bis(2, 3-dihydroxybenzoyl-l-serine) and bis(2, 3-dihydroxybenzoyl)-1, 5-pentanediamine with higher affinity than does CeuE. Both CeuE and VctP display a clear preference for the tetradentate bis(catecholates) over the tris(catecholate) siderophore enterobactin. These findings are consistent with reports that V. cholerae and C. jejuni utilise the enterobactin hydrolysis product bis(2, 3-dihydroxybenzoyl)-O-seryl serine for the acquisition of Fe(III) and suggest that the role of salmochelin S1 in the iron uptake of enteric pathogens merits further investigation.

Chemical and Biological Aspects of Nutritional Immunity-Perspectives for New Anti-Infectives that Target Iron Uptake Systems.

Upon bacterial infection, one of the defense mechanisms of the host is the withdrawal of essential metal ions, in particular iron, which leads to "nutritional immunity". However, bacteria have evolved strategies to overcome iron starvation, for example, by stealing iron from the host or other bacteria through specific iron chelators with high binding affinity. Fortunately, these complex interactions between the host and pathogen that lead to metal homeostasis provide several opportunities for interception and, thus, allow the development of novel antibacterial compounds. This Review focuses on iron, discusses recent highlights, and gives some future perspectives which are relevant in the fight against antibiotic resistance.

Interactions of the periplasmic binding protein CeuE with Fe(III) n-LICAM4- siderophore analogues of varied linker length.

Bacteria use siderophores to mediate the transport of essential Fe(III) into the cell. In Campylobacter jejuni the periplasmic binding protein CeuE, an integral part of the Fe(III) transport system, has adapted to bind tetradentate siderophores using a His and a Tyr side chain to complete the Fe(III) coordination. A series of tetradentate siderophore mimics was synthesized in which the length of the linker between the two iron-binding catecholamide units was increased from four carbon atoms (4-LICAM4-) to five, six and eight (5-, 6-, 8-LICAM4-, respectively). Co-crystal structures with CeuE showed that the inter-planar angles between the iron-binding catecholamide units in the 5-, 6- and 8-LICAM4- structures are very similar (111°, 110° and 110°) and allow for an optimum fit into the binding pocket of CeuE, the inter-planar angle in the structure of 4-LICAM4- is significantly smaller (97°) due to restrictions imposed by the shorter linker. Accordingly, the protein-binding affinity was found to be slightly higher for 5- compared to 4-LICAM4- but decreases for 6- and 8-LICAM4-. The optimum linker length of five matches that present in natural siderophores such as enterobactin and azotochelin. Site-directed mutagenesis was used to investigate the relative importance of the Fe(III)-coordinating residues H227 and Y288.

Light-Induced Activation of a Molybdenum Oxotransferase Model within a Ru(II)-Mo(VI) Dyad.

Nature uses molybdenum-containing enzymes to catalyze oxygen atom transfer (OAT) from water to organic substrates. In these enzymes, the two electrons that are released during the reaction are rapidly removed, one at a time, by spatially separated electron transfer units. Inspired by this design, a Ru(II)-Mo(VI) dyad was synthesized and characterized, with the aim of accelerating the rate-determining step in the cis-dioxo molybdenum-catalyzed OAT cycle, the transfer of an oxo ligand to triphenyl phosphine, via a photo-oxidation process. The dyad consists of a photoactive bis(bipyridyl)-phenanthroline ruthenium moiety that is covalently linked to a bioinspired cis-dioxo molybdenum thiosemicarbazone complex. The quantum yield and luminescence lifetimes of the dyad [Ru(bpy)2(L2)MoO2(solv)]2+ were determined. The major component of the luminescence decay in MeCN solution (τ = 1149 ± 2 ns, 67%) corresponds closely to the lifetime of excited [Ru(bpy)2(phen-NH2)]2+, while the minor component (τ = 320 ± 1 ns, 31%) matches that of [Ru(bpy)2(H2-L2)]2+. In addition, the (spectro)electrochemical properties of the system were investigated. Catalytic tests showed that the dyad-catalyzed OAT from dimethyl sulfoxide to triphenyl phosphine proceeds significantly faster upon irradiation with visible light than in the dark. Methylviologen acts as a mediator in the photoredox cycle, but it is regenerated and hence only required in stoichiometric amounts with respect to the catalyst rather than sacrificial amounts. It is proposed that oxidative quenching of the photoexcited Ru unit, followed by intramolecular electron transfer, leads to the production of a reactive one-electron oxidized catalyst, which is not accessible by electrochemical methods. A significant, but less pronounced, rate enhancement was observed when an analogous bimolecular system was tested, indicating that intramolecular electron transfer between the photosensitizer and the catalytic center is more efficient than intermolecular electron transfer between the separate components.

Probing linker design in citric acid-ciprofloxacin conjugates.

A series of structurally related citric acid-ciprofloxacin conjugates was synthesised to investigate the influence of the linker between citric acid and ciprofloxacin on antibacterial activities. Minimum inhibitory concentrations (MICs) were determined against a panel of reference strains and clinical isolates of bacteria associated with infection in humans and correlated with the DNA gyrase inhibitory activity. The observed trend was rationalised by computational modelling.

Probing bacterial uptake of glycosylated ciprofloxacin conjugates.

Mono- and disaccharide-functionalised conjugates of the fluoroquinolone antibiotic ciprofloxacin have been synthesised and used as chemical probes of the bacterial uptake of glycosylated ciprofloxacin. Their antimicrobial activities against a panel of clinically relevant bacteria were determined: the ability of these conjugates to inhibit their target DNA gyrase and to be transported into the bacteria was assessed by using in vivo and in vitro assays. The data suggest a lack of active uptake through sugar transporters and that although the addition of monosaccharides is compatible with the inhibition of DNA gyrase, the addition of a disaccharide results in a significant decrease in antimicrobial activity.

The background, discovery and clinical development of BCR-ABL inhibitors.

The story of the inhibition of BCR-ABL as a treatment for chronic myelogenous leukaemia serves to illustrate key aspects of the kinase drug discovery and development process. Firstly, elucidation of the disease mechanism enabled identification of the molecular target(s) which catalysed pharmaceutical research and resulted in Gleevec(®) (Novartis) as the first FDA approved BCR-ABL inhibitor. However, clinical success was soon tempered by the emergence of drug resistance through various mechanisms. Using rational drug design, several hypotheses were devised to overcome resistance issues leading to the development of second generation inhibitors, providing clinicians and patients with greater therapeutic choice.

Staphyloferrin A as siderophore-component in fluoroquinolone-based Trojan horse antibiotics.

A series of fluoroquinolone conjugates was synthesised by linking the carboxylic acid functionality of the carboxylate-type siderophore staphyloferrin A and its derivatives to the piperazinyl nitrogen of ciprofloxacin and norfloxacin via amide bond formation. Four siderophore-drug conjugates were screened against a panel of bacteria associated with infection in humans. Whilst no activity was found against ciprofloxacin- or norfloxacin-resistant bacteria, one of the conjugates retained antibacterial activity against fluoroquinolone-susceptible strains although the structure of its lysine-based siderophore component differs from that of the natural siderophore staphyloferrin A. In contrast, three ornithine-based siderophore conjugates showed significantly reduced activity against strains that are susceptible to their respective parent fluoroquinolones, regardless of the type of fluoroquinolone attached or chirality at the ornithine Cα-atom. The loss of potency observed for the (R)- and (S)-ornithine-based ciprofloxacin conjugates correlates with their reduced inhibitory activity against the target enzyme DNA gyrase.

Atropisomerisation in sterically hindered α,ß-disubstituted cyclopentenones derived from an intermolecular cobalt(0)-mediated Pauson-Khand reaction.

4-(2-Phenylethynyl)-2H-chromen-2-one reacts with norbornene and Co(2)(CO)(8) in an intermolecular Pauson-Khand reaction by focused microwave dielectric heating. Two regioisomeric products are formed; the electron-deficient coumarin moiety preferentially occupies the ß-position of the cyclopentenone ring system, whereas the phenyl occupies the α-position. The sterically hindered α,ß-(2,3)-disubstituted cyclopentenone regioisomeric products exhibit pronounced atropisomerisation, and the magnitude of the energetic barrier to interconversion between these atropisomers is dependent on the relative position of the coumarin moieties. Interconversion is slow when the coumarin is found in the α-position, whereas interconversion is relatively fast when found in the ß-position.

Synthesis of citrate-ciprofloxacin conjugates.

Two regioisomeric citrate-functionalized ciprofloxacin conjugates have been synthesized and their antimicrobial activities against a panel of clinically-relevant bacteria have been determined. Cellular uptake mechanisms were investigated using wild-type and ompF deletion strains of Escherichia coli K-12.