Enzyme-Activated, Chemiluminescent Siderophore-Dioxetane Probes Enable the Selective and Highly Sensitive Detection of Bacterial Pathogens.

The sensitive detection of bacterial infections is a prerequisite for their successful treatment. The use of a chemiluminescent readout was so far hampered by an insufficient probe enrichment at the pathogens. We coupled siderophore moieties, that harness the unique iron transport system of bacteria, with enzyme-activatable dioxetanes and obtained seven trifunctional probes with high signal-to-background ratios (S/B=426-859). Conjugates with efficient iron transport capability into bacteria were identified through a growth recovery assay. All ESKAPE pathogens were labelled brightly by desferrioxamine conjugates, while catechols were weaker due to self-quenching. Bacteria could also be detected inside lung epithelial cells. The best probe 8 detected 9.1×103  CFU mL-1 of S. aureus and 5.0×104  CFU mL-1 of P. aeruginosa, while the analogous fluorescent probe 10 was 205-305fold less sensitive. This qualifies siderophore dioxetane probes for the selective and sensitive detection of bacteria.

Filovirus Antiviral Activity of Cationic Amphiphilic Drugs Is Associated with Lipophilicity and Ability To Induce Phospholipidosis.

Several cationic amphiphilic drugs (CADs) have been found to inhibit cell entry of filoviruses and other enveloped viruses. Structurally unrelated CADs may have antiviral activity, yet the underlying common mechanism and structure-activity relationship are incompletely understood. We aimed to understand how widespread antiviral activity is among CADs and which structural and physico-chemical properties are linked to entry inhibition. We measured inhibition of Marburg virus pseudoparticle (MARVpp) cell entry by 45 heterogeneous and mostly FDA-approved CADs and cytotoxicity in EA.hy926 cells. We analyzed correlation of antiviral activity with four chemical properties: pKa, hydrophobicity (octanol/water partitioning coefficient; ClogP), molecular weight, and distance between the basic group and hydrophobic ring structures. Additionally, we quantified drug-induced phospholipidosis (DIPL) of a CAD subset by flow cytometry. Structurally similar compounds (derivatives) and those with similar chemical properties but unrelated structures (analogues) to those of strong inhibitors were obtained by two in silico similarity search approaches and tested for antiviral activity. Overall, 11 out of 45 (24%) CADs inhibited MARVpp by 40% or more. The strongest antiviral compounds were dronedarone, triparanol, and quinacrine. Structure-activity relationship studies revealed highly significant correlations between antiviral activity, hydrophobicity (ClogP > 4), and DIPL. Moreover, pKa and intramolecular distance between hydrophobic and hydrophilic moieties correlated with antiviral activity but to a lesser extent. We also showed that in contrast to analogues, derivatives had antiviral activity similar to that of the seed compound dronedarone. Overall, one-quarter of CADs inhibit MARVpp entry in vitro, and antiviral activity of CADs mostly relies on their hydrophobicity yet is promoted by the individual structure.

Molecular Signatures of the Eagle Effect Induced by the Artificial Siderophore Conjugate LP-600 in E. coli.

Achieving cellular uptake is a central challenge for novel antibiotics targeting Gram-negative bacterial pathogens. One strategy is to hijack the bacterial iron transport system by siderophore-antibiotic conjugates that are actively imported into the cell. This was realized with the MECAM-ampicillin conjugate LP-600 we recently reported that was highly active against E. coli. In the present study, we investigate a paradoxical regrowth of E. coli upon treatment of LP-600 at concentrations 16-32 times above the minimum inhibitory concentration (MIC). The phenomenon, coined "Eagle-effect" in other systems, was not due to resistance formation, and it occurred for the siderophore conjugate but not for free ampicillin. To investigate the molecular imprint of the Eagle effect, a combined transcriptome and untargeted metabolome analysis was conducted. LP-600 induced the expression of genes involved in iron acquisition, SOS response, and the e14 prophage upon regrowth conditions. The Eagle effect was diminished in the presence of sulbactam, which we ascribe to a putative synergistic antibiotic action but not to ß-lactamase inhibition. The study highlights the relevance of the Eagle effect for siderophore conjugates. Through the first systematic -omics investigations, it also demonstrates that the Eagle effect manifests not only in a paradoxical growth but also in unique gene expression and metabolite profiles.

Uptake Mechanisms and Regulatory Responses to MECAM- and DOTAM-Based Artificial Siderophores and Their Antibiotic Conjugates in Pseudomonas aeruginosa.

The development of new antibiotics against Gram-negative bacteria has to deal with the low permeability of the outer membrane. This obstacle can be overcome by utilizing siderophore-dependent iron uptake pathways as entrance routes for antibiotic uptake. Iron-chelating siderophores are actively imported by bacteria, and their conjugation to antibiotics allows smuggling the latter into bacterial cells. Synthetic siderophore mimetics based on MECAM (1,3,5-N,N',N″-tris-(2,3-dihydroxybenzoyl)-triaminomethylbenzene) and DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane) cores, both chelating iron via catechol groups, have been recently applied as versatile carriers of functional cargo. In the present study, we show that MECAM and the MECAM-ampicillin conjugate 3 transport iron into Pseudomonas aeruginosa cells via the catechol-type outer membrane transporters PfeA and PirA and DOTAM solely via PirA. Differential proteomics and quantitative real-time polymerase chain reaction (qRT-PCR) showed that MECAM import induced the expression of pfeA, whereas 3 led to an increase in the expression of pfeA and ampc, a gene conferring ampicillin resistance. The presence of DOTAM did not induce the expression of pirA but upregulated the expression of two zinc transporters (cntO and PA0781), pointing out that bacteria become zinc starved in the presence of this compound. Iron uptake experiments with radioactive 55Fe demonstrated that import of this nutrient by MECAM and DOTAM was as efficient as with the natural siderophore enterobactin. The study provides a functional validation for DOTAM- and MECAM-based artificial siderophore mimetics as vehicles for the delivery of cargo into Gram-negative bacteria.

Antibiotic Conjugates with an Artificial MECAM-Based Siderophore Are Potent Agents against Gram-Positive and Gram-Negative Bacterial Pathogens.

The development of novel drugs against Gram-negative bacteria represents an urgent medical need. To overcome their outer cell membrane, we synthesized conjugates of antibiotics and artificial siderophores based on the MECAM core, which are imported by bacterial iron uptake systems. Structures, spin states, and iron binding properties were predicted in silico using density functional theory. The capability of MECAM to function as an effective artificial siderophore in Escherichia coli was proven in microbiological growth recovery and bioanalytical assays. Following a linker optimization focused on transport efficiency, five ß-lactam and one daptomycin conjugates were prepared. The most potent conjugate 27 showed growth inhibition of Gram-positive and Gram-negative multidrug-resistant pathogens at nanomolar concentrations. The uptake pathway of MECAMs was deciphered by knockout mutants and highlighted the relevance of FepA, CirA, and Fiu. Resistance against 27 was mediated by a mutation in the gene encoding ExbB, which is involved in siderophore transport.