Structure-Activity Relationships in Supramolecular Hosts Targeting Bacterial Phosphatidylethanolamine (PE) Lipids.

The World Health Organization has described the antimicrobial resistance crisis as one of the top ten global public health threats. New antimicrobial agents that can fight infections caused by antimicrobial resistant pathogens are therefore needed. A potential strategy is the development of small molecules that can selectively interact with bacterial membranes (or membranes of other microbial pathogens), and thereby rapidly kill the bacteria. Here, we report the structure-activity relationship within a group of 22 compounds that were designed to bind the bacterial lipid phosphatidylethanolamine (PE). Liposome-based studies reveal that the lipophilicity of the compounds has the strongest effect on both the affinity and selectivity for PE. The best results were obtained for compounds with logP ≈ 3.75, which showed a 5x to 7x selectivity for bacterial PE lipids over human PC (phosphatidylcholine) lipids. Furthermore, these compounds also showed potent antibacterial activity against the Gram-positive bacterium B. cereus, with minimum inhibitory concentrations (MICs) below 10 µM, a concentration where they showed minimal hemolytic activity against human red blood cells. These results not only show the possibility of PE-binding small molecules to function as antibiotics, but also provide guidelines for the development of compounds targeting other types of biologically relevant membrane lipids.

A supramolecular host for phosphatidylglycerol (PG) lipids with antibacterial activity.

Lipids fulfill a variety of important physiological functions, such as energy storage, providing a hydrophobic barrier, and signal transduction. Despite this plethora of biological roles, lipids are rarely considered a potential target for medical applications. Here, we report a set of neutral small molecules that contain boronic acid and urea functionalities to selectively recognize the bacterial lipid phosphatidylglycerol (PG). The affinity and selectivity was determined using 1H NMR titrations and a liposome-based Alizarin Red S assay. Minimum inhibitory concentrations (MIC) were determined to assess antibacterial activity. The most potent compounds display an association constant with PG in liposomes of at least 5 × 103 M-1, function as antibacterial agents against Gram-positive bacteria (MIC = 12.5-25 µM), and show little hemolytic activity. Mode of action studies suggest that the boronic acids bind to the headgroup of the PG lipids, which leads to a change in membrane fluidity and ultimately causes membrane depolarization and cell death.

Increasing membrane permeability of carboxylic acid-containing drugs using synthetic transmembrane anion transporters.

In this manuscript, we show that small-molecule-based anion transporters can significantly increase the permeability of carboxylic acid containing drugs across lipid bilayers of model vesicles. Due to the drug-like characteristics of the transporters, this finding could not only have implications for drug delivery, but also hints towards potential drug-drug and drug-food interactions.

5'-Modifications improve potency and efficacy of DNA donors for precision genome editing.

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA-repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5'-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

An Investigation into the Potential of Targeting Escherichia coli rne mRNA with Locked Nucleic Acid (LNA) Gapmers as an Antibacterial Strategy.

The increase in antibacterial resistance is a serious challenge for both the health and defence sectors and there is a need for both novel antibacterial targets and antibacterial strategies. RNA degradation and ribonucleases, such as the essential endoribonuclease RNase E, encoded by the rne gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As rne mRNA has not been previously targeted using an antisense approach, we decided to explore using antisense oligonucleotides to target the translation initiation region of the Escherichia coli rne mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of rne mRNA translation through two mechanisms. Either LNA gapmer binding could sterically block translation and/or LNA gapmer binding could facilitate RNase H-mediated cleavage of the rne mRNA. This may prove to be an advantage over the majority of previous antibacterial antisense oligonucleotide approaches which used oligonucleotide chemistries that restrict the mode-of-action of the antisense oligonucleotide to steric blocking of translation. Using an electrophoretic mobility shift assay, we demonstrate that the LNA gapmers bind to the translation initiation region of E. coli rne mRNA. We then use a cell-free transcription translation reporter assay to show that this binding is capable of inhibiting translation. Finally, in an in vitro RNase H cleavage assay, the LNA gapmers facilitate RNase H-mediated mRNA cleavage. Although the challenges of antisense oligonucleotide delivery remain to be addressed, overall, this work lays the foundations for the development of a novel antibacterial strategy targeting rne mRNA with antisense oligonucleotides.

Bactericidal urea crown ethers target phosphatidylethanolamine membrane lipids.

An increasing number of people are infected with antibiotic-resistant bacteria each year, sometimes with fatal consequences. In this manuscript, we report a novel urea-functionalized crown ether that can bind to the bacterial lipid phosphatidylethanolamine (PE), facilitate PE flip-flop and displays antibacterial activity against the Gram-positive bacterium Bacillus cereus with a minimum inhibitory concentration comparable to that of the known PE-targeting lantibiotic duramycin.