Author Correction: Chimeric peptidomimetic antibiotics against Gram-negative bacteria.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Chimeric peptidomimetic antibiotics against Gram-negative bacteria.

There is an urgent need for new antibiotics against Gram-negative pathogens that are resistant to carbapenem and third-generation cephalosporins, against which antibiotics of last resort have lost most of their efficacy. Here we describe a class of synthetic antibiotics inspired by scaffolds derived from natural products. These chimeric antibiotics contain a ß-hairpin peptide macrocycle linked to the macrocycle found in the polymyxin and colistin family of natural products. They are bactericidal and have a mechanism of action that involves binding to both lipopolysaccharide and the main component (BamA) of the ß-barrel folding complex (BAM) that is required for the folding and insertion of ß-barrel proteins into the outer membrane of Gram-negative bacteria. Extensively optimized derivatives show potent activity against multidrug-resistant pathogens, including all of the Gram-negative members of the ESKAPE pathogens1. These derivatives also show favourable drug properties and overcome colistin resistance, both in vitro and in vivo. The lead candidate is currently in preclinical toxicology studies that-if successful-will allow progress into clinical studies that have the potential to address life-threatening infections by the Gram-negative pathogens, and thus to resolve a considerable unmet medical need.

A Peptidomimetic Antibiotic Interacts with the Periplasmic Domain of LptD from Pseudomonas aeruginosa.

The outer membrane (OM) in Gram-negative bacteria is an asymmetric bilayer with mostly lipopolysaccharide (LPS) molecules in the outer leaflet. During OM biogenesis, new LPS molecules are transported from their site of assembly on the inner membrane to the OM by seven LPS transport proteins (LptA-G). The complex formed between the integral ß-barrel OM protein LptD and the lipoprotein LptE is responsible for transporting LPS from the periplasmic side of the OM to its final location on the cell surface. Because of its essential function in many Gram-negative bacteria, the LPS transport pathway is an interesting target for the development of new antibiotics. A family of macrocyclic peptidomimetics was discovered recently that target LptD and inhibit LPS transport specifically in Pseudomonas spp. The related molecule Murepavadin is in clinical development for the treatment of life-threatening infections caused by P. aeruginosa. To characterize the interaction of these antibiotics with LptD from P. aeruginosa, we characterized the binding site by cross-linking to a photolabeling probe. We used a hypothesis-free mass spectrometry-based proteomic approach to provide evidence that the antibiotic cross-links to the periplasmic segment of LptD, containing a ß-jellyroll domain and an N-terminal insert domain characteristic of Pseudomonas spp. Binding of the antibiotic to the periplasmic segment is expected to block LPS transport, consistent with the proposed mode of action and observed specificity of these antibiotics. These insights may prove valuable for the discovery of new antibiotics targeting the LPS transport pathway in other Gram-negative bacteria.

Solution Structure and Dynamics of LptE from Pseudomonas aeruginosa.

LptE is an outer membrane (OM) lipoprotein found in Gram-negative bacteria, where it forms a complex with the ß-barrel lipopolysaccharide (LPS) transporter LptD. The LptD/E complex plays a key role in OM biogenesis, by translocating newly synthesized LPS molecules from the periplasm into the external leaflet of the asymmetric OM during cell growth. The LptD/E complex in Pseudomonas aeruginosa (Pa) is a target for macrocyclic ß-hairpin-shaped peptidomimetic antibiotics, which inhibit the transport of LPS to the cell surface. So far, the three-dimensional structure of the Pa LptD/E complex and the mode of interaction with these antibiotics are unknown. Here, we report the solution structure of a Pa LptE derivative lacking the N-terminal lipid membrane anchor, determined by multidimensional solution nuclear magnetic resonance (NMR) spectroscopy. The structure reveals a central five-stranded ß-sheet against which pack a long C-terminal and a short N-terminal α-helix, as found in homologues of LptE from other Gram-negative bacteria. One unique feature is an extended C-terminal helix in Pa LptE, which in a model of the Pa LptD/E complex appears to be long enough to contact the periplasmic domain of LptD. Chemical shift mapping experiments suggest only weak interactions occur between LptE and the oligosaccharide chains of LPS. The NMR structure of Pa LptE will be valuable for more detailed structural studies of the LptD/E complex from P. aeruginosa.

Biosynthesis and spectroscopic characterization of 2-TM fragments encompassing the sequence of a human GPCR, the Y4 receptor.

This paper presents a divide-and-conquer approach towards obtaining solution structures of G protein-coupled receptors. The human Y4 receptor was dissected into two to three transmembrane helix fragments, which were individually studied by solution NMR. We systematically compared various biosynthetic routes for the expression of the fragments in Escherichia coli and discuss purification strategies. In particular, we have compared the production of transmembrane (TM) fragments as inclusion bodies by using the ΔTrp leader sequence, with membrane-directed expression by using Mistic as the fusion partner, and developed methods for enzymatic cleavage. In addition, direct expression of two-TM fragments into inclusion bodies is a successful route in some cases. With the exception of TM13, we could produce all fragments in isotope-labeled form in quantities sufficient for NMR studies. Almost complete backbone resonance assignment was obtained for the first two helices, as well as for helices 5 and 7, and a high degree was obtained for TM6, while conformational exchange processes resulted in the disappearance of many signals from TM4. In addition, complete assignments were obtained for all residues of the N-terminal domain, as well as the extracellular and cytosolic loops (with the exception of an undecapeptide segment in the second extracellular loop, EC2) and for the complete cytosolic C-terminal tail. In total, backbone resonances of 78 % of all residues were assigned for the Y4 receptor. Predictions of secondary structure based on backbone chemical shifts indicate that most residues from the TM regions adopt helical conformations, with exception of those around polar residues or prolines. However, the domain boundaries differ slightly from those predicted for homology models. We suggest that the obtained chemical shifts might be useful in assigning the full-length receptor.