Early molecular insights into thanatin analogues binding to A. baumannii LptA

The cationic antimicrobial ß-hairpin, thanatin, was recently developed into drug-like analogues active against carbapenem-resistant Enterobacteriaceae (CRE). The analogues represent new antibiotics with a novel mode of action targeting LptA in the periplasm and disrupting LPS transport. The compounds lose antimicrobial efficacy when the sequence identity to E. coli LptA falls below 70%. We wanted to test the thanatin analogues against LptA of a phylogenetic distant organism and investigate the molecular determinants of inactivity. Acinetobacter baumannii (A. baumannii) is a critical Gram-negative pathogen that has gained increasing attention for its multi-drug resistance and hospital burden. A. baumannii LptA shares 28% sequence identity with E. coli LptA and displays an intrinsic resistance to thanatin and thanatin analogues (MIC values > 32 μg/mL) through a mechanism not yet described. We investigated the inactivity further and discovered that these CRE- optimized derivatives can bind to LptA of A. baumannii in vitro, despite the high MIC values. Herein, we present a high-resolution structure of A. baumannii LptAm in complex with a thanatin derivative 7 and binding affinities of selected thanatin derivatives. Together, these data offer structural insights into why thanatin derivatives are inactive against A. baumannii LptA, despite binding events in vitro

Peptides in BioNMR Research

Heteronuclear NMR in combination with isotope labelling is used to study folding of polypeptides induced by metals in the case of metallothioneins, binding of the peptidic allosteric modulator ?-TIA to the human G-protein coupled ?1b adrenergic receptor, the development of therapeutic drugs that interfere with the biosynthesis of the outer membrane of Gram-negative bacteria, and a system in which protein assembly is induced upon peptide addition. NMR in these cases is used to derive precise structural data and to study the dynamics

Recent progress in the discovery of macrocyclic compounds as potential anti-infective therapeutics

Novel therapeutic strategies are urgently needed for the treatment of serious diseases caused by viral, bacterial and parasitic infections, because currently used drugs are facing the problem of rapidly emerging resistance. There is also an urgent need for agents that act on novel pathogen-specific targets, in order to expand the repertoire of possible therapies. The high throughput screening of diverse small molecule compound libraries has provided only a limited number of new lead series, and the number of compounds acting on novel targets is even smaller. Natural product screening has traditionally been very successful in the anti-infective area. Several successful drugs on the market as well as other compounds in clinical development are derived from natural products. Amongst these, many are macrocyclic compounds in the 1-2 kDa size range. This review will describe recent advances and novel drug discovery approaches in the anti-infective area, focusing on synthetic and natural macrocyclic compounds for which in vivo proof of concept has been established. The review will also highlight the Protein Epitope Mimetics (PEM) technology as a novel tool in the drug discovery process. Here the structures of naturally occurring antimicrobial and antiviral peptides and proteins are used as starting points to generate novel macrocyclic mimetics, which can be produced and optimized efficiently by combinatorial synthetic methods. Several recent examples highlight the great potential of the PEM approach in the discovery of new anti-infective agents

Peptidomimetic antibiotics disrupt the lipopolysaccharide transport bridge of drug-resistant Enterobacteriaceae

The rise of antimicrobial resistance poses a substantial threat to our health system, and, hence, development of drugs against novel targets is urgently needed. The natural peptide thanatin kills Gram-negative bacteria by targeting proteins of the lipopolysaccharide transport (Lpt) machinery. Using the thanatin scaffold together with phenotypic medicinal chemistry, structural data, and a target-focused approach, we developed antimicrobial peptides with drug-like properties. They exhibit potent activity against Enterobacteriaceae both in vitro and in vivo while eliciting low frequencies of resistance. We show that the peptides bind LptA of both wild-type and thanatin-resistant Escherichia coli and Klebsiella pneumoniae strains with low-nanomolar affinities. Mode of action studies revealed that the antimicrobial activity involves the specific disruption of the Lpt periplasmic protein bridge

A Screen for Spore Wall Permeability Mutants Identifies a Secreted Protease Required for Proper Spore Wall Assembly

The ascospores of Saccharomyces cerevisiae are surrounded by a complex wall that protects the spores from environmental stresses. The outermost layer of the spore wall is composed of a polymer that contains the cross-linked amino acid dityrosine. This dityrosine layer is important for stress resistance of the spore. This work reports that the dityrosine layer acts as a barrier blocking the diffusion of soluble proteins out of the spore wall into the cytoplasm of the ascus. Diffusion of a fluorescent protein out of the spore wall was used as an assay to screen for mutants affecting spore wall permeability. One of the genes identified in this screen, OSW3 (RRT12/YCR045c), encodes a subtilisin-family protease localized to the spore wall. Mutation of the active site serine of Osw3 results in spores with permeable walls, indicating that the catalytic activity of Osw3 is necessary for proper construction of the dityrosine layer. These results indicate that dityrosine promotes stress resistance by acting as a protective shell around the spore. OSW3 and other OSW genes identified in this screen are strong candidates to encode enzymes involved in assembly of this protective dityrosine coat

Network Analysis of Differential Expression for the Identification of Disease-Causing Genes

Genetic studies (in particular linkage and association studies) identify chromosomal regions involved in a disease or phenotype of interest, but those regions often contain many candidate genes, only a few of which can be followed-up for biological validation. Recently, computational methods to identify (prioritize) the most promising candidates within a region have been proposed, but they are usually not applicable to cases where little is known about the phenotype (no or few confirmed disease genes, fragmentary understanding of the biological cascades involved). We seek to overcome this limitation by replacing knowledge about the biological process by experimental data on differential gene expression between affected and healthy individuals. Considering the problem from the perspective of a gene/protein network, we assess a candidate gene by considering the level of differential expression in its neighborhood under the assumption that strong candidates will tend to be surrounded by differentially expressed neighbors. We define a notion of soft neighborhood where each gene is given a contributing weight, which decreases with the distance from the candidate gene on the protein network. To account for multiple paths between genes, we define the distance using the Laplacian exponential diffusion kernel. We score candidates by aggregating the differential expression of neighbors weighted as a function of distance. Through a randomization procedure, we rank candidates by p-values. We illustrate our approach on four monogenic diseases and successfully prioritize the known disease causing genes

Repair at Single Targeted DNA Double-Strand Breaks in Pluripotent and Differentiated Human Cells

Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical, environmental, and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part, previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation, which are highly nonphysiologic, or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair, we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs, compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus, the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny