Covering: up to March 2013. In addition to their prominent role in basic biological and chemical research, natural products are a rich source of commercial products for the pharmaceutical and other ...industries. Industrial natural product chemistry is of fundamental importance for successful product development, as the vast majority (ca. 80%) of commercial drugs derived from natural products require synthetic efforts, either to enable economical access to bulk material, and/or to optimize drug properties through structural modifications. This review aims to illustrate issues on the pathway from lead to product, and how they have been successfully addressed by modern natural product chemistry. It is focused on natural products of current relevance that are, or are intended to be, used as pharmaceuticals.
We present the second total synthesis of (±)-acanthodoral, a sesquiterpenoid derived from the marine nudibranch Acanthodoris nanaimoensis. Our approach involves a concise three-step transformation ...from a previously reported compound, resulting in the formation of a less strained precursor of the bicyclo3.1.1heptane core and both all-carbon quaternary stereocenters characteristic of the natural product. Notably, this synthetic route incorporates two pivotal steps: a Sm(II)-induced 1,2-rearrangement and a semipinacol rearrangement.
•We developed a tool to cluster MS2 spectra and neutral loss patterns by similarity.•CluMSID enabled putative annotation of more than 30 novel alkyl quinolones.•CluMSID proved its utility also with ...other classes of metabolites.•CluMSID is open to the community and can be used by all researchers in the field.
Pseudomonas aeruginosa is an important opportunistic pathogen that produces a large arsenal of small molecule virulence factors and quorum sensing signal molecules. The annotation of these secondary metabolites in untargeted, mass spectrometry-based metabolomics is difficult, as many of them cannot be found in common metabolite databases, and as manual annotation is tedious. We therefore developed an algorithm named CluMSID that uses cosine similarities of product ion spectra and neutral loss patterns in combination with unsupervised clustering methods such as multidimensional scaling, density based clustering and hierarchical clustering to group structurally similar compounds and hence facilitate their annotation. The use of this tool allowed us to find clusters for several classes of primary and secondary metabolites, and helped identifying spectral similarities that would have gone unnoticed in standard untargeted metabolomics data analysis workflows. CluMSID enabled the annotation of 27 previously undescribed members of the canonical classes of alkyl quinolone quorum sensing signal molecules and provided evidence for the postulation of a new putative alkyl quinolone class. The CluMSID script written in R is open source and can be used by anyone in the metabolomics and natural product research community.
Infections by Gram-negative pathogens represent a major health care issue of growing concern due to a striking lack of novel antibacterial agents over the course of the last decades. The main ...scientific problem behind the rational optimization of novel antibiotics is our limited understanding of small molecule translocation into, and their export from, the target compartments of Gram-negative species. To address this issue, a versatile, label-free assay to determine the intracellular localization and concentration of a given compound has been developed for Escherichia coli and its efflux-impaired ΔTolC mutant. The assay applies a fractionation procedure to antibiotic-treated bacterial cells to obtain periplasm, cytoplasm, and membrane fractions of high purity, as demonstrated by Western Blots of compartment-specific marker proteins. This is followed by an LC-MS/MS-based quantification of antibiotic content in each compartment. Antibiotic amounts could be converted to antibiotic concentrations by assuming that an E. coli cell is a cylinder flanked by two half spheres and calculating the volumes of bacterial compartments. The quantification of antibiotics from different classes, namely ciprofloxacin, tetracycline, trimethoprim, and erythromycin, demonstrated pronounced differences in uptake quantities and distribution patterns across the compartments. For example, in the case of ciprofloxacin, a higher amount of compound was located in the cytoplasm than in the periplasm (592 ± 50 pg vs 277 ± 13 pg per 3.9 × 109 cells), but owing to the smaller volume of the periplasmic compartment, its concentration in the cytoplasm was much lower (37 ± 3 vs 221 ± 10 pg/μL for the periplasm). For erythromycin and tetracycline, differences in MICs between WT and ΔTolC mutant strains were not reflected by equal differences in uptake, illustrating that additional experimental data are needed to predict antibiotic efficacy. We believe that our assay, providing the antibiotic concentration at the compartment in which the drug target is expressed, constitutes an essential piece of information for a more rational optimization of novel antibiotics against Gram-negative infections.
Rising infection rates with multidrug-resistant pathogens calls for antibiotics with novel modes of action. Herein, we identify the inner membrane protein TonB, a motor of active uptake in ...Gram-negative bacteria, as a novel target in antimicrobial therapy. The interaction of the TonB box of outer membrane transporters with TonB is crucial for the internalization of essential metabolites. We designed TonB box peptides and coupled them with synthetic siderophores in order to facilitate their uptake into bacteria in up to 32 synthetic steps. Three conjugates repressed the growth of Pseudomonas aeruginosa cells unable to produce their own siderophores, with minimal inhibitory concentrations between 0.1 and 0.5 μM. The transporters mediating uptake of these compounds were identified as PfeA and PirA. The study illustrates a variant of cellular suicide where a transporter imports its own inhibitor and demonstrates that artificial siderophores can import cargo with molecular weights up to 4 kDa.
The opportunistic Gram-negative bacterium Pseudomonas aeruginosa is a leading pathogen for infections of immuno-compromised patients and those suffering from cystic fibrosis. Its ability to switch ...from planktonic life to aggregates, forming the so-called biofilms, is a front-line mechanism of antimicrobial resistance. The bacterial carbohydrate-binding protein LecB is an integral component and necessary for biofilm formation. Here, we report a new class of drug-like low molecular weight inhibitors of the lectin LecB with nanomolar affinities and excellent receptor binding kinetics and thermodynamics. This class of glycomimetic inhibitors efficiently blocked biofilm formation of P. aeruginosa in vitro while the natural monovalent carbohydrate ligands failed. Furthermore, excellent selectivity and pharmacokinetic properties were achieved. Notably, two compounds showed good oral bioavailability, and high compound concentrations in plasma and urine were achieved in vivo.
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 β-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.
Toll-like receptor TLR5 recognizes a conserved domain, termed D1, that is present in flagellins of several pathogenic bacteria but not in Helicobacter pylori. Highly virulent H. pylori strains ...possess a type IV secretion system (T4SS) for delivery of virulence factors into gastric epithelial cells. Here, we show that one of the H. pylori T4SS components, protein CagL, can act as a flagellin-independent TLR5 activator. CagL contains a D1-like motif that mediates adherence to TLR5
epithelial cells, TLR5 activation, and downstream signaling in vitro. TLR5 expression is associated with H. pylori infection and gastric lesions in human biopsies. Using Tlr5-knockout and wild-type mice, we show that TLR5 is important for efficient control of H. pylori infection. Our results indicate that CagL, by activating TLR5, may modulate immune responses to H. pylori.
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.
A sensitive diagnosis is crucial for the successful treatment of bacterial infections. The conjugation of bacteria‐specific, uptake‐enhancing siderophore vectors with enzyme‐inducible chemiluminescent dioxetanes enabled the broad‐spectrum detection of “ESKAPE” pathogens. The probes displayed a high sensitivity in human plasma and also detected intracellular Gram‐positive and ‐negative bacteria.
Lantibiotics are peptides, produced by bacteria, that contain the noncanonical amino acid lanthionine and many of them exhibit antibacterial activities. The labyrinthopeptin A1 (LabyA1) is a ...prototype peptide of a novel class of carbacyclic lantibiotics. Here, we extensively evaluated its broad-spectrum activity against HIV and HSV in vitro, studied its mechanism of action and evaluated potential microbicidal applications. LabyA1 exhibited a consistent and broad anti-HIV activity (EC50s: 0.70-3.3 µM) and anti-HSV activity (EC50s: 0.29-2.8 µM) in cell cultures. LabyA1 also inhibited viral cell-cell transmission between persistently HIV-infected T cells and uninfected CD4(+) T cells (EC50∶2.5 µM) and inhibited the transmission of HIV captured by DC-SIGN(+)-cells to uninfected CD4(+) T cells (EC50∶4.1 µM). Time-of-drug addition studies revealed that LabyA1 acts as an entry inhibitor against HIV and HSV. Cellular and virus binding studies combined with SPR/FLIPR technology showed that LabyA1 interacted with the HIV envelope protein gp120, but not with the HIV cellular receptors. LabyA1 also demonstrated additive to synergistic effects in its anti-HIV-1 and anti-HSV-2 activity with anti(retro)viral drugs in dual combinations such as tenofovir, acyclovir, saquinavir, raltegravir and enfuvirtide. LabyA1 can be considered as a novel lead peptide as it had profound antiviral activity against HIV and HSV. Pre-treatment of PBMCs with LabyA1 neither increased the expression of the activation markers CD69 and CD25, nor enhanced HIV replication, nor significantly induced various inflammatory cytokines/chemokines. LabyA1 also did not affect the growth of vaginal Lactobacilli populations. Based on the lack of toxicity on the vaginal Lactobacillus strains and its synergistic/additive profile in combination with clinically approved anti(retro)virals, it deserves further attention as a potential microbicide candidate in the prevention of sexual transmitted diseases.
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