Chemische Biologie

1. Improving and quantifying bacterial penetration

Antimicrobial resistance of Gram-negative bacteria has become a major issue for public health, and there is acute medical need for novel antibiotics. Especially the complex structure of the Gram-negative cell wall limits uptake of bioactive compounds. To address this problem, we are developing carrier-conjugates that facilitate and improve compound penetration into bacteria. Applied carriers are synthetic molecules that are designed based on natural templates like nutrients. The carriers will specifically mediate uptake of the conjugates through distinct outer membrane receptors in a Trojan horse strategy. For quantifying uptake of the carrier-conjugates we apply the two following approaches.

Measuring uptake by LC-MS

In order to quantify the intracellular accumulation of small molecules without relying on fluorescent or radioactive labels, we are currently implementing a broadly applicable, LC-MS-based technique. Within this approach, bacteria are incubated with the compound of interest and are then subjected to a workflow to determine uptake in whole cells or in subcellular fractions. The compound extracted from the lysates is detected, and its concentration is measured in MS-multiple reaction monitoring (MRM) mode. Using MRM, ions from a complex mixture are filtered out from those of the compound of interest, resulting in a strong increase of sensitivity even at very low concentration.

Uptake assay in subcellular fractions

Gram-negative bacteria are incubated with a selected compound, and subjected to a fractionation workflow for separating periplasmic, cytoplasmic and membrane contents. The corresponding lysates are then analyzed by LC-MS to determine effective concentrations in every fraction.

Uptake assay in whole bacterial cells

A high throughput method has been developed to screen for accumulation of known as well as novel compounds against Gram-negative bacteria, such as E. coli and P. aeruginosa. Bacteria are treated with a set of compounds at different concentrations, incubated at different time points, and subjected to a fast workflow to extract internalized compounds. Lysates are then analyzed by LC-MS to determine intracellular concentrations and kinetic behavior of every compound.

Monitoring compound uptake by conjugation to fluorogenic dyes

In order to visualize the internalization and intracellular localization of uptake mediating carriers, we make use of a conjugation-based approach. The carrier of interest is conjugated to a fluorogenic dye that only obtains fluorescent properties upon binding to a specific dye activator protein. Genetically modified bacteria that express this dye activator protein are treated with the fluorogen-conjugates. Upon uptake and intracellular binding the resulting fluorescent signal can be monitored by fluorescence microscopy, flow cytometry or fluorescence reader. By directed expression of the dye activator protein in either cytoplasm or periplasm, detailed information about the intracellular localization of a given carrier-conjugate can be obtained.

• Multivalent Siderophore DOTAM Conjugates as Theranostics for Imaging and Treatment of Bacterial Infections. Kevin Ferreira, Hai-Yu Hu, Verena Fetz, Hans Prochnow, Bushra Rais, Peter P. Müller, Mark Brönstrup, Angewandte Chemie Int. Ed. (2017), in press. DOI: 10.1002/anie.201701358R1 and 10.1002/ange.201701358R1.

2. Metabolome analysis (Metabolomics)

The overall aim of metabolomics is to obtain qualitative and quantitative insights into the metabolome, i.e. the set of small molecules involved in or resulting from metabolic processes in a biological system under given circumstances. Using analytical chemistry techniques, either the (near-)entirety of the metabolome (untargeted metabolomics) or a predefined subset (targeted metabolomics) is studied. Although it is still a rather young field struggling with the challenges imposed by high analyte diversity and pronounced dynamics in metabolome composition, metabolomics has been dubbed the “apogee of the omics trilogy” (Patti et al., Nat Rev Mol Cell Biol 2012) as it provides a direct readout of the biochemical phenotype. Recent studies have demonstrated the potential of metabolomics to decipher the mode of action of bioactive compounds (e.g. Kitagawa, Chem Biol 2010; Zlitni, Nat Chem Biol 2013), to detect metabolite switches upon induction of resistance (e.g. Derewacz, PNAS2013), or to discover novel metabolites even in well-known organisms (e.g. Srinivasan, Nature 2008; Globisch, PNAS2013). In our group,  we apply metabolomics to subjects as diverse as investigating metabolic effects of exposure to antibiotics, characterization of enzyme functions and biosynthetic pathways in genetically modified bacteria, biomarker discovery in human and animal biospecimens and metabolic phenotyping of clinical strains of pathogens like Pseudomonas aeruginosa. The knowledge gained through metabolomics experiments also supports other research activities of the group, e.g. mode of action studies and optimization of novel antibiotics.

To cope with the analytical challenges of the dynamic metabolism and the high diversity of metabolites, we use state-of-the-art LC-MS and GC-MS instruments that allow for a range of untargeted and targeted approaches and we have established a large in-house library of authentic chemical standards for metabolite identification. In addition to that, we have the possibility to use preparative LC-MS and NMR to elucidate the structure of unknown but significant compounds.

We also engage in the development of data analysis strategies and the integration of metabolomics data with information from proteomics and transcriptomics experiments to obtain system-wide perceptions of interesting processes such as bacterial adaptation to antibiotic treatment.

• Clustering of MS² spectra using unsupervised methods to aid the identification of secondary metabolites from Pseudomonas aeruginosa. Depke T, Franke R, Brönstrup M. J Chromatogr B (2017) pii: S1570-0232(17)30999-6. doi:10.1016/j.jchromb.2017.06.002.

3. Screening for novel anti infectives

The HZI S3 facility offers the opportunity to investigate the properties of pathogenic viruses and bacteria. One task addressed by the Department of Chemical Biology is the screening of (natural-) compound libraries for new anti-viral agents. This is accomplished by an automated pipetting robot which can be operated under S3 safety conditions. In an ongoing project, the malignant hepatoma cell line Huh-7 is cultivated in 96 well plates and infected with Dengue virus. After addition of compounds and viral infection, cells are fixed and immunostained for viral epitopes. Finally, the percentage of infected cells is determined by automated fluorescence microscopy.

By applying different modifications to this high-content screening approach, our group intends to identify new anti-viral compounds that block virus/host-interactions or interfere with viral replication and maturation.

• Soraphen A: a broad-spectrum antiviral natural product with potent anti-hepatitis C virus activity. Koutsoudakis, George; Romero-Brey, Inés; Berger, Carola; Pérez-Vilaró, Gemma; M. Perin, Paula; Vondran, Florian W.R.; Kalesse, Markus; Harmrolfs, Kirsten; Müller, Rolf; Martinez, Javier P.; Pietschmann, Thomas; Bartenschlager, Ralf; Bronstrup, Mark; Meyerhans, Andreas; Diez, Juana. Journal of Hepatology (2015), 63(4), 813-21. 
• The myxobacterial metabolite Soraphen A inhibits HIV-1 by reducing virus production and altering virion composition. Fleta-Soriano E, Smutná K, Martinez JP, Lorca Oró C, Sadiq SK, Mirambeau G, Lopez-Iglesias C, Bosch M, Pol A, Brönstrup M, Diez J, Meyerhans A. Antimicrobial Agents Chemotherapy (2017), pii: AAC.00739-17. doi: 10.1128/AAC.00739-17.

4. Biological profiling and mode of action studies

The characterization of the mode of action of a bioactive compound is an essential prerequisite for its application as a therapeutic. The mode of action manifests itself in terms of direct binding partners, functional perturbations as a consequence of binding, and induced downstream effects on a cellular, tissue and whole organism level. We tackle the challenge of mode of action analysis through a variety of orthogonal approaches:

Biological profiling

High content analysis

For compounds with an unknown mode of action, we apply two high-content profiling methods to obtain information about their influence on distinct cellular pathways. The first method is based on impedance spectroscopy. Under treatment, time-dependent cellular response profiles (TCRP, A) of epithelial cells are captured and mathematically described by fitting parameters. The workflow for a typical analysis is shown in B. The compound specific fitting parameters are then compared to those of a reference library of 64 compounds in a clustering analysis (C). Compounds with similar effects will group close to each other and reveal the potential mode of action. Within the second approach, phenotypic changes of mammalian cells are visualized by immunofluorescent staining and evaluated by high-content microscopy in comparison to the same reference set of compounds.

This biological profiling approach by high-content assays has been successfully applied for evaluation of paleo-soraphens and jerantinine E:

• Synthesis and biological evaluation of paleo-soraphens. Lu HH, Raja A, Franke R, Landsberg D, Sasse F, Kalesse M. Angew Chem Int Ed Engl. 2013 Dec 16;52(51):13549-52.
• Total synthesis and biological evaluation of jerantinine e. Frei R, Staedler D, Raja A, Franke R, Sasse F, Gerber-Lemaire S, Waser J., Angew Chem Int Ed Engl. 2013 Dec 9;52(50):13373-6.

Profiling of small molecules by application to a panel of organisms

For external and internal partners: CBIO offers to characterize the growth inhibitory effects of small molecules on a panel of microorganisms and eukaryotic cells using standardized protocols.

The so-called ESKAPE panel of bacteria comprises the clinically relevant Gram-positive species Enteroccocus faecium and Staphylococcus aureus and the Gram-negative species Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae, such as Escherichia coli and Enterobacter cloacae. The range of tested microorganisms is completed by yeasts and fungi, such as Saccharomyces cerevisiae and Candida albicans. In the first instance, growth inhibitory properties are evaluated by incubating the strains in either defined minimal media or in complex media to promote sufficient growth rates in (96 well or 384 well, A) microtiter plates.

In addition, a range of standard mammalian cell lines, e.g. L929, MCF-7, KB 3.1, is treated with the compounds under investigation to evaluate cytotoxic effects. Cytotoxicity is determined via tetrazolium-dye based assays, such as the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) or the WST (water soluble tetrazolium) assay. In these assays, the substrate turnover by NAD(P)H-dependent cellular oxidoreductases reflects, under defined conditions, the number of viable cells present. The exposure times of the cells with the compounds vary from 24 h for the estimation of acute cytotoxicity to 5 days for the detection of general cytotoxic effects. In subsequent assays, effects on DNA synthesis (using BrdU) or the mitochondrial membrane potential ΔΨm are probed (C, D).

Generally, all biological activities are characterized by the compound concentrations leading to either a reduction of growth or viability by 50% (IC₅₀) or to complete growth inhibition (minimal inhibitory concentration MIC, B).

• Synthesis and Biological Investigation of Δ¹²-​Prostaglandin J₃ (Δ¹²-​PGJ₃) Analogues and Related Compounds. Nicolaou, K. C.; Pulukuri, Kiran Kumar; Rigol, Stephan; Heretsch, Philipp; Yu, Ruocheng; Grove, Charles I.; Hale, Christopher R. H.; ElMarrouni, Abdelatif; Fetz, Verena; Bronstrup, Mark; Aujay, Monette; Sandoval, Joseph; Gavrilyuk, Julia. Journal of the American Chemical Society (2016), 138(20), 6550-60.
• New nitrofurans amenable by isocyanide multicomponent chemistry are active against multidrug-​resistant and poly-​resistant Mycobacterium tuberculosis. Krasavin, Mikhail; Parchinsky, Vladislav; Kantin, Grigory; Manicheva, Olga; Dogonadze, Marine; Vinogradova, Tatiana; Karge, Bianka; Bronstrup, Mark. Bioorganic & Medicinal Chemistry (2017), 25(6), 1867-1874.
• Gold(I) NHC Complexes: Antiproliferative Activity, Cellular Uptake, Inhibition of Mammalian and Bacterial Thioredoxin Reductases, and Gram-​positive directed Antibacterial Effects. Schmidt Claudia; Karge Bianka; Misgeld Rainer; Prokop Aram; Franke Raimo; Bronstrup Mark; Ott Ingo. Chem. Eur. J. (2017), 23(8), 1869-1880.

Target identification

Whole genome sequencing of resistant cells

A recently introduced method for target identification is whole genome sequencing (WGS) of resistant cell lines. For this approach, bacteria or eukaryotic cells are cultivated under increasing concentrations of the compound of interest. Resistant clones are selected and subjected to WGS. By comparison to the wildtype genome, mutations in protein encoding sequences are identified that hint to the potential target of a given compound. For further confirmation, mutant proteins are recombinantly expressed in the respective wildtype strains. Resulting resistance identifies the compound target. Verification can alternatively be done by reversing the genomic mutation. This strategy has been successfully applied for identifying the osmosensitive K⁺-channel sensor histidine kinase as a target of the antimicrobially active inhibitor vz0825 of Vibrio cholera.

• High-throughput screening and whole genome sequencing identifies an antimicrobially active inhibitor of Vibrio cholerae. Sergeev G, Roy S, Jarek M, Zapolskii V, Kaufmann DE, Nandy RK1, Tegge W. BMC Microbiol. 2014 Feb 26;14:49.

Target fishing

Protein targets of a chemical compound can be identified by a so-called target fishing approach. For this, the chemical compound has to be modified by the introduction of a functional affinity tag (i.e. biotin). The resulting conjugate consisting of affinity tag, linker and ligand is called a fishing probe. The affinity tag enables purification of compound-protein complexes that formed during incubation with cells or cell lysates. Compound binding proteins obtained from this chemical pull down are then identified by PAGE and MS.

5. Diagnostic peptide arrays

An innovative method for the generation of peptide arrays on cellulose membranes has been developed in our group (Dr. R. Frank, former head of department), which is internationally applied and known as the SPOT method. The arrays have been used in a multitude of different biological investigations like e.g. epitope mapping, enzymatic transformations, development of antimicrobial compounds and many more. The SPOT method has been extended in our department to the generation of miniaturized arrays, where up to 10,000 different peptides can be accommodated on a microscope slide and used in biological tests (SC² method). The SC² method allows the printing of several thousand copies of a microarray from one single SPOT membrane. Current projects focus on the characterization of the immune response to different viral infections in monkeys and humans.

6. Characterization of biofilm formation

The control of chronic bacterial infections is significantly affected by the formation of biofilms. In the human body, such structures enable pathogens to display an array of different adaption strategies and an increase in their resistance to drug therapies. Biofilms provide a physical barrier to the immune system and to the penetration of antibiotics. In addition, bacteria in biofilms display an altered metabolism compared to planktonic growth. In a biofilm, bacteria are embedded in a self-produced matrix of extracellular polymeric substances (EPS), which consist of DNA, proteins and polysaccharides. Since bacteria in biofilms are much less susceptible towards antibiotics, antimicrobial treatment in the presence of biofilms is often inefficient.

In our group, we are addressing biofilm formation in particular with two clinically very relevant pathogens: Pseudomonas aeruginosa, which is a Gram-negative bacterium, and Staphylococcus aureus, a Gram-positive pathogen. A new method based on impedance spectroscopy is being developed that aims at the real-time determination of biofilm growth dynamics. Pseudomonas aeruginosa was taken as a first model strain. Other ESKAPE microorganisms will also be addressed (Figure A). This opens up the opportunity for further miniaturization of the method with the goal to develop a microchip-based diagnostic tool, which can be used as a high-throughput assay system for drug research, stratification of clinical bacterial isolates as well as the optimization of individualized medical treatments.

For Staphyloccocus aureus we are trying to identify compounds that (i) prevent the formation of biofilms or (ii) are able to kill the bacteria inside established biofilms. In collaboration with a major pharmaceutical company we have investigated approximately 20,000 natural compounds in a primary screening assay for their effect on biofilm formation. Primary actives were further characterized by laser-scanning fluorescence microscopy at the Twincore in Hannover (Prof. S. Häußler). In an example shown in Figure B (below), the bacteria have been visualized by fluorescent dyes that stain living bacteria green and dead ones red following compound treatment. Compound 1 killed bacteria at increasing concentrations and also caused a reduction of the amount of biofilm (left panel). Compound 2 left the bacteria alive, but reduced the amount of biofilm (right panel). Especially the profile of compound 2 is desirable as part of a pathoblocker concept.

• High-throughput screening and whole genome sequencing identifies an antimicrobially active inhibitor of Vibrio cholerae. Sergeev G, Roy S, Jarek M, Zapolskii V, Kaufmann DE, Nandy RK1, Tegge W. BMC Microbiol. 2014 Feb 26;14:49.

• Use of Single-Frequency Impedance Spectroscopy to Characterize the Growth Dynamics of Biofilm Formation in Pseudomonas aeruginosa. Jozef B. J. H. van Duuren, Mathias Müsken, Bianka Karge, Jürgen Tomasch, Christoph Wittmann, Susanne Häussler, Mark Brönstrup. Scientific Reports (2017), DOI:10.1038/s41598-017-05273-5