Chemical Biology

In their ongoing quest for new therapies against pathogens, scientists are focusing primarily on chemical and biological agents. These can function as antibiotics or stimulate the immune system. Discovering new drugs, characterising their functionality and optimising their properties are the three main goals of the department “Chemical Biology” at the HZI.  

1. 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-wells and subjected to 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 blocking virus/host-interactions or interfering with viral replication and viral maturation.

(A) Changes in the standardized impedance during biofilm growth of Pseudomonas aeruginosa. (B) Laser scanning fluorescence microscopy of biofilm forming Staphylococcus aureus. After fluorescence staining living bacteria appear green and dead bacteria red.

3. 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. 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.

Such biological profiling 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.
Impedance profiling

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 the growth or the viability by 50% (IC50) or to complete growth inhibition (minimal inhibitory concentration MIC, B).

(A) UPLC-ESI-Q-TOF-MS: Ultimate 3000 (Dionex) coupled to Maxis HD mass spectrometer (Bruker Daltonic). This LC/MS system is used for non-targeted analysis for polar and non-polar metabolites. The mass spectrometry can also be coupled with gas chromatography via an APCI source.

(B) UPLC-ESI-QQQ-MS: 1290 Infinity (Agilent) coupled to QTrap 6500 (ABSciex). This LC/MS system is used for high-throughput screening analysis for selected metabolites and compound classes.

(C) GC-EI-iontrap-MS: Trace GC Ultra coupled to ITQ900 MS (Thermo Fisher Scientific). This GC/MS system is mainly used for analysis of polar compounds of primary metabolism

6. 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 accomplish this 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 LCMS

In order to quantify the intracellular accumulation of small molecules without relying on fluorescent or radioactive labels we are currently implementing a broadly applicable, LCMS-based technique combined with subcellular fractionation. Within this approach, bacteria are incubated with the compound of interest and are then subjected to a fractionation workflow for separating periplasmic, cytoplasmic and membrane contents. Upon compound extraction out of the lysates their identity and concentration is measured by highly sensitive mass spectrometric analysis.

Leader

  • Prof Dr Mark Brönstrup

    Mark Broenstrup

    Head of the department chemical biology

    +49 531 6181-3400

    Contact

Bachelor & Master
Are you interested in a bachelor or master thesis? We are looking forward to your request!

  • Epothilon: Ein HZI-Molekül wird zum Krebsmedikament
    Wissenschaftler an der ehemaligen Gesellschaft für Biotechnologische Forschung (GBF), dem heutigen HZI, haben in den 1980er Jahren Epothilon B entdeckt. Dieser Naturstoff stört die Teilung von Krebszellen: Tumore werden durch ihn im Wachstum gebremst, schrumpfen und verschwinden. Das Pharmaunternehmen Bristol-Myers Squibb (BMS) hat jetzt auf der Basis von Epothilon B ein Krebsmedikament auf den US-amerikanischen Markt gebracht. Seine Entwicklung ist so spannend wie ein Krimi...
    Länge: 11:34

Audio Podcast

  • Kleine Moleküle ganz groß – Mit Naturstoffen gegen Hepatitis C
    Mit weltweit 130 Millionen Infizierten ist Hepatitis C eine der häufigsten Infektionskrankheiten. Die Therapien, die es derzeit gibt, sind langwierig, haben starke Nebenwirkungen und helfen nicht jedem Patienten. Florenz Sasse vom Braunschweiger Helmholtz-Zentrum für Infektionsforschung und Thomas Pietschmann vom TWINCORE in Hannover suchen gemeinsam nach neuen Wirkstoffen gegen das Hepatitis C-Virus. Hören Sie hier von ihren ersten Treffern und folgen Sie Florenz Sasse zu einer Bibliothek der anderen Art…