Natural products chemistry: Total syntheses and analysis of structure-activity-relationships
Secondary metabolites have manifold biological activities in living organisms, and have undergone optimization by nature over thousands of years. Hence, due to their bioactivity, but also their structural diversity natural products represent a most attractive source of lead structures for drug development.
We work out total syntheses of natural products with cytotoxic and/or antimicrobial activities (e.g. β-carbolines, polycyclic aromatic alkaloids, steroid alkaloids) from tropical plants (e.g. Annonaceae) and from marine organisms (tunicates, sea anemones, sponges). We focus on natural products with drug-like structures, i.e. manageable structural complexity and promising physico-chemical properties (Lipinski’s rule of five). Compounds with high biological activities are further modified in order to optimize their activity and selectivity, and to gain deeper insight into structure-activity relationships.
A number of these natural products served as lead structures for the development of bioactive compounds for epigenetic targets (kinase inhibitors, bromodomain inhibitors, histone deacetylase inhibitors, …).
For a compilation of all natural products total syntheses of the Bracher group, see Total Syntheses Bracher
Epigenetic targets: Kinases, bromodomains, macrodomains and histone deacetylases
The regulation of transcription of genetically encoded information is essential for all living organisms. Numerous posttranslational modifications of DNA and proteins (e.g. histones) are part of this regulation. These processes open new and attractive targets for new drugs for treating different diseases (cancer, inflammatory diseases, infections).
In cooperation with the Structural Genomics Consortium (Oxford, UK) and other partners we develop new bioactive compounds for epigenetic targets. In many cases natural products from our own research (see above) acted as lead structures for these investigations.
Our most important targets are:
A number of natural products for which total syntheses had been developed in our group were found to be inhibitors of kinases. Starting from the β-carboline alkaloids bauerine C and annomontine we developed potent and selective inhibitors of the kinases CLK1, CLK3, PIM1, DYRK1A, and BMP2K.
Further characterization of these inhibitors was performed in cooperation with the following groups: Prof. Dr. S. Knapp (Oxford/Frankfurt), Prof. Dr. P. Filippakopoulos (Oxford), Prof. Dr. J. Schwaller (Basel), Dr. L. Meijer (ManRos, Roscoff), Prof. Dr. W. Becker (Aachen), Prof. Dr. T. Meyer (MPI für Infektionsbiologie, Berlin), Prof. Dr. A. Vollmar (LMU), Prof. Dr. S. Zahler (LMU), Prof. Dr. A. Aigner (Leipzig), Prof. Dr. U. Rauch-Kroehnert (Berlin), Prof. Dr. Robert Fürst (Frankfurt/Main).
Below you find structures of selected kinase inhibitors from our projects:
Co-crystallizations of inhibitors with kinases were performed at the Structural Genomics Consortium (Oxford, UK). Data are published in the Protein Data Bank (PDB) (see „Publications“).
An outstanding result of these investigations was the discovery of a novel, uncommon binding mode of chlorinated indole-type inhibitors to the hinge region of kinases by halogen bonding (Huber et al., 2012).
Bromodomains are epigenetic „readers“ which are involved in the regulation of N-acetylation of lysine residues in histones, and consequently in gene regulation. Bromodomain inhibitors represent potential drugs for treatment of different tumors, but also other diseases.
In cooperation with the Structural Genomics Consortium (Oxford, UK) we develop inhibitors of several types of bromodomains (BRD, CREBBP, …).
Some bromodomain inhibitors from the Bracher lab:
Macrodomains are involved in the regulation of ADP-ribosylation of proteins and in gene transcription.
In cooperation with the Structural Genomics Consortium (Oxford, UK) we develop new types of macrodomain inhibitors.
The interaction of DNA and histones is controlled on an epigenetic level by modifications of histone proteins (methylation, acetylation, …). The extent of lysine acetylation is a factor determining gene regulation. Inhibitors of histone deacetylases open new opportunities in tumor therapy, but also in fighting microbial and protozoal infections.
In cooperation with Prof. Dr. Manfred Jung (Freiburg) and Prof. Dr. W. Sippl (Halle) we develop novel inhibitors of histone deacetylases (sirtuins, HDAC6).
In cooperation with Prof. W. de Souza (Rio de Janeiro) and Prof. Dr. R. Pierce (Lille) we could demonstrate the potential of sirtuin inhibitors for treatment of protozoal infections.
Synthesis and analysis of steroids, development of sterol biosynthesis inhibitors
Inhibitors of ergosterol biosynthesis as new antifungals
Substances inhibiting enzymes in the biosynthesis of ergosterol, the main sterol in membranes of fungi, represent the most important class of antifungals. Until now only four of these enzymes are used as drug targets for antifungals. Our research is mainly aimed at the imitation of cationic “high energy intermediates“ of crucial steps of ergosterol biosynthesis, what results in inhibition of selected enzymes (e.g., Δ14-reductase, Δ8,Δ7-isomerase, C24-sterol methyltransferase, oxidosqualene cyclase, C22-desaturase, ...).
We worked out a cell-based assay for the identification of target enzymes in ergosterol biosynthesis in fungi, based on incubation of model strains with the potential enzyme inhibitors, followed by cell lysis and analysis of the modifications in the sterol patterns by GLC-MS/MS. For quantitation of the inhibitory activity we developed a GC-MS/MS protocol which does not afford the use of radioactive material.
A collection of ergosterol biosynthesis inhibitors:
Inhibitors of cholesterol biosynthesis as new antilipidemic drugs
In analogy to the screening system described above, we worked out a whole cell assay for the identification of inhibitors of cholesterol biosynthesis in human cells. For the quantitation of the inhibitory effect we once again use 13C-isotope labeling and GC-MS/MS analysis.
In the course of this research the first selective inhibitor of the enzyme lathosterol oxidase was developed. So the last gap concerning inhibitors of enzymes of the post-squalene part in cholesterol biosynthesis was closed.
A collection of cholesterol biosynthesis inhibitors:
For these assays we need intermediates of the biosynthesis pathways as reference standards. If necessary, new approaches to these sterols are worked out in our lab (Giera & Bracher 2008, Dittrich & Bracher 2015).
Analysis of steroids
Based on the expertise we gained in the installation of the sterol biosynthesis assays, we have outstanding experience in the analysis of steroids by means of high-end GC-MS/MS, including modern sample preparation techniques from complex matrices (SPE, QuEChERS, SPME). This expertise is highly appreciated by numerous cooperating groups for whom we identify and analyze steroids (ergosterol, cholesterol and metabolites, cholic acids, phytosterols) in cells, cell cultures, blood, tissues, feces, plant material, extracts, and drug preparations.
Our analytical experience is also applied to some cooperative projects for ultra trace analysis of drugs, toxins (e.g. nicotine), and pesticides in surface water and food samples.
Prof. Dr. Manfred Jung, Pharmazeutisches Institut, Universität Freiburg (inhibitors of sirtuins and und histone deacetylases)
Prof. Dr. Erika von Mutius, Kinderklinik und Kinderpoliklinik im Dr. von Haunerschen Kinderspital, LMU München (pediatry)
Prof. Dr. Robert Fürst, Institut für Pharmazeutische Biologie, Universität Frankfurt (antiinflammatory kinase inhibitors)
Dr. Fabio Gsaller, Universität Manchester (UK), Institute of Inflammation and Repair (mechanisms of fungal resistance)
Prof. Dr. Oded Livnah, Department of Chemical Biology, Hebrew University of Jerusalem (co-crystallization with kinases)