Download Project 1 Pradimicine Derivates as new Antiviral Drug Leads

Project 1 Pradimicine Derivates as new Antiviral Drug Leads
Ph.D. Supervisor:
Prof. Dr. M. Boysen (LUH)
http://www.akoci.uni-hannover.de/AK_Boysen/index_english.htm
Project Partner:
Prof. Dr. T. Pietschmann (Twincore)
Starting date:
01.10.2014
Project description: Viral infections in humans range from a harmless cold to fatal diseases
like AIDS. Compared to methods of antibacterial treatment, the means of addressing viral
infections are severely limited. Most antiviral drugs target a single enzyme encoded by viral
nucleic acids and therefore, they rarely have useful activity against more than one viral
species. The Pradimicins, a family of natural products isolated from actinomycetes,
selectively interact with oligosaccharides of glycoproteins and are known to target a highly
mannosylated surface protein on the envelope of HIV 1 & 2, inhibiting viral entry into host
cells in vitro. Moreover, resistance development of HIV against Pradimicins proceeds slowly
and by loss of oligosacchrides in the envelope glycoproteins. This exposes hitherto masked
viral protein motifs, which might stimulate an immune reaction in vivo. This unique mode of
action makes Pradimicins potentially active against more viral pathogens carrying
glycoproteins, and further studies revealed activity against the hepatic virus C (HCV).
In order to develop the Pradimicins into antiviral drug leads, improvement of their biological
activity and reduction of their structural complexity is required, but to date, no studies to this
end have been performed. The aim of this project is to prepare a series of new Pradimicin
derivatives with reduced complexity and enhanced physiologic stability and to test them
regarding their activity against HCV. NMR studies on Pradimicin binding showed close
interactions of the D-amino acid moiety and the A-C rings of the aromatic system with a
model mannoside. Therefore, these motifs will be retained in new derivatives, while the Dand E-rings are omitted. Further variations will be made on the pattern of hydrogen bond
donors and acceptors on the aromatic scaffold and on the residue of the amino acid. The
disaccharide, contributing to activity by binding or by enhancing solubility, will be replaced by
a more simple carbohydrate structures or will be attached as a C-glycoside to improve
metabolic stability.
Projekt 2 Glucosinolate turnover in Arabidopsis thaliana
Ph.D. Supervisor:
Prof. Dr. H.-P. Braun (LUH); http://www.genetik.uni-hannover.de/braun.html
Starting date:
01.01.2015
Project description: Glucosinolates are a group of thioglucosides produced by plants of the
Brassicaceae family, which includes cruciferous vegetables such as broccoli, cauliflower and
turnips. Upon tissue damage, e.g. during food preparation and digestion, the glucosinolates
are broken down to form biologically active compounds such as indoles, nitriles,
epithionitriles, thiocyanates, and isothiocyanates. Several indoles and isothiocyanates have
been found to inhibit carcinogenesis in rats and mice. They induce cytoprotective genes,
inhibit angiogenesis, and stimulate apoptosis in tumor cells. Recent studies suggest that
besides the classical pathway of glucosinolate breakdown upon tissue disruption,
glucosinolates are also metabolized in undamaged tissue, and there is evidence for a diurnal
circle of glucosinolate turnover within intact plants. Thus, the glucosinolate profile and organ
distribution in a plant depends not only on rates of biosynthesis but also on turnover.
However, the enzymatic pathways of internal glucosinolate catabolism have not been
established yet.
We will investigate glucosinolate turnover in the model plant Arabidopsis thaliana using a
knockdown mutant that shows changes in glucosinolate degradation indicating a specific
block in one of the catabolic pathways. The PhD student will analyze the proteome of the
mutant plants in order to identify additional components involved in glucosinolate turnover. In
addition, metabolite profiles will be taken with a special focus on glucosinolates as well as
products and intermediates of glucosinolate degradation such as isothiocyanates,
thiocyanates and nitriles. We will use these datasets to develop hypotheses about enzymatic
steps involved in glucosinolate degradation in undamaged plant tissue, which will be tested
with additional Arabidopsis mutants deficient in the respective enzymes.
A better understanding of glucosinolate turnover in plants will help to exploit the beneficial
effects of brassica vegetables for human health. Metabolism and tissue distribution
determines the concentrations of biologically active phytochemicals in the edible parts of the
plants. A detailed knowledge about the pathways involved will make it possible to selectively
produce glucosinolates that can be used as drugs against cancer.
Projekt 3 Synthesis and Profiling of Natural Product Conjugates
Ph.D. Supervisor:
Prof. Dr. M. Brönstrup (HZI/LUH); http://www.helmholtzhzi.de/en/research/research_topics/anti_infectives/chemical_biology/ou
r_research/
Project Partner:
Prof. Dr. M. Kalesse (LUH), Prof. Andreas Kirschning (LUH), Prof.
Mark Stadler (HZI)
Starting date:
01.10.2014
Project description: A key issue in the development of natural products as antitumor drugs
concerns their small therapeutic window, as the often highly potent compounds exert their
effects not only against cancer cells, but also against healthy cells. We would like to address
this issue by the synthesis of hybrid molecules that consist of (i) a targeting moiety that
selectively attaches to cancer cells and (ii) an effector moiety that kills the targeted cells. The
concept has been successfully realized in form of antibody drug conjugates (e.g.
Brentuximab Vedotin, Trastuzumab-DM1). However, the far majority of effectors act via only
two mechanisms, i.e. tubulin binding or DNA binding.
The subject of the Ph.D. Thesis will be to synthesize and characterize conjugates carrying
effectors against essential, but hitherto unexplored intracellular targets.
Three different targets have been selected that can be addressed by highly potent natural
product inhibitors. The natural products will be made available to us through Prof. Marc
Stadler and modified by multistep semisynthesis. One example is outlined below: Ratjadon, a
highly potent inhibitor of the nuclear export protein CRM1, fulfills all requirements for
conjugation with respect to potency and biological relevance. Ratjadon will be produced by
fermentation and derivatized through one of its secondary alcohol functions, utilizing the
extensive experience of the Kalesse group with Ratjadon chemistry.
Ratjadon
In a first step, the well-accessible and validated targeting moiety folate will be employed.
In later phases of the Thesis, an extension to other targeting moieties (e.g. peptides,
protein binding domains or nanoparticles) is conceivable.
A first target molecule is depicted below:
Ratjadon-Folate conjugate
The focus of the Thesis will be on preparative organic chemistry. However, synthetic efforts
are directed to the generation of functional molecules. The incumbent will therefore be wellexposed to the biological and medical context, in particular to profiling assays and cellular
biology.
Projekt 4 The Tat system as novel target for antibiotics
Ph.D. Supervisor:
Prof. Dr. T. Brüser (LUH); http://www.ifmb.uni-hannover.de
Project Partner:
Prof. Dr. M. Brönstrup (HZI/LUH), Prof. Dr. M. Kalesse (LUH)
Starting date:
01.10.2014
Project description:
The prokaryotic Tat protein translocation system is a promising
target for novel antibiotics. Bacteria with compromised Tat transport show manifold
phenotypes, including diminished virulence of most pathogens, such as Pseudomonas
aeruginosa and Mycobacterium tuberculosis. In many cases, these virulence-affecting
phenotypes are due to effects on the transport of proteins involved in respiratory electron
transport, biofilm formation, iron acquisition, lipolysis or motility. The advantage of Tatspecific inhibitors would be the low probability of harmful adverse reactions, as Tat systems
do not exist in humans. However, the Tat system depends on the proton motive force and
thus sensitivity of Tat transport against uncouplers and other compounds with general toxicity
impedes the identification of useful compounds by standard negative screens. In this project
we will develop a reliable positive selection screen for Tat inhibitory compounds that can
serve as lead compounds for new antibiotics. This screen is based on the production of
conditionally essential cytoplasmic proteins that are recombinantly fused to Tat signal
peptides. Specific inhibition of Tat transport thereby promotes growth, whereas functional Tat
transport inhibits growth. Using this test system, we will analyze a broad range of Tat
systems from different pathogenic bacteria in order to assess the possible species- or more
broad range specificity of the compounds. In parallel, we will assess the inhibitory
mechanism and possible cross-species effect of inhibitors that have been already identified
by standard screens. In co-operation with the Chemistry groups, we intend to develop
optimized inhibitors with enhanced properties.
Scheme: Principle of the positive selection screen for Tat inhibitors.
Projekt 5 Development of methodologies for structure-based drug
design
Ph.D. Supervisor:
Prof. Dr. T. Carlomagno (LUH); http://www.kalesse.uni-hannover.de
Project Partner:
Synthetic Chemistry groups (HZI/LUH)
Starting date:
01.07.2015
Project description:
Within the last decades structure-based drug design (SBDD)
has evolved to a powerful tool for the optimization of many low molecular weight lead
compounds to highly potent drugs. The principle of SBDD lies in the combination of different
chemical moieties with the aim of obtaining a molecule that, while possessing the
pharmacological properties necessary for a drug, is complementary in shape to the receptorbinding pocket. This process requires knowledge of the exact structure of the receptor-ligand
complex.
The new NMR-based methodology INPHARMA, developed by my group, provides
access to the relative binding mode of low-affinity ligands to a common target. The method
requires two competitively binding ligands and a model of the structure of the apo-receptor.
INPHARMA is based on the observation of interligand, spin-diffusion mediated, transferredNOE (Nuclear Overhauser Effect) data, between the two ligands L1 and L2. As the ligands
are competitive binders, such NOEs do not originate from direct transfer of magnetization
between L1 and L2, but rather from a spin-diffusion process mediated by the protons of the
receptor binding pocket and are, therefore, dependent on the specific interactions of each of
the two ligands with the protein. Thus, a number of such intermolecular NOE peaks describe
the relative orientation of the two ligands in the receptor-binding pocket. In accordance with
existing SBDD workflows, the experimental information derived from the INPHARMA NOEs
is used to select the correct binding mode among many possible binding orientations
obtained by molecular docking.
In this project we will develop a method to use INPHARMA data to directly calculate the
structure of the protein-ligand complex starting from a low-resolution model of the aporeceptor. To reach this objective, we will develop NMR measurement schemes as well as
data analysis and data fitting protocols. Starting from a low-resolution model of the complex,
the protocol will explore the complex multidimensional energy landscape defined by the
experimental INPHARMA data, and will find the global minimum, that is the complex
structure that uniquely fits the experimental data. A number of test cases will be analysed,
spanning from soluble proteins to membrane receptors (kinases, GPCR receptors). In
addition, collaborations with BMWZ and HZI/HIPS scientists will be instrumental to identify
additional targets and ligands.
Projekt 6 Investigating the Biosynthesis and Synthesis of the
Communesins: Multipotent Natural Products from Fungi.
Ph.D. Supervisor:
Prof. Dr. R. J. Cox (LUH); http://www.cox-group.uni-hannover.de/377.html
Project Partner:
Dr. T. Gaich (LUH)
Starting date:
01.10.2014
Project description: The communesins A-H are complex fungal alkaloids possessing a
diversity of biological activities icluding cytotoxic, insecticidal and antiparasitic properties
arising from the densely functionalised indole-derived core. The complex structures have
stimulated the interests of many synthetic chemists, but the biosynthesis has been almost
totally overlooked. This project focusses on investigating the biosynthesis of the
communesins in fungi using a combination of the latest biological and chemical methods with
the aim of generating new potent bioactive molecules. This project will focus on obtaining
genome sequences from fungi known to make various bioactive communesisns. Genome
mining will be used to find and compare the gene clusters responsible for the biosynthesis.
We will then use gene knockout strategies to block the pathway at key points to generate
intermediate and shunt metabolites. These compounds will be purified and their structures
determined by NMR and crystallography. The structures will help delineate the order of the
biological synthetic steps, but intermediates are also likely possess interesting biological
properties and these will be tested in collaboration with other BMWZ and HSBDR colleagues.
Scheme 1. Possible biosynthetic precursors of the communesins.
Projekt 7 Isolation and structural analysis of new secondary
metabolites from strains and extracts of south east India
Ph.D. Supervisor:
Dr. G. Dräger (LUH); http://www.oci.unihannover.de/de/arbeitskreise/draeger/index.php
Project Partner:
Prof. Dr. T. Scheper (LUH), Prof. Dr. J. Papenbrock (LUH); Prof. R. J.
Cox (LUH)
Starting date:
01.12.2014
Project description: Within this highly interdisziplinary project we continue and strengthen
our existing collaboration (isolation of marine natural products) with the CAS in Marine
Biology (Annamalai University, south east India). Samples from this region have only been
sparsely studied so far. We envision to collect samples (e.g. soil, wood, mud; collection will
be performed during a visit this fall), isolate bacteria (with a focus on streptomyces and
actinomyces; other bacteria and fungi could be isolated in collaboration with Prof. Stadler
and/or Prof. Cox; organisms from marine sources will be isolated and extracted by Prof.
Arumugam Muthuvel, CAS), ferment the isolated species and undergo a chemical and
activity based screening to isolate biological active and/or chemical interesting metabolites.
We strongly believe that this approach will lead to the identification of new natural products.
This project will also produce enriched fractions and small molecules for the screening
groups. The combination of organisms from highly competitive biological niches,
fermentation capacities and knowledge, a full equipped set of chromatographic techniques
and a strong analytical (MS / NMR) unit will still guarantee a high success rate although we
always have to face the problem of reisolating known compounds
Project objectives:
Isolation of microorganisms from samples
Fermentation, Extraction, enrichment and isolation of compounds
Screening of fractionated extracts and pure compounds
Structure elucidation of the active compounds
Projekt 8 Total Synthesis and Target Identification of Haprolid
Ph.D. Supervisor:
Prof. Dr. M. Kalesse (LUH); http://www.kalesse.uni-hannover.de
Project Partner:
Prof. Dr. M. Brönstrup (HZI/LUH), Prof. Dr. T. Pietschmann (Twincore)
Starting date:
01.10.2014
Project description: Haprolid, a novel natural product isolated at the HZI shows antiviral
effects against hepatic virus C (HCV). At this stage it is not clear whether haprolide targets a
specific viral protein or a host protein that is essential for viral replication. However, no
synthetic contributions, structure-activity investigations or efforts for target identification have
been reported. The aim of this project is to provide the total synthesis of haprolide and to
perform SAR-studies in order to identify the phamacophoric groups. Based on these SAR
studies it is planned to construct a chemical probe based on haprolide in order to identify its
cellular target. The synthesis takes advantage of coupling between the peptidic and
polyketide portion of the molecule. In contrast to the tetrapeptide the construction of the
polyketide is non-trivial and we plan to construct the Z-configured double bond from a
sequence of Birch reduction followed ozonolysis. A stereoselective alkylation should finally
complete the synthesis of the polyketide segment (Scheme 1).
Scheme 1 Retrosynthetic analysis.
The planned synthesis of the chemical probe uses the position of the methyl ether as linkage to the
reactivity and sorting function.
Scheme 2 Molecular probe based on haprolide.
Projekt 9 Probing the biosynthesis of the GABA-receptor inhibitor
Xenovulene A by chemical synthesis
Ph.D. Supervisor:
Prof. Dr. A. Kirschning (LUH); http://www.kirschning-group.com/
Project Partner:
Prof. Dr. R. Cox (LUH), Prof. Dr. T. Scheper (LUH)
Starting date:
01.10.2014
Project description: Xenovulene A is a terpenpoid first isolated from cultures of
Acremonium strictum which shows strong activity on benzodiazepine binding to the GABAbenzodiazepine receptor. It contains an unusual furocyclopentenone moiety fused to a
humulene derived 11-membered ring.
A number of minor co-metabolites of 1 have been isolated3 in which the cyclopentenone
moiety is replaced by a trioxygenated benzene 2 or highly oxygenated tropolone rings, 3 and
4. While fungal metabolites containing all of these types of rings are known, the presence of
all three in a single family is unique. Biosynthetically, an unusual cascade of ring expansion
and ring contraction steps has been proposed which was based on feeding experiments with
labeled precursors. So far no total synthesis has been reported nor have these unique
biotransformations been mimicked by chemical synthesis. In a collaboration between the two
research groups the
biosynthetic route is planned to be established by a biomimetic
approach thereby enriching the chemistry of ring expansion and ring contraction chemistry
around the tropolone moiety unsing model systems like A and B.
Projekt 10 Small-Molecule Modulators of -Cardiac Myosin Stability and
Function
Ph.D. Supervisor:
Prof. Dr. D.J. Manstein (MHH); http://www.mh-hannover.de/bpc.html
Project Partner:
Prof. Dr. M. Kalesse (LUH); http://www.kalesse.uni-hannover.de
Starting date:
01.10.2014
Project description: To promote myocardial regeneration, we aim to use pharmacological
chaperones and myosin activators. Suitable compounds will be identified and their influence
on the molecular mechanisms controlling reverse cardiac remodeling and atrophy will be
examined. Degenerative processes in the heart include irreversible changes in structure with
extracellular matrix accumulation, impairment of cytoskeleton and sarcomeric structures and
general loss of cardiac cells due to death or transformation. In patients with heart failure,
these are accurate predictors of morbidity and mortality. The scarcity of donor organs limits
the number of heart transplantations, and the waiting list by far exceeds the number of
possible transplants. Therefore, we aim to increase the number of transplantable hearts by
developing small molecule-based approaches that protect donor organs during
transplantation and improve the long-term performance of suboptimal donor organs or grafts.
We recently showed that drug-like molecules as well as metabolites and common food
ingredients such as poly-unsaturated fatty acids (Figure 1) can act as potent modulators of
actomyosin-based contractility of the heart. The thiadiazinone derivatives EMD 57033 and
EMD 60263 are members of a new class of pharmacological chaperones that stabilize,
enhance the activity, and correct stress-induced misfolding of -cardiac myosin (Radke et al.,
elife 2014). EMD 57033 binds to an allosteric pocket that is conserved in most members of
the myosin family. The site is close to the site where cardiac myosin activator omecamtiv
mecarbil (Malik et al., Science 2011) and arachidonic acid are predicted to bind. We aim to
dissect these binding pockets and the associated allosteric communication pathways. Based
on the resulting information, we expect to gain a better understanding of the mechanisms
contribution to motor protein stabilization, activation, and refolding by small molecules. We
will combine computer-aided design, chemical synthesis of candidate compounds, and
functional analysis to test the extent to which the biological activity and the specificity of the
compound can be optimized for therapeutic applications.
Figure 1: Common metabolites and food ingredients such as arachidonic acid can greatly affect actomyosin function. (A) In
the presence of ATP, arachidonic acid increases the affinity of -cardiac myosin for actin (KM(actin)) more than 10-fold. The
maximal rate of ATP turnover (Kcat) is increased approximately 2-fold. (B) Histogram showing the velocity distribution for
actin filaments moving on a lawn of -cardiac myosin. Addition of arachidonic acid to the same flow-cell increases the
number of -cardiac myosin motors that productively interact with actin filaments to produce force and movement.
REFERENCES:
F. I. Malik, J. J. Hartman, et al. and, D. J. Morgans, Science 2011, 331, 1439-1443.
M. B. Radke, M. H. Taft, B. Stapel, D. Hilfiker-Kleiner, M. Preller, D. J. Manstein, eLife 2014, DOI: 10.7554/eLife.01603.
Projekt 11 Analysis of the bioactive compounds of seagrasses and
mangroves: composition, identification of compounds and their role in
biofilm inhibition
Ph.D. Supervisor:
Project Partner:
Starting date:
Prof. Dr. J. Papenbrock (LUH); http://www.botanik.unihannover.de/109.html
Dr. Dräger (LUH/BMWZ), Prof. Kirschning (LUH/BMWZ), Prof. Häußler
(HZI/Twincore)
01.01.2015
Project description: Seagrasses, a unique group of submerged flowering plants, and
mangroves profoundly influence the physical, chemical and biological environments of
coastal waters through their high primary productivity. The phytochemical contents of
seagrasses and mangroves are gaining importance for medicine and biotechnology because
several studies report their bioactive value. There are indications that seagrasses and
mangroves may possess some rare and new compounds that are not reported from their
terrestrial relatives. It is well known that seagrasses decay very slowly, maybe indicating that
they contain compounds inhibiting the degradation by microorganisms. One example is
zosteric acid, a biofilm inhibiting compound isolated from Zostera marina. Many seagrasses
and mangroves are covered by epiphytes but in an intact environment they are not damaged
by these epiphytes indicating a balance controlled by attractants and repellents. The very
promising results obtained with zosteric acid demonstrate the potential of seagrasses for the
isolation of further biofilm-inhibiting compounds also from other seagrass species. Also for
mangroves high concentrations of secondary compounds with medicinal use known from
ethnobotany are reported, however, the structure of these compounds has not been
investigated in detail so far. First results demonstrate that extracts from seagrasses and
mangroves and fractions thereof show biofilm-inhibiting activity in bioassays using
Escherichia coli and Candida albicans.
Scientific objectives:
 Establishment of cultivation and induction conditions to produce reproducibly large
amounts of secondary compounds
 Extraction, enrichment and isolation of compounds inhibiting biofilm formation from
seagrasses and mangrove species
 Identification of putative compounds inhibiting biofilm formation from seagrasses
 Evaluation of the anti-biofilm activity and unravelling the mode of action of isolated
compounds
Working plan
Optimization of the fractionation protocol (HPLC)
Establishment of more bioassays to test biofilm-inhibiting
activity (screening)
Screening of fractions from several seagrass and mangrove
species
Identification of known (bioactive) compounds using standards
and libraries (LC-MS)
Identification of unknown bioactive compounds (LC-MS, NMR)
Testing of isolated bioactive compounds in more advanced
biofilm tests
Year 1
x
x
Year 2
Year 3
x
x
x
x
x
Projekt 12 Clinically approved T-type calcium channel blockers as
inhibitors of Hepatitis C Virus (HCV) membrane fusion
Ph.D. Supervisor:
Project Partner:
Starting date:
Prof. Dr. Thomas Pietschmann, TWINCORE
Prof. Dr. Andrease Kirschning, LUH
01.10.2014
Project description: Seagrasses, a unique group of submerged flowering plants, and
mangroves profoundly influence the physical
Previous work:
To identify compounds with anti-HCV activity and novel modes of action, we screened a
library of FDA-approved ion channel inhibitors. Among the most potent molecules,
flunarizine, a T-type calcium channel inhibitor used to treat migraine, was selected for
functional studies. Flunarizine inhibited HCV entry (IC50 of 388,2 nM) in Huh7-Lunet cells
and primary human hepatocytes, without influence on RNA replication, assembly or release.
Interestingly, genotype 2a (GT2a) cell culture-derived particles (HCVcc), but not HCV
pseudoparticles (HCVpp) or HCVcc particles from non-GT2a were susceptible to flunarizine.
Shuffling structural proteins between GT1a, GT2b and GT2a chimeric viruses revealed that
GT2a-derived E1-E2 genes confer susceptibility to flunarizine. Long-term culture of HCV in
the presence of this compound yielded resistance mutations mapping to the viral envelope,
thus indicating that flunarizine targets glycoprotein function(s). Pretreatment of target cells,
but not of virus particles inhibited HCV entry and time of addition experiments indicated that
flunarizine acts at a late stage of entry. Since flunarizine interfered with HCV infection
through low-pH-triggered fusion at the plasma membrane, we conclude it inhibits HCV
independent of endocytosis. Interestingly, when flunarizine was added merely during the
brief wash with low pH buffer, virus infection was fully blocked, suggesting that it specifically
interferes with viral membrane fusion. Finally, trans-complemented HCV particles (HCVTCP) carrying glycoproteins from GT2a patients were inhibited by flunarizine and flunarizineresistant viruses were more prone to neutralization by patient-derived antibodies. Therefore,
and considering the clinical use of this compound, flunarizine may be an alternative
treatment option for GT2a-infected individuals.
Aims of the project:
1. Identification of the pharmacophoric group(s) that confer antiviral activity
2. Modulation of antiviral activity with regard to breadth of antiviral activity (towards additional
HCV genotypes)
3. Identification of viral/host target of flunarizine
Work plan:
In collaboration with Prof. Andreas Kirschning, a number of derivatives of flunarizine will be
synthesized and tested with regard to antiviral activity against HCV (potency,antiviral activity
against different genotypes, cross resistance conferred by resistance mutations selected with
flunarizine). These assays will also include a spectrum of other licensed T-Type Ca channel
inhibitors and they are geard to identify molecules with superior antiviral activity and to
identify the characteristic features responsible for the antiviral activity of this class of
compounds. Based on these studies, a derivative that retains antiviral activity and that can
be coupled to a solid support will be synthesized. This molecule will be used to identify
proteins that interact with flunarizine in HCV infected cells in order to identify host or viral
factors responsible for the antiviral activity of flunarizine. In parallel we will attempt to
synthesize the viral peptide with and without flunarizine resistence mutations. In collaboration
with NMR experts within the HSBDR graduate school we wish to explore if there is a direct
interaction between flunarizine and this viral protein domain.
Projekt 13 Targeted Modulation of the Enzymatic and Mechanical
Function of Molecular Motors
Ph.D. Supervisor:
Prof. Dr. M. Preller (MHH);
http://www.mh-hannover.de/bpc_structbioinf.html
Project Partner:
Prof. Dr. D.J. Manstein (MHH), Prof. Dr. M Kalesse (LUH)
Starting date:
01.10.2014
Project description: Myosins represent a diverse class of molecular motors and play a role
in a wide range of biological processes. However, less is yet known about the roles different
myosin isoforms from various classes play in diverse molecular processes, and the dys- or
hyperfunction of specific isoforms are implicated in the development of severe diseases,
including cancer, cardiovascular failures, and disorders of sensory organs and the central
nervous system. Therefore, targeted strategies to modulate the motor properties of specific
myosin isoforms hold on the one hand the potential as tools for the analysis of complex
biological processes, and on the other hand the possibility to develop therapeutic agents to
treat myosin-associated diseases. The aim of this project is to develop cell-permeable small
molecule modulators of myosin function, with the goal to both decrease the motor activity
(inhibition) as well as stimulating force production by myosin motors (activation) in a
controlled manner. In particular, for the development and optimization of myosin activators
we found the thiadiazinone scaffold to represent a promising moiety. Starting with (Q)SAR
[(quantitative) structure-activity-relationship] studies of known thiadiazinones, it is planned to
detect favored and disfavored properties and thus, to generate a pharmacophore model for
the design process. Synthetic access to new thiadiazinone derivatives will be gained by
condensation reaction of 2-chloro-1-(1,2,3,4-tetrahydroquinolin-6-yl)propan-1-one with Oethyl hydrazinethioformate and subsequent N-acylation (scheme 1). Complementing
structural and computational studies will be employed to gain information about their
mechanisms of action.
O
O
1.) AlCl3 Cl
Cl
Cl
N
O
2.) HCl
CH 3
CH 3
N
H
CH 3
H
N
EtO
H
N
O
O
N
S
N
S
S
O
Cl
N
Y
O
Scheme 1:
H
N
Y
Synthesis of new thiadiazinones.
N
H
NH 2
Projekt 14 Cellular screening assays for liposome-based drug delivery
systems
Ph.D. Supervisor:
Prof. Dr. Thomas Scheper (LUH);
Project Partner:
Prof. Dr. Kalesse, Prof. Dr. Kirschning, PD Dr. Carsten Zeilinger
Starting date:
01.10.2014
Project background:
Huge libraries of small molecule drug candidates are readily available and the number of candidates is
steadily increasing. These substances need to be screened for their effectiveness as well as for
possible side effects. Today different cell-based screening platforms are used in which the cells are
directly contacted with the drug candidate in the surrounding cell-culture medium. Therefore, these
systems can be seen as tools to mimic systemic administration of the drug. Systemic administration of
drugs often involves serious limitations. It is usually associated with non-specific delivery of the drug to
all cells, including diseased ones as well as healthy cells, which may be a source of serious sideeffects. Additionally, the potential drug has to conquer the barrier of the cells´ membrane to induce
cytotoxicity. To overcome these limitations, targeted drug delivery is an upcoming alternative to
systemic administration. In this context, liposomes are promising candidates for targeted delivery
based on their good biocompatibility and biodegradability. They offer a surface which can be modified
with biomolecules to promote specificity and cellular uptake; the surface and the volume encapsulated
by the liposomes membrane, can be used as a carrier of the drug. Based on high specificity in
combination with enhanced cellular uptake and high local drug concentration, targeted drug delivery is
expected to emerge in the future. Thus, cell-based test systems to investigate targeted drug delivery
and to screen drug candidates for their effectiveness in targeted delivery approaches are needed.
Objectives:
The aim of this project is the development of cell-based assay systems enabling the screening of
drugs administered via targeted delivery. As a model system, multi-functional liposomes will be
established and used to develop the cell-based screening assay. To develop multi-functional
liposomes different building blocks will be combined:
1. The carrier: The liposome will be produced and the incorporation of potential drugs will be
optimized.
2. Aptamers: In order to provide specificity, liposomes will be modified with aptamers. Comparative
experiments will be performed using antibodies.
3. Peptides: To further enhance cellular uptake of liposomes – even in the case that the targeted cell
surface receptor does not promote endocytosis – the liposomes will be modified with RGD peptides
4. Membrane channel proteins: Besides endocytosis and peptide-mediated cellular uptake the use of
membrane channel proteins for the delivery of drugs will be investigated.. This part of the project will
be performed in close cooperation with PD Dr. Carsten Zeilinger.
Based on the toolbox-like design of the liposomes, different mechanisms of cellular internalization
(endocytosis, peptide-mediated internalization, connexon-mediated uptake) can be investigated.
Preliminary studies:
Modification of nanoparticles with biomolecules
Aptamer-based targeting of cells
Development of liposome microarrays (with PD Dr. Zeilinger)
Development of cytotoxicity assays
Working plan:
Year 1
Year 2 Year 3
Production and mono-functionalization of liposomes
x
Production of multi-functional liposomes
x
x
Characterization of liposomes via microarrays
x
x
Development and of cellular assay
x
Screening of compounds using the developed assays
x
x
Projekt 15 Signaling aptamers for specific cellular targeting and medical
applications
Ph.D. Supervisor:
Prof. Dr. Thomas Scheper (LUH);
Starting date:
01.10.2014
Project background:
Specific targeting of cells is urgently needed in diagnostics as well as in the targeted delivery of drugs. In these
applications, the targeting molecule must provide a number of properties: (i) It must bind the cells of interest
with high specificity even in the presence of a vast amount of other cells, e.g. in tissue sections or the human
body. (ii) It must be stable in biological fluids and (iii) should show no significant immunogenicity.Aptamers
can fulfil these requirements and are expected to become widely used in diagnostics and drug delivery
applications.
Objectives:
The aim of this project is the development of signalling aptamers that allow the direct detection of the binding of
the aptamer to its target cell. This would enable the fast assessment of binding assays and the development of
rapid diagnostic assays that do not require any washing steps. Moreover, false-positive signals will be
eliminated. This will be ensured by the development of signalling aptamers based on a mechanism termed
“target-induced dissociation of complementary oligonucleotides (TID). In this TID mechanism, the aptamer is
labelled with a fluorophore and hybridized with a complementary oligonucleotide which is modified with a
quencher. In the absence of the target, the oligonucleotide binds to the portion of the aptamer that is responsible
for binding of the target and thereby quenches the fluorescence. Target binding results in the release of the
oligonucleotide, and thus in increased fluorescence. Consequently the signalling aptamer emits fluorescence
solely after specific binding to the target, while non-bound and non-specifically bound aptamers remain in the
quenched state. Besides direct diagnostics, signalling aptamers will also be helpful to observe the cellular
targeting in the development of targeted drug delivery.
Figure 1: TID Mechanism. F: Fluorophore, Q: Quencher.
As a model system, aptamer S15 targeting human adenocarcinoma lung cancer cells will be used. To develop
signalling aptamers, the following tasks have to be solved:
1. The target binding site of aptamer S15 will be identified by systematic truncations of the aptamer.
2. Complementary oligonucleotides will be designed and investigated using aptamer microarrays.
3. TID assay will be developed on microarrays and transferred to flow cytometry.
4. Diagnostic applicability will be evaluated via cell culture experiments and tissue microarrays.
5. Potential use in drug delivery will be investigated using liposomes.
Preliminary studies:
Aptamer Microarrays
Flow cytometry using Aptamers
Staining of cells using aptamer-modified nanoparticles
Working plan:
Year 1
Year 2
Year 3
Identification of target binding site
x
Design of complementary oligonucleotides
x
x
Development of TID assay
x
x
Diagnostic application
x
Drug delivery
x
x
Projekt 16 Synthesis of Sigillin Derivatives and their Biological Activity
Ph.D. Supervisor:
Prof. Dr. S. Schulz (TUBS); http://www.oc.tu-bs.de/schulz/index.html
Project Partner:
Prof. Dr. M. Brönstrup (HZI/LUH), Prof. Dr. T. Pietschmann (Twincore)
Starting date:
01.10.2014
Project description: Sigillin is a natural product isolated by our group from springtails
(collembola), which produce the compound in relatively large amounts. It is the first member
of a new class of natural products, polychlorinated benzopyranone derivatives carrying 4 to 6
chlorine atoms. Sigillin shows high activity in anti-feeding assays using ants, and moderate
activity in some antibacterial assays. The total synthesis of sigillin is complicated. One route
developed in our group led to desoxygenated sigillin derivatives and an unprecedented
tricyclus, WS-121. No total synthesis of this compound exists to date.
In the project new synthetic approaches to sigillin and WS-121 will be explored. Experience
in the chemistry of these compounds exists in our group. Promising entries into the synthesis
of sigillin derivatives are the lactone 2, available from precursor 1, and the SmI2 mediated
intraspecific Reformatzky-reaction leading from 3 to 4. Previous approaches by us used
lactone 5 as starting material. Difficulties arise especially from the chlorine substituents that
give rise to unexpected reaction pathways.
The cooperating partner groups of Prof. Brönstrup (HZI, Braunschweig) and Prof. Pietschmann
(Twincore, Hannover) will profile the biological activity of sigillin and its derivatives. In favorable
cases the molecular target of sigillin will be addressed by target fishing or pulldown experiments.
Projekt 17 Use of novel natural compounds for the alleviation of T helper
(Th) 2- and Th17-driven inflammatory diseases
Ph.D. Supervisor:
Prof. Dr. med. Tim Sparwasser (Twincore)
Project Partner:
Prof. Dr. M. Kalesse (LUH)
Starting date:
01.10.2014
Project description: CD4+ T cells coordinate immune responses by differentiating into
distinct lineages of effector T cells, each defined by their own signature production of
cytokines and by their distinctive functions in host defense against pathogens. These subsets
include Th1, Th2 and Th17 cells. Proper differentiation of naïve T cells and execution of the
resulting adaptive immune response is tightly regulated. However, under certain conditions,
these mechanisms can be dysregulated. For instance, a dysregulated Th2 response could
lead to allergic asthma, meanwhile Th1 or Th17 hyper-response could lead to autoimmune
diseases. After initial screenings of a library of secondary metabolites from Myxobacteria
(HZI-Braunschweig) for their effect on Th2 differentiation, we identified P2B8, P2A7 and
P1A9 as strong modulators. P2A7 and P1A9 have also shown to inhibit Th17 differentiation,
with the latter recently being reported to affect T cell metabolism, favoring regulatory T cell
subset differentiation (Nature Medicine, in press).
The aim of this project is to provide candidate molecules for the alleviation of Th2 or Th17driven inflammatory responses.
To address this aim, we plan to perform in vitro assays to validate the first results, such as:
dose optimization, cell proliferation assays, expression of key transcription factors for Th2
and Th17 differentiation, and measurement of hallmark cytokine secretion. Further in vitro
studies will seek to determine molecular targets of candidate compounds. Interaction with
experts in structural chemistry (HSBDR) will be of great value for a better understanding of
the molecular structure-biological function relationship, therefore contributing to the finding of
cellular targets, water soluble derivatives and to overcome toxicity problems, if any.
Finally, we will examine the impact of P2A9 and P2B8 administration during well-established
in vivo mouse models of Th2 and Th17-driven diseases in our laboratory. To address this
point OVA- induced asthma (Th2), citrobacter-induced colitis and experimental autoimmune
encephalomyelitis (Th17) will be used as read-outs.
Projekt 18 Full Exploitation of the secondary metabolome of the
ubiquitous fungal saprotroph and endophyte, Hypoxylon fragiforme
Ph.D. Supervisor:
Prof. Dr. M. Stadler (HZI/TU Braunschweig);
Project Partner:
Prof. Dr. Russell Cox (LUH)
Starting date:
01.01.2015
Project description:
Hypoxylon fragiforme is one of the most frequent ascomycetes of the Northern hemisphere.
Its saprotrophic, stromatal life stage (including the stromata, that develop the sexual state) is
omnipresent in beech forests, but the fungus has been very frequently reported as
endophyte of various seed plants. This species has already been subjected to extensive
studies of its secondary metabolism in the past. Nevertheless, HPLC-MS profiling during
various stages in the life cycle of this fungus have revealed ca.70 metabolites that could not
be assigned to any of the known structures. In certain stages of development, not only
prolific secondary metabolite production, but extremely strong antibiotic and other biological
activities have been observed in extracts from the fungus. The active principles largely
remain to be identified, as they could not yet be isolated in sufficient quantities.
The objectives of the proposed project are


to identify the above mentioned antibiotics and make them available for biological
characterisation and as templates for total synthesis or chemical modification.
to find parameters suitable for large scale production of the most interesting
compounds by using a variety of methods ranging from artificial production of
fruitbodies in the lab on solid substrates to regular fermentation in liquid culture up
to 350 litre scale
In parallel, the genome of the producing organism will be sequenced and annotated, in order
to identify the relevant gene clusters encoding for the biosynthesis of the above mentioned
compounds in collaboration with the group of Russell Cox.
All compounds isolated will undergo extensive studies on their biological activities, either inhouse at the department MWIS, or in collaboration with other groups at HZI and Hannover
Medical School.
Once interesting lead compounds have been found, it will be possible to further optimise
them (e.g. in collaboration with the department of M. Brönstrup, HZI) by means of medicinal
chemistry, or to attempt their total synthesis in collaboration with the chemistry at LUH.
Candidates for this project should be interested in or –even better- have relevant experience
in analytical/natural product chemistry (especially preparative chromatography),
microbiology, mycology and biotechnology. The various methods that will be used for this
interdisciplinary project have been described in our ongoing studies of Hypoxylon and other
xylariacaeous fungi cited below.
Projekt 19 Hsp100/Clp protease complexes as targets for antibiotics: a
novel bacteriocidal mechanism
Ph.D. Supervisor:
Prof. Dr. K. Turgay (LUH); http://www.ifmb.uni-hannover.de/turgay.html
Project Partner:
Prof. Dr. E. Charpentier (HZI/MHH), Prof. Dr. R. Müller (HIPS/HZI)
Starting date:
01.10.2014
Project description: Protein degradation is necessary to maintain cellular protein
homeostasis. In bacteria Hsp100/Clp protease complexes are part of the cellular protein
quality control system and can remove misfolded or damaged protein species from the
cellular environment by degradation. Importantly, the same Hsp100/Clp protease complexes
are concurrently involved in the signal transduction and control of cellular and developmental
processes by regulatory proteolysis of e.g. transcription factors. Interestingly, mutations in
Hsp100/Clp genes of many pathogens affect their virulence capabilities. The full Hsp100/Clp
protease complex is a molecular machine, which consists of two components: a hexameric
ring of Hsp100/Clp ATPases such as ClpC or ClpX, which associates with a barrel-like
compartmentalized protease complex, like ClpP. The hexameric Hsp100/Clp protein is a
chaperone, which can unfold and translocate the recognized substrate protein for
degradation into the associated ClpP protease complex1. These molecular machines were
identified as new targets for bactericidal antibiotics, which kill dormant, non-dividing bacterial
cells by a new mechanism 2-8.
Aims of this project include: (1) Establish a screen to identify and subsequently characterize
new bactericidal natural compounds or small molecules targeting Hsp100Clp protease
complexes such as ClpCP. (2) We want to elucidate the molecular mechanism, by which
new antibacterial compounds, identified in the established screen, or known compounds
such as Cyclomarin change the activity and substrate recognition of bacterial ClpCP
protease complexes, resulting in the recognition and degradation of intracellular proteins
necessary for cellular function of dormant non-dividing bacterial cells.
1
6
2
7
Kirstein, J. et al. (2009) Nat. Rev. Microbiol. 7:589-99.
Brötz-Oesterhelt, H. et al. (2005) Nat. Med. 11, 1082–1087.
3
Sass, P. et al. (2011) Proc. Natl. Acad. Sci. U. S. A. 108, 17474–
17479.
4
Kirstein, J. et al. (2009) EMBO Mol. Med. 1, 37–49.
5
Lee, B.-G. et al. (2010) Nat. Struct. Mol. Biol. 17, 471–478.
Conlon, B. P. et al. (2013) Nature 503, 365–370.
Schmitt, E. K. et al. (2011) Angew. Chem. Int. Ed Engl. 50, 5889–
5891.
8
Vasudevan, D., Rao, S. P. S. & Noble, C. G. (2013) J. Biol. Chem.
288, 30883–30891.
9
Gavrish, E. et al. (2014) Chem. Biol. 21, 509–518.