FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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Design and Stereoselective Synthesis of ProM-2: A Spirocyclic Diproline Mimetic with Polyproline Type II (PPII) Helix Conformation
Reuter(*), C., Opitz, R., Soicke(*), A., Dohmen(*), S., Barone, M., Chiha(*), S., Klein(*), M. T., Neudörfl(*), J. M., Kühne, R.; Schmalz(*), H. G.
Chemistry, 21:8464-8470

Tags: Computational Chemistry and Protein Design (Kühne)

Abstract: With the aim of developing polyproline type II helix (PPII) secondary-structure mimetics for the modulation of prolin-rich-mediated protein-protein interactions, the novel diproline mimetic ProM-2 was designed by bridging the two pyrrolidine rings of a diproline (Pro-Pro) unit through a Z-vinylidene moiety. This scaffold, which closely resembles a section of a PPII helix, was then stereoselectively synthesized by exploiting a ruthenium-catalyzed ring-closing metathesis (RCM) as a late key step. The required vinylproline building blocks, that is, (R)-N-Boc-2-vinylproline (Boc=tert-butyloxycarbonyl) and (S,S)-5-vinylproline-tert-butyl ester, were prepared on a gram scale as pure stereoisomers. The difficult peptide coupling of the sterically demanding building blocks was achieved in good yield and without epimerization by using 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)/N,N-diisopropylethylamine (DIPEA). The RCM proceeded smoothly in the presence of the Grubbs II catalyst. Stereostructural assignments for several intermediates were secured by X-ray crystallography. As a proof of concept, it was shown that certain peptides containing ProM-2 exhibited improved (canonical) binding towards the Ena/VASP homology 1 (EVH1) domain as a relevant protein interaction target.

Sensitivity and resolution of proton detected spectra of a deuterated protein at 40 and 60 kHz magic-angle-spinning
Nieuwkoop, A. J., Franks, W. T., Rehbein, K., Diehl, A., Akbey, Ü., Engelke(*), F., Emsley(*), L., Pintacuda(*), G.; Oschkinat, H.
J Biomol NMR, 61:161-171

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: The use of small rotors capable of very fast magic-angle spinning (MAS) in conjunction with proton dilution by perdeuteration and partial reprotonation at exchangeable sites has enabled the acquisition of resolved, proton detected, solid-state NMR spectra on samples of biological macromolecules. The ability to detect the high-gamma protons, instead of carbons or nitrogens, increases sensitivity. In order to achieve sufficient resolution of the amide proton signals, rotors must be spun at the maximum rate possible given their size and the proton back-exchange percentage tuned. Here we investigate the optimal proton back-exchange ratio for triply labeled SH3 at 40 kHz MAS. We find that spectra acquired on 60 % back-exchanged samples in 1.9 mm rotors have similar resolution at 40 kHz MAS as spectra of 100 % back-exchanged samples in 1.3 mm rotors spinning at 60 kHz MAS, and for (H)NH 2D and (H)CNH 3D spectra, show 10-20 % higher sensitivity. For 100 % back-exchanged samples, the sensitivity in 1.9 mm rotors is superior by a factor of 1.9 in (H)NH and 1.8 in (H)CNH spectra but at lower resolution. For (H)C(C)NH experiments with a carbon-carbon mixing period, this sensitivity gain is lost due to shorter relaxation times and less efficient transfer steps. We present a detailed study on the sensitivity of these types of experiments for both types of rotors, which should enable experimentalists to make an informed decision about which type of rotor is best for specific applications.

Discovery of N-[4-(1H-Pyrazolo[3,4-b]pyrazin-6-yl)-phenyl]-sulfonamides as Highly Active and Selective SGK1 Inhibitors
Halland(*), N., Schmidt(*), F., Weiss(*), T., Saas(*), J., Li(*), Z., Czech(*), J., Dreyer(*), M., Hofmeister(*), A., Mertsch(*), K., Dietz(*), U., Strübing(*), C.; Nazare, M.
Acs Med Chem Lett, 6:73-78

Tags: Medicinal Chemistry (Nazare)

Abstract: From a virtual screening starting point, inhibitors of the serum and glucocorticoid regulated kinase 1 were developed through a combination of classical medicinal chemistry and library approaches. This resulted in highly active small molecules with nanomolar activity and a good overall in vitro and ADME profile. Furthermore, the compounds exhibited unusually high kinase and off-target selectivity due to their rigid structure.

DOTAM derivatives as active cartilage-targeting drug carriers for the treatment of osteoarthritis
Hu(*), H. Y., Lim(*), N. H., Ding-Pfennigdorff(*), D., Saas(*), J., Wendt(*), K. U., Ritzeler(*), O., Nagase(*), H., Plettenburg(*), O., Schultz(*), C.; Nazare, M.
Bioconjug Chem, 26:383-388

Tags: Medicinal Chemistry (Nazare)

Abstract: Targeted drug-delivery methods are crucial for effective treatment of degenerative joint diseases such as osteoarthritis (OA). Toward this goal, we developed a small multivalent structure as a model drug for the attenuation of cartilage degradation. The DOTAM (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid amide)-based model structure is equipped with the cathepsin D protease inhibitor pepstatin A, a fluorophore, and peptide moieties targeting collagen II. In vivo injection of these soluble probes into the knee joints of mice resulted in 7-day-long local retention, while the drug carrier equipped with a scrambled peptide sequence was washed away within 6-8 h. The model drug conjugate successfully reduced the cathepsin D protease activity as measured by release of GAG peptide. Therefore, these conjugates represent a promising first drug conjugate for the targeted treatment of degenerative joint diseases.

In vivo visualization of osteoarthritic hypertrophic lesions
Hu(*), H. Y., Lim(*), N. H., Juretschke(*), H. P., Ding-Pfennigdorff(*), D., Florian(*), P., Kohlmann(*), M., Kandira(*), A., von Kries(*), J. P., Saas(*), J., Rudolphi(*), K. A., Wendt(*), K. U., Nagase(*), H., Plettenburg(*), O., Nazare, M.; Schultz(*), C.
Chem Sci, 6:6256-6261

Tags: Medicinal Chemistry (Nazare)

Abstract: Osteoarthritis (OA) is one of the most common diseases in the aging population. While disease progress in humans is monitored indirectly by X-ray or MRI, small animal OA lesions detection always requires surgical intervention and histology. Here we introduce bimodal MR/NIR probes based on cartilage-targeting 1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid amide (DOTAM) that are directly administered to the joint cavity. We demonstrate applications in healthy and diseased rat joints by MRI in vivo. The same joints are inspected post-mortem by fluorescence microscopy, showing not only the precise location of the reagents but also revealing details such as focal cartilage damage and chondrophyte or osteophyte formation. This allows for determining the distinct pathological state of the disease and the regeneration capability of the animal model and will help to correctly assess the effect of potential disease modifying OA drugs (DMOADs) in the future.

Specific binding of Clostridium perfringens enterotoxin fragment to Claudin-b and modulation of zebrafish epidermal barrier
Zhang(*), J., Ni(*), C., Yang(*), Z., Piontek, A., Chen(*), H., Wang(*), S., Fan(*), Y., Qin(*), Z.; Piontek(*), J.
Exp Dermatol, 24:605-610

Tags: Structural Bioinformatics and Protein Design (Krause, G.)

Abstract: Claudins (Cldn) are the major components of tight junctions (TJs) sealing the paracellular cleft in tissue barriers of various organs. Zebrafish Cldnb, the homolog of mammalian Cldn4, is expressed at epithelial cell-cell contacts and is important for regulating epidermal permeability. The bacterial toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to a subset of mammalian Cldns. In this study, we used the Cldn-binding C-terminal domain of CPE (194-319 amino acids, cCPE 194-319 ) to investigate its functional role in modulating zebrafish larval epidermal barriers. In vitro analyses show that cCPE 194-319 removed Cldn4 from epithelial cells and disrupted the monolayer tightness, which could be rescued by the removal of cCPE 194-319. Incubation of zebrafish larvae with cCPE 194-319 removed Cldnb specifically from the epidermal cell membrane. Dye diffusion analysis with 4-kDa fluorescent dextran indicated that the permeability of the epidermal barrier increased due to cCPE 194-319 incubation. Electron microscopic investigation revealed reversible loss of TJ integrity by Cldnb removal. Collectively, these results suggest that cCPE 194-319 could be used as a Cldnb modulator to transiently open the epidermal barrier in zebrafish. In addition, zebrafish might be used as an in vivo system to investigate the capability of cCPE to enhance drug delivery across tissue barriers.

Covalent attachment of cyclic TAT peptides to GFP results in protein delivery into live cells with immediate bioavailability
Nischan, N., Herce(*), H. D., Natale(*), F., Bohlke(*), N., Budisa(*), N., Cardoso(*), M. C.; Hackenberger, C. P.
Angew Chem Int Ed Engl, 54:1950-1953

Tags: Chemical Biology II (Hackenberger)

Abstract: The delivery of free molecules into the cytoplasm and nucleus by using arginine-rich cell-penetrating peptides (CPPs) has been limited to small cargoes, while large cargoes such as proteins are taken up and trapped in endocytic vesicles. Based on recent work, in which we showed that the transduction efficiency of arginine-rich CPPs can be greatly enhanced by cyclization, the aim was to use cyclic CPPs to transport full-length proteins, in this study green fluorescent protein (GFP), into the cytosol of living cells. Cyclic and linear CPP-GFP conjugates were obtained by using azido-functionalized CPPs and an alkyne-functionalized GFP. Our findings reveal that the cyclic-CPP-GFP conjugates are internalized into live cells with immediate bioavailability in the cytosol and the nucleus, whereas linear CPP analogues do not confer GFP transduction. This technology expands the application of cyclic CPPs to the efficient transport of functional full-length proteins into live cells.

Stonin1 mediates endocytosis of the proteoglycan NG2 and regulates focal adhesion dynamics and cell motility
Feutlinske, F., Browarski, M., Ku(*), M. C., Trnka, P., Waiczies(*), S., Niendorf(*), T., Stallcup(*), W. B., Glass(*), R., Krause, E.; Maritzen, T.
Nat Commun, 6:8535

Tags: Membrane Traffic and Cell Motility (Maritzen), Mass Spectrometry (Krause, E.)

Abstract: Cellular functions, ranging from focal adhesion (FA) dynamics and cell motility to tumour growth, are orchestrated by signals cells receive from outside via cell surface receptors. Signalling is fine-tuned by the exo-endocytic cycling of these receptors to control cellular responses such as FA dynamics, which determine cell motility. How precisely endocytosis regulates turnover of the various cell surface receptors remains unclear. Here we identify Stonin1, an endocytic adaptor of unknown function, as a regulator of FA dynamics and cell motility, and demonstrate that it facilitates the internalization of the oncogenic proteoglycan NG2, a co-receptor of integrins and platelet-derived growth factor receptor. Embryonic fibroblasts obtained from Stonin1-deficient mice display a marked surface accumulation of NG2, increased cellular signalling and defective FA disassembly as well as altered cellular motility. These data establish Stonin1 as a specific adaptor for the endocytosis of NG2 and as an important factor for FA dynamics and cell migration.

Anchoring dipalmitoyl phosphoethanolamine to nanoparticles boosts cellular uptake and fluorine-19 magnetic resonance signal
Waiczies(*), S., Lepore(*), S., Sydow, K., Drechsler(*), S., Ku(*), M. C., Martin(*), C., Lorenz, D., Schütz, I., Reimann(*), H. M., Purfurst(*), B., Dieringer(*), M. A., Waiczies(*), H., Dathe, M., Pohlmann(*), A.; Niendorf(*), T.
Sci Rep, 5:8427

Tags: Peptide-Lipid-Interaction/ Peptide Transport (Dathe), Molecular Pharmacology and Cell Biology (Haucke), Cellular Imaging (Wiesner)

Abstract: Magnetic resonance (MR) methods to detect and quantify fluorine ((19)F) nuclei provide the opportunity to study the fate of cellular transplants in vivo. Cells are typically labeled with (19)F nanoparticles, introduced into living organisms and tracked by (19)F MR methods. Background-free imaging and quantification of cell numbers are amongst the strengths of (19)F MR-based cell tracking but challenges pertaining to signal sensitivity and cell detection exist. In this study we aimed to overcome these limitations by manipulating the aminophospholipid composition of (19)F nanoparticles in order to promote their uptake by dendritic cells (DCs). As critical components of biological membranes, phosphatidylethanolamines (PE) were studied. Both microscopy and MR spectroscopy methods revealed a striking (at least one order of magnitude) increase in cytoplasmic uptake of (19)F nanoparticles in DCs following enrichment with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). The impact of enriching (19)F nanoparticles with PE on DC migration was also investigated. By manipulating the nanoparticle composition and as a result the cellular uptake we provide here one way of boosting (19)F signal per cell in order to overcome some of the limitations related to (19)F MR signal sensitivity. The boost in signal is ultimately necessary to detect and track cells in vivo.

Different inhibition of Gbetagamma-stimulated class IB phosphoinositide 3-kinase (PI3K) variants by a monoclonal antibody. Specific function of p101 as a Gbetagamma-dependent regulator of PI3Kgamma enzymatic activity
Shymanets(*), A., Vadas(*), O., Czupalla(*), C., LoPiccolo(*), J., Brenowitz(*), M., Ghigo(*), A., Hirsch(*), E., Krause, E., Wetzker(*), R., Williams(*), R. L., Harteneck(*), C.; Nürnberg(*), B.
Biochem J, 469:59-69

Tags: Mass Spectrometry (Krause, E.)

Abstract: Class IB phosphoinositide 3-kinases gamma (PI3Kgamma) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled receptors (GPCRs). PI3Kgamma variants have one catalytic p110gamma subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110gamma and p101-p110gamma allowing integration into distinct signalling pathways. Pharmacological tools enabling distinct modulation of the two variants are missing. The ability of an anti-p110gamma monoclonal antibody [mAb(A)p110gamma] to block PI3Kgamma enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110gamma interface, hydrogen-deuterium exchange coupled to MS (HDX-MS) measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110gamma, which was accompanied by conformational changes in the helical domain harbouring the Gbetagamma-binding site. We then studied the modulation of phospholipid vesicles association of PI3Kgamma by the antibody. p87-p110gamma showed a significantly reduced Gbetagamma-mediated phospholipid recruitment as compared with p101-p110gamma. Concomitantly, in the presence of mAb(A)p110gamma, Gbetagamma did not bind to p87-p110gamma. These data correlated with the ability of the antibody to block Gbetagamma-stimulated lipid kinase activity of p87-p110gamma 30-fold more potently than p101-p110gamma. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific Gbetagamma-dependent regulation of p101 in PI3Kgamma activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3Kgamma variants downstream of GPCRs.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)

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