FMP Publications

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

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Hydrogen bonding involving side chain exchangeable groups stabilizes amyloid quarternary structure
Agarwal, V., Linser, R., Dasari, M., Fink, U., del Amo, J. M.; Reif, B.
Phys Chem Chem Phys, 15:12551-12557

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: The amyloid beta-peptide (Abeta) is the major structural component of amyloid fibrils in the plaques of brains of Alzheimer's disease patients. Numerous studies have addressed important aspects of secondary and tertiary structure of fibrils. In electron microscopic images, fibrils often bundle together. The mechanisms which drive the association of protofilaments into bundles of fibrils are not known. We show here that amino acid side chain exchangeable groups like e.g. histidines can provide useful restraints to determine the quarternary assembly of an amyloid fibril. Exchangeable protons are only observable if a side chain hydrogen bond is formed and the respective protons are protected from exchange. The method relies on deuteration of the Abeta peptide. Exchangeable deuterons are substituted with protons, before fibril formation is initiated.

Cryogenic solid state NMR studies of fibrils of the Alzheimer's disease amyloid-beta peptide: perspectives for DNP
del Amo, J. M. L., Schneider(*), D., Loquet(*), A., Lange(*), A.; Reif, B.
J. Biomol. NMR, 56:359-363

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: Dynamic Nuclear Polarization solid-state NMR holds the potential to enable a dramatic increase in sensitivity by exploiting the large magnetic moment of the electron. However, applications to biological solids are hampered in uniformly isotopically enriched biomacromolecules due to line broadening which yields a limited spectral resolution at cryogenic temperatures. We show here that high magnetic fields allow to overcome the broadening of resonance lines often experienced at liquid nitrogen temperatures. For a fibril sample of the Alzheimer's disease beta-amyloid peptide, we find similar line widths at low temperature and at room temperature. The presented results open new perspectives for structural investigations in the solid-state.

The mechanism of denaturation and the unfolded state of the alpha-helical membrane-associated protein Mistic
Jacso, T., Bardiaux, B., Broecker(*), J., Fiedler(*), S., Baerwinkel(*), T., Mainz, A., Fink, U., Vargas(*), C., Oschkinat, H., Keller(*), S.; Reif, B.
J Am Chem Soc, 135:18884-18891

Tags: Solid-State NMR Spectroscopy (Reif), NMR-Supported Structure Biology (Oschkinat)

Abstract: In vitro protein-folding studies using chemical denaturants such as urea are indispensible in elucidating the forces and mechanisms determining the stability, structure, and dynamics of water-soluble proteins. By contrast, alpha-helical membrane-associated proteins largely evade such approaches because they are resilient to extensive unfolding. We have used optical and NMR spectroscopy to provide an atomistic-level dissection of the effects of urea on the structure and dynamics of the alpha-helical membrane-associated protein Mistic as well as its interactions with detergent and solvent molecules. In the presence of the zwitterionic detergent lauryl dimethylamine oxide, increasing concentrations of urea result in a complex sequence of conformational changes that go beyond simple two-state unfolding. Exploiting this finding, we report the first high-resolution structural models of the urea denaturation process of an alpha-helical membrane-associated protein and its completely unfolded state, which contains almost no regular secondary structure but nevertheless retains a topology close to that of the folded state.

NMR Spectroscopy of Soluble Protein Complexes at One Mega-Dalton and Beyond
Mainz, A., Religa, T. L., Sprangers, R., Linser, R., Kay, L. E.; Reif, B.
Angew Chem Int Edit, 52:8746-8751

Tags: Solid-State NMR Spectroscopy (Reif)

Alzheimer's Disease: Identification and Development of -Secretase (BACE-1) Binding Fragments and Inhibitors by Dynamic Ligation Screening (DLS)
Fernandez-Bachiller, M. I., Horatscheck, A., Lisurek, M.; Rademann, J.
Chemmedchem, 8:1041-1056

Tags: Medicinal Chemistry (Rademann)

Abstract: The application of dynamic ligation screening (DLS), a methodology for fragment-based drug discovery (FBDD), to the aspartic protease -secretase (BACE-1) is reported. For this purpose, three new fluorescence resonance energy transfer (FRET) substrates were designed and synthesized. Their kinetic parameters (Vmax, KM, and kcat) were determined and compared with a commercial substrate. Secondly, a peptide aldehyde was designed as a chemically reactive inhibitor (CRI) based on the Swedish mutation substrate sequence. Incubation of this CRI with the protease, a FRET substrate, and one amine per well taken from an amine library, which was assembled by a maximum common substructure (MCS) approach, revealed the fragment 3-(3-aminophenyl)-2H-chromen-2-one (1) to be a competitive BACE-1 inhibitor that enhanced the activity of the CRI. Irreversibly formed fragment combination products of 1 with the initial peptide sequence were active and confirmed the targeting of the active site through the ethane-1,2-diamine isostere. Finally, structure-assisted combination of fragment 1 with secondary fragments that target the S1 site in hit optimization yielded novel, entirely fragment-based BACE-1 inhibitors with up to 30-fold improved binding affinity. Interactions with the protein were explained by molecular modeling studies, which indicate that the new fragment combinations interact with the catalytic aspartic acid dyad, as well as with the adjacent binding sites required for potency.

Highly functionalized terpyridines as competitive inhibitors of AKAP-PKA interactions
Schäfer(*), G., Milic(*), J., Eldahshan, A., Götz(*), F., Zühlke(*), K., Schillinger, C., Kreuchwig, A., Elkins(*), J. M., Abdul Azeez(*), K. R., Oder(*), A., Moutty(*), M. C., Masada(*), N., Beerbaum, M., Schlegel, B., Niquet(*), S., Schmieder, P., Krause, G., von Kries, J. P., Cooper(*), D. M., Knapp(*), S., Rademann, J., Rosenthal(*), W.; Klussmann(*), E.
Angew Chem Int Ed Engl, 52:12187-12191

Tags: Medicinal Chemistry (Rademann), Screening Unit (von Kries), Solution NMR (Schmieder)

What Goes around Comes around-A Comparative Study of the Influence of Chemical Modifications on the Antimicrobial Properties of Small Cyclic Peptides
Scheinpflug, K., Nikolenko, H., Komarov(*), I. V., Rautenbach(*), M.; Dathe, M.
Pharmaceuticals (Basel), 6:1130-1144

Tags: Peptide-Lipid-Interaction/ Peptide Transport (Dathe)

Abstract: Tryptophan and arginine-rich cyclic hexapeptides of the type cyclo-RRRWFW combine high antibacterial activity with rapid cell killing kinetics, but show low toxicity in human cell lines. The peptides fulfil the structural requirements for membrane interaction such as high amphipathicity and cationic charge, but membrane permeabilisation, which is the most common mode of action of antimicrobial peptides (AMPs), could not be observed. Our current studies focus on elucidating a putative membrane translocation mechanism whereupon the peptides might interfere with intracellular processes. These investigations require particular analytical tools: fluorescent analogues and peptides bearing appropriate reactive groups were synthesized and characterized in order to be used in confocal laser scanning microscopy and HPLC analysis. We found that minimal changes in both the cationic and hydrophobic domain of the peptides in most cases led to significant reduction of antimicrobial activity and/or changes in the mode of action. However, we were able to identify two modified peptides which exhibited properties similar to those of the cyclic parent hexapeptide and are suitable for subsequent studies on membrane translocation and uptake into bacterial cells.

Small-molecule screening identifies modulators of aquaporin-2 trafficking
Bogum, J., Faust(*), D., Zühlke, K., Eichhorst, J., Moutty, M. C., Furkert, J., Eldahshan(*), A., Neuenschwander, M., von Kries, J. P., Wiesner, B., Trimpert(*), C., Deen(*), P. M., Valenti(*), G., Rosenthal(*), W.; Klussmann(*), E.
Journal of the American Society of Nephrology : JASN, 24:744-758

Tags: Cellular Imaging (Wiesner), Screening Unit (von Kries), Anchored Signaling (Klussmann)

Abstract: In the principal cells of the renal collecting duct, arginine vasopressin (AVP) stimulates the synthesis of cAMP, leading to signaling events that culminate in the phosphorylation of aquaporin-2 water channels and their redistribution from intracellular domains to the plasma membrane via vesicular trafficking. The molecular mechanisms that control aquaporin-2 trafficking and the consequent water reabsorption, however, are not completely understood. Here, we used a cell-based assay and automated immunofluorescence microscopy to screen 17,700 small molecules for inhibitors of the cAMP-dependent redistribution of aquaporin-2. This approach identified 17 inhibitors, including 4-acetyldiphyllin, a selective blocker of vacuolar H(+)-ATPase that increases the pH of intracellular vesicles and causes accumulation of aquaporin-2 in the Golgi compartment. Although 4-acetyldiphyllin did not inhibit forskolin-induced increases in cAMP formation and downstream activation of protein kinase A (PKA), it did prevent cAMP/PKA-dependent phosphorylation at serine 256 of aquaporin-2, which triggers the redistribution to the plasma membrane. It did not, however, prevent cAMP-induced changes to the phosphorylation status at serines 261 or 269. Last, we identified the fungicide fluconazole as an inhibitor of cAMP-mediated redistribution of aquaporin-2, but its target in this pathway remains unknown. In conclusion, our screening approach provides a method to begin dissecting molecular mechanisms underlying AVP-mediated water reabsorption, evidenced by our identification of 4-acetyldiphyllin as a modulator of aquaporin-2 trafficking.

Culturing Primary Rat Inner Medullary Collecting Duct Cells
Faust(*), D., Geelhaar(*), A., Eisermann(*), B., Eichhorst, J., Wiesner, B., Rosenthal(*), W.; Klussmann(*), E.
Jove-J Vis Exp,

Tags: Cellular Imaging (Wiesner)

Abstract: Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion. Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Different intra- and intermolecular activation mechanisms at the human lutropin receptor: Lutropin induces only cis- and choriogonadotropin also trans-activation
Grzesik, P., Teichmann, A., Furkert, J., Rutz, C., Wiesner, B., Kleinau(*), G., Schülein, R., Gromoll(*), J.; Krause, G.
Exp Clin Endocr Diab, 121

Tags: Structural Bioinformatics and Protein Design (Krause, G.), Cellular Imaging (Wiesner), Protein Trafficking (Schülein)

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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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