FMP Publications

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

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Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC
Voss, F. K., Ullrich, F., Münch, J., Lazarow, K., Lutter, D., Mah(*), N., Andrade-Navarro(*), M. A., von Kries, J. P., Stauber, T.; Jentsch, T. J.
Science, 344:634-638

Tags: Physiology and Pathology of Ion Transport (Jentsch), Screening Unit (von Kries)

Abstract: Regulation of cell volume is critical for many cellular and organismal functions, yet the molecular identity of a key player, the volume-regulated anion channel VRAC, has remained unknown. A genome-wide small interfering RNA screen in mammalian cells identified LRRC8A as a VRAC component. LRRC8A formed heteromers with other LRRC8 multispan membrane proteins. Genomic disruption of LRRC8A ablated VRAC currents. Cells with disruption of all five LRRC8 genes required LRRC8A cotransfection with other LRRC8 isoforms to reconstitute VRAC currents. The isoform combination determined VRAC inactivation kinetics. Taurine flux and regulatory volume decrease also depended on LRRC8 proteins. Our work shows that VRAC defines a class of anion channels, suggests that VRAC is identical to the volume-sensitive organic osmolyte/anion channel VSOAC, and explains the heterogeneity of native VRAC currents.

Small-molecule inhibitors of AF6 PDZ-mediated protein-protein interactions
Vargas, C., Radziwill(*), G., Krause, G., Diehl, A., Keller, S., Kamdem, N., Czekelius(*), C., Kreuchwig, A., Schmieder, P., Doyle(*), D., Moelling(*), K., Hagen, V., Schade(*), M.; Oschkinat, H.
Chemmedchem, 9:1458-1462

Tags: NMR-Supported Structural Biology (Oschkinat), Solution NMR (Schmieder), Synthetic Organic Biochemistry (Hagen), Structural Bioinformatics and Protein Design (Krause, G.), Biophysics of Membrane Proteins (Keller), Synthetic Organic Biochemistry (Hagen)

Abstract: PDZ (PSD-95, Dlg, ZO-1) domains are ubiquitous interaction modules that are involved in many cellular signal transduction pathways. Interference with PDZ-mediated protein-protein interactions has important implications in disease-related signaling processes. For this reason, PDZ domains have gained attention as potential targets for inhibitor design and, in the long run, drug development. Herein we report the development of small molecules to probe the function of the PDZ domain from human AF6 (ALL1-fused gene from chromosome 6), which is an essential component of cell-cell junctions. These compounds bind to AF6 PDZ with substantially higher affinity than the peptide (Ile-Gln-Ser-Val-Glu-Val) derived from its natural ligand, EphB2. In intact cells, the compounds inhibit the AF6-Bcr interaction and interfere with epidermal growth factor (EGF)-dependent signaling.

Cell penetrating peptides and cationic antibacterial peptides: two sides of the same coin
Rodriguez Plaza(*), J. G., Morales-Nava(*), R., Diener(*), C., Schreiber(*), G., Gonzalez(*), Z. D., Lara Ortiz(*), M. T., Ortega Blake(*), I., Pantoja(*), O., Volkmer, R., Klipp(*), E., Herrmann(*), A.; Del Rio(*), G.
J Biol Chem, 289:14448-14457

Tags: Peptide Synthesis (Hackenberger/Volkmer)

Abstract: Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs.

Multivalent presentation of the cell-penetrating peptide nona-arginine on a linear scaffold strongly increases its membrane-perturbing capacity
Chakrabarti(*), A., Witsenburg(*), J. J., Sinzinger(*), M. D., Richter, M., Wallbrecher(*), R., Cluitmans(*), J. C., Verdurmen(*), W. P., Tanis(*), S., Adjobo-Hermans(*), M. J., Rademann, J.; Brock(*), R.
Biochim Biophys Acta, 1838:3097-3106

Tags: Medicinal Chemistry (Rademann)

Abstract: Arginine-rich cell-penetrating peptides (CPP) are widely employed as delivery vehicles for a large variety of macromolecular cargos. As a mechanism-of-action for induction of uptake cross-linking of heparan sulfates and interaction with lipid head groups have been proposed. Here, we employed a multivalent display of the CPP nona-arginine (R9) on a linear dextran scaffold to assess the impact of heparan sulfate and lipid interactions on uptake and membrane perturbation. Increased avidity through multivalency should potentiate molecular phenomena that may only play a minor role if only individual peptides are used. To this point, the impact of multivalency has only been explored for dendrimers, CPP-decorated proteins and nanoparticles. We reasoned that multivalency on a linear scaffold would more faithfully mimic the arrangement of peptides at the membrane at high local peptide concentrations. On average, five R9 were coupled to a linear dextran backbone. The conjugate displayed a direct cytoplasmic uptake similar to free R9 at concentrations higher than 10muM. However, this uptake was accompanied by an increased membrane disturbance and cellular toxicity that was independent of the presence of heparan sulfates. In contrast, for erythrocytes, the multivalent conjugate induced aggregation, however, showed only limited membrane perturbation. Overall, the results demonstrate that multivalency of R9 on a linear scaffold strongly increases the capacity to interact with the plasma membrane. However, the induction of membrane perturbation is a function of the cellular response to peptide binding.

Efficient modification of alpha-synuclein serine 129 by protein kinase CK1 requires phosphorylation of tyrosine 125 as a priming event
Kosten, J., Binolfi, A., Stuiver, M., Verzini, S., Theillet, F. X., Bekei, B., van Rossum, M.; Selenko, P.
Acs Chem Neurosci, 5:1203-1208

Tags: In-Cell NMR (Selenko)

Abstract: S129-phosphorylated alpha-synuclein (alpha-syn) is abundantly found in Lewy-body inclusions of Parkinson's disease patients. Residues neighboring S129 include the alpha-syn tyrosine phosphorylation sites Y125, Y133, and Y136. Here, we use time-resolved NMR spectroscopy to delineate atomic resolution insights into the modification behaviors of different serine and tyrosine kinases targeting these sites and show that Y125 phosphorylation constitutes a necessary priming event for the efficient modification of S129 by CK1, both in reconstituted kinase reactions and mammalian cell lysates. These results suggest that alpha-syn Y125 phosphorylation augments S129 modification under physiological in vivo conditions.

Development of 1,8-naphthalimides as clathrin inhibitors
MacGregor(*), K. A., Robertson(*), M. J., Young(*), K. A., von Kleist(*), L., Stahlschmidt, W., Whiting(*), A., Chau(*), N., Robinson(*), P. J., Haucke, V.; McCluskey(*), A.
Journal of medicinal chemistry, 57:131-143

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: We reported the first small molecule inhibitors of the interaction between the clathrin N-terminal domain (TD) and endocyctic accessory proteins (i.e., clathrin inhibition1). Initial screening of a approximately 17 000 small molecule ChemBioNet library identified 1. Screening of an existing in-house propriety library identified four substituted 1,8-napthalimides as approximately 80-120 muM clathrin inhibitors. Focused library development gave 3-sulfo-N-(4-aminobenzyl)-1,8-naphthalimide, potassium salt (18, IC50 approximately 18 muM). A second library targeting the 4-aminobenzyl moiety was developed, and four analogues displayed comparable activity (26, 27, 28, 34 with IC50 values of 22, 16, 15, and 15 muM respectively) with a further four (24, 25, 32, 33) more active than 18 with IC50 values of 10, 6.9, 12, and 10 muM, respectively. Docking studies rationalized the structure-activity relationship (SAR) with the biological data. 3-Sulfo-N-benzyl-1,8-naphthalimide, potassium salt (25) with an IC50 approximately 6.9 muM, is the most potent clathrin terminal domain-amphiphysin inhibitor reported to date.

Quadruple-resonance magic-angle spinning NMR spectroscopy of deuterated solid proteins
Akbey, Ü., Nieuwkoop, A. J., Wegner, S., Voreck, A., Kunert, B., Bandara, P., Engelke, F., Nielsen, N. C.; Oschkinat, H.
Angew Chem Int Ed Engl, 53:2438-2442

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: (1)H-detected magic-angle spinning NMR experiments facilitate structural biology of solid proteins, which requires using deuterated proteins. However, often amide protons cannot be back-exchanged sufficiently, because of a possible lack of solvent exposure. For such systems, using (2)H excitation instead of (1)H excitation can be beneficial because of the larger abundance and shorter longitudinal relaxation time, T1, of deuterium. A new structure determination approach, "quadruple-resonance NMR spectroscopy", is presented which relies on an efficient (2)H-excitation and (2)H-(13)C cross-polarization (CP) step, combined with (1)H detection. We show that by using (2)H-excited experiments better sensitivity is possible on an SH3 sample recrystallized from 30 % H2O. For a membrane protein, the ABC transporter ArtMP in native lipid bilayers, different sets of signals can be observed from different initial polarization pathways, which can be evaluated further to extract structural properties.

Reporter assay for endo/lysosomal escape of toxin-based therapeutics
Gilabert-Oriol(*), R., Thakur(*), M., von Mallinckrodt(*), B., Bhargava(*), C., Wiesner, B., Eichhorst, J., Melzig(*), M. F., Fuchs(*), H.; Weng(*), A.
Toxins (Basel), 6:1644-1666

Tags: Cellular Imaging (Wiesner)

Abstract: Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters-horseradish peroxidase (HRP), Alexa Fluor 488 (Alexa) and ricin A-chain (RTA)-were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates-saporin-HRP, (Alexa)saporin and saporin-KQ-RTA-were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of (Alexa)saporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10-1000 nM.

Disrupting MLC1 and GlialCAM and ClC-2 interactions in leukodystrophy entails glial chloride channel dysfunction
Hoegg-Beiler, M. B., Sirisi(*), S., Orozco, I. J., Ferrer(*), I., Hohensee, S., Auberson, M., Gödde, K., Vilches(*), C., de Heredia(*), M. L., Nunes(*), V., Estevez(*), R.; Jentsch, T. J.
Nat Commun, 5:3475

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Defects in the astrocytic membrane protein MLC1, the adhesion molecule GlialCAM or the chloride channel ClC-2 underlie human leukoencephalopathies. Whereas GlialCAM binds ClC-2 and MLC1, and modifies ClC-2 currents in vitro, no functional connections between MLC1 and ClC-2 are known. Here we investigate this by generating loss-of-function Glialcam and Mlc1 mouse models manifesting myelin vacuolization. We find that ClC-2 is unnecessary for MLC1 and GlialCAM localization in brain, whereas GlialCAM is important for targeting MLC1 and ClC-2 to specialized glial domains in vivo and for modifying ClC-2's biophysical properties specifically in oligodendrocytes (OLs), the cells chiefly affected by vacuolization. Unexpectedly, MLC1 is crucial for proper localization of GlialCAM and ClC-2, and for changing ClC-2 currents. Our data unmask an unforeseen functional relationship between MLC1 and ClC-2 in vivo, which is probably mediated by GlialCAM, and suggest that ClC-2 participates in the pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts.

Design of a General-Purpose European Compound Screening Library for EU-OPENSCREEN
Horvath(*), D., Lisurek, M., Rupp, B., Kühne, R., Specker, E., von Kries, J., Rognan(*), D., Andersson(*), C. D., Almqvist(*), F., Elofsson(*), M., Enqvist(*), P. A., Gustavsson(*), A. L., Remez(*), N., Mestres(*), J., Marcou(*), G., Varnek(*), A., Hibert(*), M., Quintana(*), J.; Frank, R.
Chemmedchem, 9:2309-2326

Tags: Chemical Systems Biology (Frank), Screening Unit (von Kries), Computational Chemistry and Protein Design (Kühne)

Abstract: This work describes a collaborative effort to define and apply a protocol for the rational selection of a general-purpose screening library, to be used by the screening platforms affiliated with the EU-OPENSCREEN initiative. It is designed as a standard source of compounds for primary screening against novel biological targets, at the request of research partners. Given the general nature of the potential applications of this compound collection, the focus of the selection strategy lies on ensuring chemical stability, absence of reactive compounds, screening-compliant physicochemical properties, loose compliance to drug-likeness criteria (as drug design is a major, but not exclusive application), and maximal diversity/coverage of chemical space, aimed at providing hits for a wide spectrum of drugable targets. Finally, practical availability/cost issues cannot be avoided. The main goal of this publication is to inform potential future users of this library about its conception, sources, and characteristics. The outline of the selection procedure, notably of the filtering rules designed by a large committee of European medicinal chemists and chemoinformaticians, may be of general methodological interest for the screening/medicinal chemistry community. The selection task of 200K molecules out of a pre-filtered set of 1.4M candidates was shared by five independent European research groups, each picking a subset of 40K compounds according to their own in-house methodology and expertise. An in-depth analysis of chemical space coverage of the library serves not only to characterize the collection, but also to compare the various chemoinformatics-driven selection procedures of maximal diversity sets. Compound selections contributed by various participating groups were mapped onto general-purpose self-organizing maps (SOMs) built on the basis of marketed drugs and bioactive reference molecules. In this way, the occupancy of chemical space by the EU-OPENSCREEN library could be directly compared with distributions of known bioactives of various classes. This mapping highlights the relevance of the selection and shows how the consensus reached by merging the five different 40K selections contributes to achieve this relevance. The approach also allows one to readily identify subsets of target-or target-class-oriented compounds from the EU-OPENSCREEN library to suit the needs of the diverse range of potential users. The final EU-OPENSCREEN library, assembled by merging five independent selections of 40K compounds from various expert groups, represents an excellent example of a Europe-wide collaborative effort toward the common objective of building best-in-class European open screening platforms.

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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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