FMP Publications

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

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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.

Removal of albumin and immunoglobulins from canine cerebrospinal fluid using depletion kits: a feasibility study
Günther, R., Krause, E., Schümann, M., Ausseil(*), J., Heard(*), J. M., Blasig, I. E.; Haseloff, R. F.
Fluids and barriers of the CNS, 11:14

Tags: Molecular Cell Physiology (Blasig, I.E.), Mass Spectrometry (Krause, E.)

Abstract: BACKGROUND: Highly abundant proteins in biological fluids such as serum or cerebrospinal fluid (CSF) can hinder the detection of proteins in lower abundance, e.g., potential biomarkers. Commercial products are available for the depletion of albumin and immunoglobulins (Igs), although most of these kits have not been validated for dog samples. The present study therefore examines the use of different types of depletion kits for dog CSF. FINDINGS: Three kits, with different mechanisms for the depletion of albumin and Igs, were tested with dog CSF specimens. One product significantly decreased the amount of albumin; with all kits, IgG was less efficiently removed than albumin. Mass spectrometry of the fractions eluted from the depletion columns revealed considerable co-depletion of other CSF proteins. CONCLUSIONS: A commercially available depletion kit was identified which depletes albumin and (to a lower extent) immunoglobulins from dog CSF. However, the limited efficacy and the concomitant loss of other proteins from the sample should be taken into account when using this product.

Neurotransmission: spontaneous and evoked release filing for divorce
Walter, A. M., Haucke, V.; Sigrist(*), S. J.
Curr Biol, 24:R192-194

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Neurotransmitter release can be evoked by action potentials or occur spontaneously, but the relationship between those modes has been unclear. The direct visualization of release events has now shown that individual synapses display preferences for evoked versus spontaneous transmission that are determined by the release machinery at active zones.

Clathrin terminal domain-ligand interactions regulate sorting of mannose 6-phosphate receptors mediated by AP-1 and GGA adaptors
Stahlschmidt, W., Robertson(*), M. J., Robinson(*), P. J., McCluskey(*), A.; Haucke, V.
J Biol Chem, 289:4906-4918

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Clathrin plays important roles in intracellular membrane traffic including endocytosis of plasma membrane proteins and receptors and protein sorting between the trans-Golgi network (TGN) and endosomes. Whether clathrin serves additional roles in receptor recycling, degradative sorting, or constitutive secretion has remained somewhat controversial. Here we have used acute pharmacological perturbation of clathrin terminal domain (TD) function to dissect the role of clathrin in intracellular membrane traffic. We report that internalization of major histocompatibility complex I (MHCI) is inhibited in cells depleted of clathrin or its major clathrin adaptor complex 2 (AP-2), a phenotype mimicked by application of Pitstop(R) inhibitors of clathrin TD function. Hence, MHCI endocytosis occurs via a clathrin/AP-2-dependent pathway. Acute perturbation of clathrin also impairs the dynamics of intracellular clathrin/adaptor complex 1 (AP-1)- or GGA (Golgi-localized, gamma-ear-containing, Arf-binding protein)-coated structures at the TGN/endosomal interface, resulting in the peripheral dispersion of mannose 6-phosphate receptors. By contrast, secretory traffic of vesicular stomatitis virus G protein, recycling of internalized transferrin from endosomes, or degradation of EGF receptor proceeds unperturbed in cells with impaired clathrin TD function. These data indicate that clathrin is required for the function of AP-1- and GGA-coated carriers at the TGN but may be dispensable for outward traffic en route to the plasma membrane.

Synthesis of the Pitstop family of clathrin inhibitors
Robertson(*), M. J., Deane(*), F. M., Stahlschmidt, W., von Kleist, L., Haucke, V., Robinson(*), P. J.; McCluskey(*), A.
Nat Protoc, 9:1592-1606

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: This protocol describes the synthesis of two classes of clathrin inhibitors, Pitstop 1 and Pitstop 2, along with two inactive analogs that can be used as negative controls (Pitstop inactive controls, Pitnot-2 and Pitnot-2-100). Pitstop-induced inhibition of clathrin TD function acutely interferes with clathrin-mediated endocytosis (CME), synaptic vesicle recycling and cellular entry of HIV, whereas clathrin-independent internalization pathways and secretory traffic proceed unperturbed; these reagents can, therefore, be used to investigate clathrin function, and they have potential pharmacological applications. Pitstop 1 is synthesized in two steps: sulfonation of 1,8-naphthalic anhydride and subsequent reaction with 4-amino(methyl)aniline. Pitnot-1 results from the reaction of 4-amino(methyl)aniline with commercially available 4-sulfo-1,8-naphthalic anhydride potassium salt. Reaction of 1-naphthalene sulfonyl chloride with pseudothiohydantoin followed by condensation with 4-bromobenzaldehyde yields Pitstop 2. The synthesis of the inactive control commences with the condensation of 4-bromobenzaldehyde with the rhodanine core. Thioketone methylation and displacement with 1-napthylamine affords the target compound. Although Pitstop 1-series compounds are not cell permeable, they can be used in biochemical assays or be introduced into cells via microinjection. The Pitstop 2-series compounds are cell permeable. The synthesis of these compounds does not require specialist equipment and can be completed in 3-4 d. Microwave irradiation can be used to reduce the synthesis time. The synthesis of the Pitstop 2 family is easily adaptable to enable the synthesis of related compounds such as Pitstop 2-100 and Pitnot-2-100. The procedures are also simple, efficient and amenable to scale-up, enabling cost-effective in-house synthesis for users of these inhibitor classes.

A presynaptic role for the cytomatrix protein GIT in synaptic vesicle recycling
Podufall, J., Tian(*), R., Knoche(*), E., Puchkov, D., Walter, A. M., Rosa(*), S., Quentin(*), C., Vukoja, A., Jung(*), N., Lampe, A., Wichmann(*), C., Böhme(*), M., Depne(*)r, H., Zhang(*), Y. Q., Schmoranzer, J., Sigrist(*), S. J.; Haucke, V.
Cell Rep, 7:1417-1425

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Neurotransmission involves the exo-endocytic cycling of synaptic vesicles (SVs) within nerve terminals. Exocytosis is facilitated by a cytomatrix assembled at the active zone (AZ). The precise spatial and functional relationship between exocytic fusion of SVs at AZ membranes and endocytic SV retrieval is unknown. Here, we identify the scaffold G protein coupled receptor kinase 2 interacting (GIT) protein as a component of the AZ-associated cytomatrix and as a regulator of SV endocytosis. GIT1 and its D. melanogaster ortholog, dGIT, are shown to directly associate with the endocytic adaptor stonin 2/stoned B. In Drosophila dgit mutants, stoned B and synaptotagmin levels are reduced and stoned B is partially mislocalized. Moreover, dgit mutants show morphological and functional defects in SV recycling. These data establish a presynaptic role for GIT in SV recycling and suggest a connection between the AZ cytomatrix and the endocytic machinery.

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.

Imaging of doxorubicin release from theranostic macromolecular prodrugs via fluorescence resonance energy transfer
Krüger(*), H. R., Schütz, I., Justies(*), A., Licha(*), K., Welker(*), P., Haucke, V.; Calderon(*), M.
J Control Release, 194:189-196

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Herein we present a FRET-based theranostic macromolecular prodrug (TMP) composed of (a) dendritic polyglycerol (PG) as polymeric nanocarrier, (b) doxorubicin (Dox) linked via a pH-sensitive hydrazone to (c) a tri-functional linker, and (d) an indodicarbocyanine dye (IDCC) attached in close proximity to Dox. The drug fluorescence is quenched via intramolecular FRET until the pH-sensitive hydrazone bond between the TMP and Dox is cleaved at acidic pH. By measuring its fluorescence, we characterized the TMP cleavage kinetics at different pH values in vitro. The intracellular release of Dox from the carrier was monitored in real time in intact cancer cells, giving more insight into the mode of action of a polymer drug conjugate.

A novel twist in membrane dePHormation
Krauss, M.; Haucke, V.
Dev Cell, 31:3-4

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Bin-Amphiphysin-Rvs (BAR) domain-containing proteins form oligomeric assemblies that aid membrane remodeling. In this issue of Developmental Cell, Pang et al. (2014) show that the BAR domain of ACAP1, although architecturally similar to other BAR domains, cooperates with its neighboring pleckstrin homology domain to deform membranes and facilitate endosomal recycling.

Clathrin/AP-2 mediate synaptic vesicle reformation from endosome-like vacuoles but are not essential for membrane retrieval at central synapses
Kononenko, N. L., Puchkov, D., Classen, G. A., Walter, A. M., Pechstein, A., Sawade, L., Kaempf, N., Trimbuch(*), T., Lorenz, D., Rosenmund(*), C., Maritzen, T.; Haucke, V.
Neuron, 82:981-988

Tags: Molecular Pharmacology and Cell Biology (Haucke), Membrane Traffic and Cell Motility (Maritzen), Cellular Imaging (Wiesner, Puchkov)

Abstract: Neurotransmission depends on presynaptic membrane retrieval and local reformation of synaptic vesicles (SVs) at nerve terminals. The mechanisms involved in these processes are highly controversial with evidence being presented for SV membranes being retrieved exclusively via clathrin-mediated endocytosis (CME) from the plasma membrane or via ultrafast endocytosis independent of clathrin. Here we show that clathrin and its major adaptor protein 2 (AP-2) in addition to the plasma membrane operate at internal endosome-like vacuoles to regenerate SVs but are not essential for membrane retrieval. Depletion of clathrin or conditional knockout of AP-2 result in defects in SV reformation and an accumulation of endosome-like vacuoles generated by clathrin-independent endocytosis (CIE) via dynamin 1/3 and endophilin. These results together with theoretical modeling provide a conceptual framework for how synapses capitalize on clathrin-independent membrane retrieval and clathrin/AP-2-mediated SV reformation from endosome-like vacuoles to maintain excitability over a broad range of stimulation frequencies.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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