Leibnizinstitut For Molekulare Pharmakologie Fmp

Berlin, Germany

Leibnizinstitut For Molekulare Pharmakologie Fmp

Berlin, Germany

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Gilabert-Oriol R.,Charité - Medical University of Berlin | Thakur M.,Charité - Medical University of Berlin | von Mallinckrodt B.,Charité - Medical University of Berlin | Bhargava C.,Charité - Medical University of Berlin | And 6 more authors.
Toxins | Year: 2014

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, Alexasaporin 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 Alexasaporin 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. © 2014 by the authors; licensee MDPI, Basel, Switzerland.


Gilabert-Oriol R.,Charité - Medical University of Berlin | Thakur M.,Charité - Medical University of Berlin | Von Mallinckrodt B.,Charité - Medical University of Berlin | Hug T.,Charité - Medical University of Berlin | And 5 more authors.
Molecular Pharmaceutics | Year: 2013

Monoclonal antibody-based therapy is one of the most successful strategies for treatment of cancer. However, the insufficient cell killing activity of monoclonal antibodies limits their therapeutic potential. These limitations can be overcome by the application of immunotoxins, which consist of a monoclonal antibody that specifically delivers a toxin into the cancer cell. An ideal immunotoxin combines the functionality of the monoclonal antibody (antagonistic binding to targeted receptors and interaction with the innate immune system) with the cell-killing activity of the toxic moiety. In addition, it should be sensitive for certain triterpenoid saponins that are known to lead to a tremendous augmentation of the antitumoral efficacy of the immunotoxin. In this study, the monoclonal antibodies trastuzumab (Herceptin) and cetuximab (Erbitux) were conjugated via cleavable disulfide bonds to the plant derived toxin saporin. The ability of the modified tumor-specific therapeutic antibodies to deliver their toxic payload into the target cells was investigated by impedance-based real-time viability assays and confocal live cell imaging. We further provide evidence that the immunotoxins retained their ability to trigger antibody-dependent cell-mediated cytotoxicity. They specifically bound to their target cell receptor, and their cell-killing activity was drastically augmented in the presence of triterpenoid saponins. Further mechanistic studies indicated a specific saponin-mediated endo/lysosomal release of the toxin moiety. These results open a promising avenue to overcome the present limitations of therapeutic antibodies and to achieve a higher antitumoral efficacy in cancer therapy. © 2013 American Chemical Society.


Lopez Del Amo J.M.,Helmholtz Center Munich | Lopez Del Amo J.M.,TU Munich | Schmidt M.,Martin Luther University of Halle Wittenberg | Fink U.,Leibnizinstitut For Molekulare Pharmakologie Fmp | And 5 more authors.
Angewandte Chemie - International Edition | Year: 2012

Two-faced culprit: Fibrils of recombinantly produced amyloid β peptides (Aβs; residues 1-40) gave well-resolved solid-state NMR spectra. Two sets of resonances corresponding to residues 12-40 and 21-38 of the Aβ primary sequence were observed (see picture). Statistical analysis of electron microscopy data revealed that it was composed of a single Aβ polymorph, thus indicating that this Aβ fibril is composed of an asymmetric dimer. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Chevelkov V.,Max Planck Institute for Chemistry | Chevelkov V.,Leibnizinstitut For Molekulare Pharmakologie Fmp | Xiang S.,Max Planck Institute for Chemistry | Giller K.,Max Planck Institute for Chemistry | And 6 more authors.
Journal of Biomolecular NMR | Year: 2015

In this work, we show how the water flip-back approach that is widely employed in solution-state NMR can be adapted to proton-detected MAS solid-state NMR of highly deuterated proteins. The scheme allows to enhance the sensitivity of the experiment by decreasing the recovery time of the proton longitudinal magnetization. The method relies on polarization transfer from non-saturated water to the protein during the inter-scan delay. © 2015 European Union.


Fiebig J.E.,University of Würzburg | Weidauer S.E.,University of Würzburg | Qiu L.-Y.,University of Würzburg | Bauer M.,University of Würzburg | And 6 more authors.
Molecules | Year: 2013

Bone Morphogenetic Proteins (BMPs) are secreted protein hormones that act as morphogens and exert essential roles during embryonic development of tissues and organs. Signaling by BMPs occurs via hetero-oligomerization of two types of serine/threonine kinase transmembrane receptors. Due to the small number of available receptors for a large number of BMP ligands ligand-receptor promiscuity presents an evident problem requiring additional regulatory mechanisms for ligand-specific signaling. Such additional regulation is achieved through a plethora of extracellular antagonists, among them members of the Chordin superfamily, that modulate BMP signaling activity by binding. The key-element in Chordin-related antagonists for interacting with BMPs is the von Willebrand type C (VWC) module, which is a small domain of about 50 to 60 residues occurring in many different proteins. Although a structure of the VWC domain of the Chordin-member Crossveinless 2 (CV2) bound to BMP-2 has been determined by X-ray crystallography, the molecular mechanism by which the VWC domain binds BMPs has remained unclear. Here we present the NMR structure of the Danio rerio CV2 VWC1 domain in its unbound state showing that the key features for high affinity binding to BMP-2 is a pre-oriented peptide loop. © 1996-2013 MDPI AG.

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