Sondersammelgebiets-Volltexte
Refine
Year of publication
Document Type
- Article (119)
- Conference Proceeding (3)
Has Fulltext
- yes (122) (remove)
Is part of the Bibliography
- no (122)
Keywords
- rna (6)
- base pairing (3)
- IAPs (2)
- NMR spectroscopy (2)
- XIAP (2)
- adenine (2)
- helix (snails) (2)
- 3D EM (1)
- BPTI (1)
- Biophysical chemistry (1)
Institute
- Biochemie und Chemie (122) (remove)
We present a computational method for the reaction-based de novo design of drug-like molecules. The software DOGS (Design of Genuine Structures) features a ligand-based strategy for automated ‘in silico’ assembly of potentially novel bioactive compounds. The quality of the designed compounds is assessed by a graph kernel method measuring their similarity to known bioactive reference ligands in terms of structural and pharmacophoric features. We implemented a deterministic compound construction procedure that explicitly considers compound synthesizability, based on a compilation of 25'144 readily available synthetic building blocks and 58 established reaction principles. This enables the software to suggest a synthesis route for each designed compound. Two prospective case studies are presented together with details on the algorithm and its implementation. De novo designed ligand candidates for the human histamine H4 receptor and γ-secretase were synthesized as suggested by the software. The computational approach proved to be suitable for scaffold-hopping from known ligands to novel chemotypes, and for generating bioactive molecules with drug-like properties.
Ubiquitin ligases and beyond
(2012)
First paragraph (this article has no abstract): In a review published in 2004 [1] and that still repays reading today, Cecile Pickart traced the evolution of research on ubiquitination from its origins in the proteasomal degradation of proteins through the revelation that it has a central role in cell cycle regulation and the recognition of regulatory roles for ubiquitin in intracellular membrane transport, cell signalling, transcription, translation, and DNA repair.
Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway.
Bacterial porin disrupts mitochondrial membrane potential and sensitizes host cells to apoptosis
(2009)
The bacterial PorB porin, an ATP-binding beta-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (delta psi m). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of beta-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of delta psi m. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce delta psi m loss and apoptosis, demonstrating that dissipation of delta psi m is a requirement for cell death caused by neisserial infection.
Background: Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available. Methodology/Findings: Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high-throughput microscopy platform (Z'factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamideand 2-benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor. Conclusions: The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance.
Background: ClC-7 is a ubiquitous transporter which is broadly expressed in mammalian tissues. It is implied in the pathogenesis of lysosomal storage disease and osteopetrosis. Because of its endosomal/lysosomal localization it is still poorly characterized. Methodology/Principal Findings: An electrophysiological characterization of rat ClC-7 using solid-supported membrane-based electrophysiology is presented. The measured currents show the characteristics of ClC-7 and confirm its function as a Cl−/H+-antiporter. We have used rat ClC-7 in CHO cells as a model system to investigate the functionality and cellular localization of the wt transporter and its variant G213R ClC-7 which is the analogue of human G215R ClC-7 responsible for autosomal dominant osteopetrosis type II. Our study shows that rat G213R ClC-7 is functional but has a localization defect in CHO cells which prevents it from being correctly targeted to the lysosomal membrane. The electrophysiological assay is tested as a tool for drug discovery. The assay is validated with a number of drug candidates. It is shown that ClC-7 is inhibited by DIDS, NPPB and NS5818 at micromolar concentrations. Conclusions/Significance: It is suggested that the scenario found in the CHO model system also applies to the human transporter and that mislocalization rather than impaired functionality of G215R ClC-7 is the primary cause of the related autosomal dominant osteopetrosis type II. Furthermore, the robust solid-supported membrane-based electrophysiological assay is proposed for rapid screening for potential ClC-7 inhibitors which are discussed for treatment of osteoporosis.
Reciprocal t(9;22) ABL/BCR fusion proteins: leukemogenic potential and effects on B cell commitment
(2009)
Background: t(9;22) is a balanced translocation, and the chromosome 22 breakpoints (Philadelphia chromosome – Ph+) determine formation of different fusion genes that are associated with either Ph+ acute lymphatic leukemia (Ph+ ALL) or chronic myeloid leukemia (CML). The "minor" breakpoint in Ph+ ALL encodes p185BCR/ABL from der22 and p96ABL/BCR from der9. The "major" breakpoint in CML encodes p210BCR/ABL and p40ABL/BCR. Herein, we investigated the leukemogenic potential of the der9-associated p96ABL/BCR and p40ABL/BCR fusion proteins and their roles in the lineage commitment of hematopoietic stem cells in comparison to BCR/ABL. Methodology: All t(9;22) derived proteins were retrovirally expressed in murine hematopoietic stem cells (SL cells) and human umbilical cord blood cells (UCBC). Stem cell potential was determined by replating efficiency, colony forming - spleen and competitive repopulating assays. The leukemic potential of the ABL/BCR fusion proteins was assessed by in a transduction/transplantation model. Effects on the lineage commitment and differentiation were investigated by culturing the cells under conditions driving either myeloid or lymphoid commitment. Expression of key factors of the B-cell differentiation and components of the preB-cell receptor were determined by qRT-PCR. Principal Findings: Both p96ABL/BCR and p40ABL/BCR increased proliferation of early progenitors and the short term stem cell capacity of SL-cells and exhibited own leukemogenic potential. Interestingly, BCR/ABL gave origin exclusively to a myeloid phenotype independently from the culture conditions whereas p96ABL/BCR and to a minor extent p40ABL/BCR forced the B-cell commitment of SL-cells and UCBC. Conclusions/Significance: Our here presented data establish the reciprocal ABL/BCR fusion proteins as second oncogenes encoded by the t(9;22) in addition to BCR/ABL and suggest that ABL/BCR contribute to the determination of the leukemic phenotype through their influence on the lineage commitment.
Background: The human pathogen Helicobacter pylori (H. pylori) is a main cause for gastric inflammation and cancer. Increasing bacterial resistance against antibiotics demands for innovative strategies for therapeutic intervention. Methodology/Principal Findings: We present a method for structure-based virtual screening that is based on the comprehensive prediction of ligand binding sites on a protein model and automated construction of a ligand-receptor interaction map. Pharmacophoric features of the map are clustered and transformed in a correlation vector (‘virtual ligand’) for rapid virtual screening of compound databases. This computer-based technique was validated for 18 different targets of pharmaceutical interest in a retrospective screening experiment. Prospective screening for inhibitory agents was performed for the protease HtrA from the human pathogen H. pylori using a homology model of the target protein. Among 22 tested compounds six block E-cadherin cleavage by HtrA in vitro and result in reduced scattering and wound healing of gastric epithelial cells, thereby preventing bacterial infiltration of the epithelium. Conclusions/Significance: This study demonstrates that receptor-based virtual screening with a permissive (‘fuzzy’) pharmacophore model can help identify small bioactive agents for combating bacterial infection.
Chlamydia are obligate intracellular bacteria that cause variety of human diseases. Host cells infected with Chlamydia are protected against many different apoptotic stimuli. The induction of apoptosis resistance is thought to be an important immune escape mechanism allowing Chlamydia to replicate inside the host cell. Infection with C. trachomatis activates the Raf/MEK/ERK pathway and the PI3K/AKT pathway. Here we show that inhibition of these two pathways by chemical inhibitors sensitized C. trachomatis infected cells to granzyme B-mediated cell death. Infection leads to the Raf/MEK/ERK-mediated up-regulation and PI3K-dependent stabilization of the anti-apoptotic Bcl-2 family member Mcl-1. Consistently, interfering with Mcl-1 up-regulation sensitized infected cells for apoptosis induced via the TNF receptor, DNA damage, granzyme B and stress. Our data suggest that Mcl-1 up-regulation is primarily required to maintain apoptosis resistance in C. trachomatis-infected cells.