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Herbert Waldmann

Publications and source records attributed to Herbert Waldmann.

At least 55 records · Page 3Linked to original sources
PubMed2005

Protein structure similarity clustering and natural product structure as guiding principles in drug discovery.

The identification of new chemical entities that are capable of altering protein function lies at the heart of the hit and lead finding process, for which combinatorial chemistry has emerged as a powerful tool. Following the maturation of combinatorial chemistry and compound library development, it was soon recognized that biological relevance, design and diversity of a library are more important than library size. The universe of conceivable compounds is almost infinite, therefore, the decisive question arises: where is a biologically validated starting point in structural space from which to build a compound library to be found? As a new approach to address this complex problem, a synergistic strategy is presented, which is based on protein structure similarity clustering and natural product structure as guiding rationales.

Biological Factors↗
PubMed2005

Modulation of MRP-1-mediated multidrug resistance by indomethacin analogues.

Multidrug resistance (MDR) is a major limiting factor in the development and application of drug candidates. MDR caused by MRP-1 is known to be modulated by the nonsteroidal antiinflammatory drug indomethacin. We have synthesized and biologically evaluated a library of indomethacin analogues. The indomethacin-derived compound library was synthesized employing the Fischer-indole synthesis as the key transformation and making use of a "resin-capture-release" strategy. Sixty representative members of the library were evaluated in a cell biological cytotoxicity assay employing the MRP-1 expressing human glioblastoma cell line T98G as a model system. Nine of the 60 tested derivatives increased the doxorubicin-mediated cytotoxicity at a comparable or higher level than indomethacin itself. Analysis of these derivatives revealed an interesting structure-function relationship. Most remarkably, two substances increased the toxicity, when doxorubicin was used at clinically relevant low concentrations, at a higher degree than indomethacin.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2005

Chemical biology of protein lipidation: semi-synthesis and structure elucidation of prenylated RabGTPases.

Rab/Ypt guanosine triphosphatases (GTPases) represent a family of key membrane traffic regulators in eukaryotic cells. For their function Rab/Ypt proteins require double modification with two covalently bound geranylgeranyl lipid moieties at the C-terminus. Generally, prenylated proteins are very difficult to obtain by recombinant or enzymatic methods. We generated prenylated RabGTPases using a combination of chemical synthesis and protein engineering. This semi-synthesis depends largely on the availability of functionalized prenylated peptides corresponding to the proteins' native structure or modifications. We developed solution phase and solid phase strategies for the generation of peptides corresponding to the prenylated C-terminus of Rab7 GTPase in preparative amounts enabling us to crystallize the mono-prenylated Ypt1:RabGDI complex. The structure of the complex provides a structural basis for the ability of RabGDI to inhibit the release of nucleotide by Rab proteins and a molecular basis for understanding a RabGDI mutant that causes mental retardation in humans.

Models, Molecular↗
PubMed2005

An acylation cycle regulates localization and activity of palmitoylated Ras isoforms.

We show that the specific subcellular distribution of H- and Nras guanosine triphosphate-binding proteins is generated by a constitutive de/reacylation cycle that operates on palmitoylated proteins, driving their rapid exchange between the plasma membrane (PM) and the Golgi apparatus. Depalmitoylation redistributes farnesylated Ras in all membranes, followed by repalmitoylation and trapping of Ras at the Golgi, from where it is redirected to the PM via the secretory pathway. This continuous cycle prevents Ras from nonspecific residence on endomembranes, thereby maintaining the specific intracellular compartmentalization. The de/reacylation cycle also initiates Ras activation at the Golgi by transport of PM-localized Ras guanosine triphosphate. Different de/repalmitoylation kinetics account for isoform-specific activation responses to growth factors.

Acylation↗
PubMed2005

Synthesis and application of fluorescent ras proteins for live-cell imaging.

Semisynthetic Ras proteins are efficient probes for cell-biology experiments. With a Bodipy FL fluorophore introduced at an appropriate site on the Ras peptide by solid-phase synthesis, the resulting Ras chimera is processed by the cellular machinery and the intracellular localization of the protein can then be visualized by means of confocal laser fluorescence microscopy at relatively low concentrations. The absence of a large N-terminal protein tag overcomes possible interferences in the interaction with cellular partner proteins. The fluorescence emission from Bodipy FL is continuous and disappears only after irreversible bleaching. These characteristics make Ras proteins with nonprotein fluorophores suitable for biophysical analysis. The easy accessibility of the lipopeptide moiety by chemical synthesis opens up numerous options for further biological investigations.

Amino Acid Sequence↗
PubMed2005

Protein structure similarity clustering (PSSC) and natural product structure as inspiration sources for drug development and chemical genomics.

Finding small molecules that modulate protein function is of primary importance in drug development and in the emerging field of chemical genomics. To facilitate the identification of such molecules, we developed a novel strategy making use of structural conservatism found in protein domain architecture and natural product inspired compound library design. Domains and proteins identified as being structurally similar in their ligand-sensing cores are grouped in a protein structure similarity cluster (PSSC). Natural products can be considered as evolutionary pre-validated ligands for multiple proteins and therefore natural products that are known to interact with one of the PSSC member proteins are selected as guiding structures for compound library synthesis. Application of this novel strategy for compound library design provided enhanced hit rates in small compound libraries for structurally similar proteins.

Biological Factors↗
PubMed2005

A genetically encodable microtag for chemo-enzymatic derivatization and purification of recombinant proteins.

Efficient separation of recombinant polypeptides from proteins of the expression host and their subsequent derivatisation with functional chemical groups is essential for the success of many biological applications. Numerous tag systems have been developed to facilitate the purification procedure but only limited progress has been made in development of generic methods for targeted modification of proteins with functional groups. In this work, we present a novel 6 amino acid long C-terminal protein tag that can be selectively modified with functionalized derivatives of farnesyl isoprenoids by protein farnesyltransferase. The reaction could be performed in complex protein mixtures without detectable unspecific labeling. We demonstrate that this modification can be used to purify the target protein by over 800-fold in a single purification step using phase partitioning. Moreover, we show that the fluorescent group could be used to monitor the interaction of the derivatized proteins with other polypeptides.

Electrophoresis, Polyacrylamide Gel↗
PubMed2005

Application of protein semisynthesis for the construction of functionalized posttranslationally modified rab GTPases.

Rab GTPases represent a family of key membrane traffic regulators in eukaryotic cells. To exert their function, Rab proteins must be modified with one or two geranylgeranyl moieties. This modification enables them to reversibly associate with intracellular membranes. In vivo the newly synthesized Rab proteins are recruited by Rab escort protein (REP) that presents them to the Rab geranylgeranyl transferase (Rab GGTase), which transfers one or two geranylgeranyl moieties to the C-terminal cysteines. Detailed understanding of the mechanism of prenylation reaction and subsequent membrane delivery of Rab proteins to the target membranes were hampered by lack of efficient technologies for the generation of preparative amounts of prenylated Rab GTPases. To circumvent this problem, we developed an approach that combines recombinant protein production, chemical synthesis of lipidated peptides with precisely designed and readily alterable structures, and a technique for peptide-to-protein ligation. Using this approach, we generated a number of semisynthetic prenylated Rab GTPases. Some of the proteins were also supplemented with fluorophores, which enabled us to develop a fluorescence-based in vitro prenylation assay. The approach described allows production of preparative amounts of prenylated GTPases, which was demonstrated by generation and crystallization of a monoprenylated YPT1:Rab GDI complex.

Crystallization↗
PubMed2004

Synthesis of fluorescently labeled mono- and diprenylated Rab7 GTPase.

Modification of proteins with isoprenoid lipids is a widespread phenomenon in eukaryotic organisms that has received much attention due to its involvement in the progression of several diseases including cancer. Progress in studies of prenylated proteins has been hampered by difficulties associated with isolation of these proteins from native or recombinant sources. Small GTPases of the Rab family represent a particularly difficult example since they are doubly C-terminally geranylgeranylated and in some cases methylated. Here, we report an efficient and versatile strategy for the synthesis of mono- and digeranylgeranylated fluorescent RabGTPases using a combination of chemical synthesis and expressed protein ligation. Using this approach we generated fluorescent mono- and diprenylated Rab7 proteins that display near-native properties and form stoichiometric complexes with their natural chaperone REP-1. We demonstrate that the complex formed from semisynthetic monoprenylated Rab7 and REP-1 represents a genuine intermediate of the Rab prenylation reaction and thus provides a unique tool for studies of the Rab prenylation mechanism. Semisynthetic Rab7 proteins were used to develop a novel fluorescence-based in vitro prenylation assay. Using this assay we dissected the mechanism of the Rab7 double-geranylgeranylation reaction mediated by Rab geranylgeranyl transferase. We conclude that the reaction follows a random sequential mechanism. These results highlight the usefulness of the semisynthetic reaction intermediates in the study of protein posttranslational modification.

Dipeptides↗
PubMed2004

Compound library development guided by protein structure similarity clustering and natural product structure.

To identify biologically relevant and drug-like protein ligands for medicinal chemistry and chemical biology research the grouping of proteins according to evolutionary relationships and conservation of molecular recognition is an established method. We propose to employ structure similarity clustering of the ligand-sensing cores of protein domains (PSSC) in conjunction with natural product guided compound library development as a synergistic approach for the identification of biologically prevalidated ligands with high fidelity. This is supported by the concepts that (i) in nature spatial structure is more conserved than amino acid sequence, (ii) the number of fold types characteristic for all protein domains is limited, and (iii) the underlying frameworks of natural product classes with multiple biological activities provide evolutionarily selected starting points in structural space. On the basis of domain core similarity considerations and irrespective of sequence similarity, Cdc25A phosphatase, acetylcholinesterase, and 11beta-hydroxysteroid dehydrogenases type 1 and type 2 were grouped into a similarity cluster. A 147-member compound collection derived from the naturally occurring Cdc25A inhibitor dysidiolide yielded potent and selective inhibitors of the other members of the similarity cluster with a hit rate of 2-3%. Protein structure similarity clustering may provide an experimental opportunity to identify supersites in proteins.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2004

Synthesis of GTP-derived Ras ligands.

A practical and convenient method for the synthesis of acid- and base-sensitive GTP analogues carrying a further substituent at the terminal phosphate has been developed. Key to the successful synthesis of these potential ligands of the Ras protein is the use of Pd0-sensitive allyl protecting groups in a one-pot synthesis that avoids evaporation steps. Initial biochemical analysis of a representative compound revealed that such GTP analogues can bind to Ras and might open up the possibility of developing small molecules that can act as deactivators of oncogenic Ras.

Binding, Competitive↗
PubMed2004

Partitioning of dual-lipidated peptides into membrane microdomains: lipid sorting vs peptide aggregation.

The lateral membrane organization and phase behavior of the lipid mixture DMPC(di-C(14))/DSPC(di-C(18))/cholesterol (0-33 mol %) with and without an incorporated fluorescence-labeled palmitoyl/farnesyl dual-lipidated peptide, BODIPY-Gly-Cys(Pal)-Met-Gly-Leu-Pro-Cys(Far)-OMe, which represents a membrane recognition model system for Ras proteins, was studied by two-photon excitation fluorescence microscopy. Measurements were performed on giant unilamellar vesicles (GUVs) over a large temperature range, ranging from 30 to 80 degrees C to cover different lipid phase states (all-gel, fluid/gel, liquid-ordered, all-fluid). At temperatures where the fluid-gel coexistence region of the pure binary phospholipid system occurs, large-scale concentration fluctuations appear. Incorporation of cholesterol levels up to 33 mol % leads to a significant increase of conformational order in the membrane system and a reduction of large domain structures. Adding the peptide leads to dramatic changes in the lateral organization of the membrane. With cholesterol present, a phase separation is induced by a lipid sorting mechanism owing to the high affinity of the lipidated peptide to a fluid, DMPC-rich environment. This phase separation leads to the formation of peptide-containing domains with high fluorescence intensity that become progressively smaller with decreasing temperature. As a result, the local concentration of the peptide increases steadily within the confines of the shrinking domains. At the lowest temperatures, where the acyl-chain order parameter of the membrane has already drastically increased and the membrane achieves a liquid-ordered character, an efficient lipid sorting mechanism is no longer supported and aggregation of the peptide into small clusters prevails. We can conclude that palmitoyl/farnesyl dual-lipidated peptides do not associate with liquid-ordered or gel-like domains in phase-separated bilayer membranes. In particular, the study shows the interesting ability of the peptide to induce formation of fluid microdomains at physiologically relevant cholesterol concentrations, and this effect very much depends on the concentration of fluid vs ordered lipid molecules.

Cholesterol↗