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Gerd Folkers

Publications and source records attributed to Gerd Folkers.

35 records · Page 2Linked to original sources
PubMed2004

Synthesis, 18F-radiolabelling and biological evaluations of C-6 alkylated pyrimidine nucleoside analogues.

Synthesis of pyrimidine derivatives with a side-chain attached to the C-6 of pyrimidine ring (6-14) is reported. Target compounds 8 and 12 were subjected to in vitro phosphorylation tests, determination of their binding affinities to herpes simplex virus (HSV-1) thymidine kinase (TK) and catalytic turnover constants. Fluorinated pyrimidine derivative 12 (40 microM) exhibited better binding affinity for HSV-1 TK than acyclovir (ACV, 170 microM) and ganciclovir (GCV, 48 microM). Catalytic turnover constant (k(cat)) of 12 (0.08 s(-1)) was close to the k(cat) values of ACV (0.10 s(-1)) and GCV (0.10 s(-1)). Furthermore, compounds 8 and 12 showed no cytotoxic effects in HSV-1 TK-transduced and non-transduced cell lines. Besides, compounds 8 and 12 did not exhibit antiviral or cytostatic activities against several viruses and malignant tumor cell lines that were evaluated. The new fluorinated pyrimidine derivative 16 that is phosphorylated by HSV-1 TK could be developed as non-toxic PET-tracer molecule. Thus, 18F labelling of the precursor 14 was performed by nucleophilic substitution using [18F] tetrabutylammonium fluoride as the fluorinating reagent.

Alkylation↗
PubMed2004

Thermodynamics of protein-ligand interactions: history, presence, and future aspects.

The understanding of molecular recognition processes of small ligands and biological macromolecules requires a complete characterization of the binding energetics and correlation of thermodynamic data with interacting structures involved. A quantitative description of the forces that govern molecular associations requires determination of changes of all thermodynamic parameters, including free energy of binding (deltaG), enthalpy (deltaH), and entropy (deltaS) of binding and the heat capacity change (deltaCp). A close insight into the binding process is of significant and practical interest, since it provides the fundamental know-how for development of structure-based molecular design-strategies. The only direct method to measure the heat change during complex formation at constant temperature is provided by isothermal titration calorimetry (ITC). With this method one binding partner is titrated into a solution containing the interaction partner, thereby generating or absorbing heat. This heat is the direct observable that can be quantified by the calorimeter. The use of ITC has been limited due to the lack of sensitivity, but recent developments in instrument design permit to measure heat effects generated by nanomol (typically 10-100) amounts of reactants. ITC has emerged as the primary tool for characterizing interactions in terms of thermodynamic parameters. Because heat changes occur in almost all chemical and biochemical processes, ITC can be used for numerous applications, e.g., binding studies of antibody-antigen, protein-peptide, protein-protein, enzyme-inhibitor or enzyme-substrate, carbohydrate-protein, DNA-protein (and many more) interactions as well as enzyme kinetics. Under appropriate conditions data analysis from a single experiment yields deltaH, K(B), the stoichiometry (n), deltaG and deltaS of binding. Moreover, ITC experiments performed at different temperatures yield the heat capacity change (deltaCp). The informational content of thermodynamic data is large, and it has been shown that it plays an important role in the elucidation of binding mechanisms and, through the link to structural data, also in rational drug design. In this review we will present a comprehensive overview to ITC by giving some historical background to calorimetry, outline some critical experimental and data analysis aspects, discuss the latest developments, and give three recent examples of studies published with respect to macromolecule-ligand interactions that have utilized ITC technology.

Animals↗
PubMed2004

A molecular dynamics study of reovirus attachment protein sigma1 reveals conformational changes in sigma1 structure.

Molecular dynamics simulations were performed using the recently determined crystal structure of the reovirus attachment protein, sigma1. These studies were conducted to improve an understanding of two unique features of sigma1 structure: the protonation state of Asp(345), which is buried in the sigma1 trimer interface, and the flexibility of the protein at a defined region below the receptor-binding head domain. Three copies of aspartic acids Asp(345) and Asp(346) cluster in a solvent-inaccessible and hydrophobic region at the sigma1 trimer interface. These residues are hypothesized to mediate conformational changes in sigma1 during viral attachment or cell entry. Our results indicate that protonation of Asp(345) is essential to the integrity of the trimeric structure seen by x-ray crystallography, whereas deprotonation induces structural changes that destabilize the trimer interface. This finding was confirmed by electrostatic calculations using the finite difference Poisson-Boltzmann method. Earlier studies show that sigma1 can exist in retracted and extended conformations on the viral surface. Since protonated Asp(345) is necessary to form a stable, extended trimer, our results suggest that protonation of Asp(345) may allow for a structural transition from a partially detrimerized molecule to the fully formed trimer seen in the crystal structure. Additional studies were conducted to quantify the previously observed flexibility of sigma1 at a defined region below the receptor-binding head domain. Increased mobility was observed for three polar residues (Ser(291), Thr(292), and Ser(293)) located within an insertion between the second and third beta-spiral repeats of the crystallized portion of the sigma1 tail. These amino acids interact with water molecules of the solvent bulk and are responsible for oscillating movement of the head of approximately 50 degrees during 5 ns of simulations. This flexibility may facilitate viral attachment and also function in cell entry and disassembly. These findings provide new insights about the conformational dynamics of sigma1 that likely underlie the initiation of the reovirus infectious cycle.

Amino Acids↗
PubMed2003

Synthesis and biological evaluation of iodinated and fluorinated 9-(2-hydroxypropyl) and 9-(2-hydroxyethoxy)methyl purine nucleoside analogues.

The novel fluorinated and iodinated purine derivatives containing 9-(2-hydroxypropyl) (1a-7a and 9a-13a) and 9-(2-hydroxyethoxymethyl) (1b-3b, 5b, and 7b-12c) side chains were synthesized by a multistep synthetic route involving Baltz-Schiemann's fluorination and diazotation/iodination as key reactions. An unequivocal proof for the stereostructure of 5b was obtained by X-ray structure analysis. New compounds were evaluated for their cytostatic activity against murine leukemia (L1210); mammary carcinoma (FM3A); and human T-lymphocytes (Molt4/C8 and CEM), melanoma (HBL), cervical carcinoma (HeLa), colon carcinoma (HT29 and SW620), laryngeal carcinoma (Hep2), and pancreatic carcinoma (MiaPaCa2) as well as diploid fibroblasts (WI38). Of all the compounds, the 2-aminopurin-6-thione derivative 9a showed the most pronounced inhibitory activity against human SW620 cells. The 2-aminopurin-6-thione derivative 9b exhibited the most selective inhibitory activity against human HeLa, Hep2, SW620, and murine L1210 cell proliferation as compared to normal fibroblast (WI38) cell proliferation. None of the compounds showed inhibitory activities against HIV-1, HIV-2, HSV-1, and HSV-2, vaccinia, vesicular stomatitis, parainfluenza-3, reovirus-1, Sindbis, Coxsackie B4, or respiratory syncytial virus. The new purine derivatives, and particularly 9a and 9b, appear to demonstrate sufficient cytostatic potency and selectivity to justify further evaluation of their potential.

Animals↗
PubMed2003

Calpain-1 regulates Bax and subsequent Smac-dependent caspase-3 activation in neutrophil apoptosis.

In the absence and in the resolution of inflammatory responses, neutrophils rapidly undergo spontaneous apoptosis. Here we report about a new apoptosis pathway in these cells that requires calpain-1 activation and is essential for the enzymatic activation of the critical effector caspase-3. Decreased levels of calpastatin, a highly specific intrinsic inhibitor of calpain, resulted in activation of calpain-1, but not calpain-2, in neutrophils undergoing apoptosis, a process that was blocked by a specific calpain-1 inhibitor or by intracellular delivery of a calpastatin peptide. Further support for the importance of the calpastatin-calpain system was obtained by analyzing neutrophils from patients with cystic fibrosis that exhibited delayed apoptosis, associated with markedly increased calpastatin and decreased calpain-1 protein levels compared with neutrophils from control individuals. Additional studies were designed to place calpain-1 into the hierarchy of biochemical events leading to neutrophil apoptosis. Pharmacological calpain inhibition during spontaneous and Fas receptor-induced neutrophil apoptosis prevented cleavage of Bax into an 18-kDa fragment unable to interact with Bcl-xL. Moreover, calpain blocking prevented the mitochondrial release of cytochrome c and Smac, which was indispensable for caspase-3 processing and enzymatic activation, both in the presence and absence of agonistic anti-Fas receptor antibodies. Taken together, calpastatin and calpain-1 represent critical proximal elements in a cascade of pro-apoptotic events leading to Bax, mitochondria, and caspase-3 activation, and their altered expression appears to influence the life span of neutrophils under pathologic conditions.

Apoptosis↗
PubMed2003

Synthesis and biological evaluation of 5-substituted derivatives of the potent antiherpes agent (north)-methanocarbathymine.

The conformationally locked nucleoside, (north)-methanocarbathymine (1a), is a potent and selective anti-herpes agent effective against herpes simplex type 1 (HSV1) and type 2 (HSV2) viruses. Hereby, we report on the synthesis and biological evaluation of a small set of 5-substituted pyrimidine nucleosides belonging to the same class of bicyclo[3.1.0]hexane nucleosides. Both the 5-bromovinyl (4) and the 5-bromo analogue (3) appeared to be exclusive substrates of HSV1 thymidine kinase (TK), contrasting with the 5-iodo analogue (2), which was significantly phosphorylated by the human cytosolic TK. The binding affinity constant and catalytic turnover for HSV1 TK were measured to assess the influence of the substitution on these parameters. In the plaque reduction and cytotoxicity assays, the 5-bromo analogue (3) showed good activity against HSV1 and HSV2 with less general toxicity than 1a. Against varicella-zoster virus (VZV), the north-locked 5-bromovinyl analogue (4) proved to be as potent as its conformationally unlocked 2'-deoxyriboside equivalent BVDU. The three compounds were also tested in vitro as prodrugs used in a gene therapy context on three osteosarcoma cell lines, either deficient in TK (TK(-)), nontransduced, or stably transduced with HSV1 TK. The 5-iodo compound (2, CC(50) 25 +/- 7 microM) was more efficient than ganciclovir (GCV, CC(50) 75 +/- 35 microM) in inhibiting growth of HSV1-TK transfected cells and less inhibitory than GCV toward TK(-) cells, whereas compound 3 inhibited transfected and nontransfected cell lines in a relatively similar dose-dependent manner.

Antiviral Agents↗
PubMed2003

Protonation states of methionine aminopeptidase and their relevance for inhibitor binding and catalytic activity.

We have performed a computational study of different protomeric states of the methionine aminopeptidase active site using a combined quantum-mechanical/molecular mechanical simulation approach. The aim of this study was to clarify the native protonation state of the enzyme, which is needed for the development of novel irreversible inhibitors that can possibly be used as antiangiogenic and antibiotic drugs by virtual screening and other drug design methods. The results of the simulations indicated that two protonation states are possible without disturbing the overall geometry of the active site. We then verified experimentally the presence of the two protonation states by studying the substrate hydrolysis and inhibitor binding reactions at different pH values and come to the conclusion that one of the protomeric states is relevant for inhibitor binding, whereas the other is relevant for substrate hydrolysis. This result has implications for the development of other inhibitors of this class of enzymes and adds a new perspective to the pharmacological properties of the antiangiogenic drug fumagillin, which is an irreversible inhibitor of the human methionine aminopeptidase type II.

Aminopeptidases↗
PubMed2003

Homology modelling and docking studies on Varicella Zoster Virus Thymidine kinase.

Thymidine kinase (TK) is the key enzyme in antiviral and suicide gene therapies. While herpes simplex virus type 1 thymidine kinase has been widely studied and crystallised less is known on Varicella Zoster Virus thymidine kinase (VZV TK) and its three-dimensional structure. In this paper we report the model of the three-dimensional structure of VZV TK resulting from a homology modelling study. Subsequent docking studies of the natural substrate deoxythymidine (dT) and known antiviral drugs were performed and shaded new light on the binding characteristics of the enzyme.

Acyclovir↗
PubMed2003

Tautomerism in computer-aided drug design.

Tautomers are often disregarded in computer-aided molecular modeling applications. Little is known about the different tautomeric states of a molecule and they are rarely registered in chemical databases. Tautomeric forms of a molecule differ in shape, functional groups, surface, and hydrogen-bonding pattern. Calculation of physical-chemical properties and molecular descriptors differ from one tautomeric state to the other as it is demonstrated with an example of the log P calculation, similarity index, and the complementarity pattern to the targeted protein. Considering tautomery in ligand-protein interactions therefore has a significant impact on the prediction of the ligand binding using various docking techniques. This article points on hitherto unaddressed issue of tautomerism in computer-aided drug design.

Binding Sites↗
PubMed2002

Probing the cysteine-34 position of endogenous serum albumin with thiol-binding doxorubicin derivatives. Improved efficacy of an acid-sensitive doxorubicin derivative with specific albumin-binding properties compared to that of the parent compound.

We have recently proposed a macromolecular prodrug strategy for improved cancer chemotherapy based on two features (Kratz, F.; et al. J. Med. Chem 2000, 43, 1253-1256.): (a) rapid and selective binding of thiol-reactive prodrugs to the cysteine-34 position of endogenous albumin after intravenous administration and (b) release of the albumin-bound drug in the acidic environment at the tumor site due to the incorporation of an acid-sensitive bond between the drug and the carrier. To investigate this therapeutic strategy in greater depth, four (maleinimidoalkanoyl)hydrazone derivatives of doxorubicin were synthesized differing in the length of the aliphatic spacer (1, -(CH(2))(2)-; 2, -(CH(2))(3)-; 3, -(CH(2))(5)-; 4, -(CH(2))(7)-). The albumin-binding doxorubicin prodrugs, especially the (6-maleimidocaproyl)hydrazone derivative of doxorubicin (3), are rapidly and selectively bound to the cysteine-34 position of endogenous albumin. 3 was distinctly superior to the parent compound doxorubicin in three animal tumor models (RENCA, MDA-MB 435, and MCF-7) with respect to antitumor efficacy and toxicity.

Animals↗
PubMed2002

Bovine pancreatic polypeptide (bPP) undergoes significant changes in conformation and dynamics upon binding to DPC micelles.

The pancreatic polypeptide (PP), a 36-residue, C-terminally amidated polypeptide hormone is a member of the neuropeptide Y (NPY) family. Here, we have studied the structure and dynamics of bovine pancreatic polypeptide (bPP) when bound to DPC-micelles as a membrane-mimicking model as well as the dynamics of bPP in solution. The comparison of structure and dynamics of bPP in both states reveals remarkable differences. The overall correlation time of 5.08ns derived from the 15N relaxation data proves unambiguously that bPP in solution exists as a dimer. Therein, intermolecular as well as intramolecular hydrophobic interactions from residues of both the amphiphilic helix and of the back-folded N terminus contribute to the stability of the PP fold. The overall rigidity is well-reflected in positive values for the heteronuclear NOE for residues 4-34. The membrane-bound species displays a partitioning into a more flexible N-terminal region and a well-defined alpha-helical region comprising residues 17-31. The average RMSD value for residues 17-31 is 0.22(+/-0.09)A. The flexibility of the N terminus is compatible with negative values of the heteronuclear NOE observed for the N-terminal residues 4-12 and low values of the generalized order parameter S(2). The membrane-peptide interface was investigated by micelle-integrating spin-labels and H,2H exchange measurements. It is formed by those residues which make contacts between the C-terminal alpha-helix and the polyproline helix. In contrast to pNPY, also residues from the N terminus display spatial proximity to the membrane interface. Furthermore, the orientation of the C terminus, that presumably contains residues involved in receptor binding, is different in the two environments. We speculate that this pre-positioning of residues could be an important requirement for receptor activation. Moreover, we doubt that the PP fold is of functional relevance for binding at the Y(4) receptor.

Animals↗
PubMed2002

Comparison of the crystal structures of the human manganese superoxide dismutase and the homologous Aspergillus fumigatus allergen at 2-A resolution.

Manganese superoxide dismutase (MnSOD) of Aspergillus fumigatus, a fungus involved in many pulmonary complications, has been identified as IgE-binding protein. It has been shown also that MnSODs from other organisms, including human, are recognized by IgE Abs from individuals sensitized to A. fumigatus MnSOD. Comparison of the fungal and the human crystal structure should allow the identification of structural similarities responsible for IgE-mediated cross-reactivity. The three-dimensional structure of A. fumigatus MnSOD has been determined at 2-A resolution by x-ray diffraction analysis. Crystals belonged to space group P2(1)2(1)2(1) with unit cell dimensions of a = 65.88 A, b = 98.7 A, and c = 139.28 A. The structure was solved by molecular replacement using the structure of the human MnSOD as a search model. The final refined model included four chains of 199-200 amino acids, four manganese ions, and 745 water molecules, with a crystallographic R-factor of 19.4% and a free R-factor of 23.3%. Like MnSODs of other eukaryotic organisms, A. fumigatus MnSOD forms a homotetramer with the manganese ions coordinated by three histidines, one aspartic acid, and one water molecule. The fungal and the human MnSOD share high similarity on the level of both primary and tertiary structure. We identified conserved amino acids that are solvent exposed in the fungal and the human crystal structure and are therefore potentially involved in IgE-mediated cross-reactivity.

Allergens↗
PubMed2002

The production of macrophage inflammatory protein-2 induced by soluble intercellular adhesion molecule-1 in mouse astrocytes is mediated by src tyrosine kinases and p42/44 mitogen-activated protein kinase.

Severe traumatic brain injury stimulates the release of soluble intercellular adhesion molecule-1 (sICAM-1) into CSF. Studies in cultured mouse astrocytes suggest that sICAM-1 induces the production of macrophage inflammatory protein-2 (MIP-2). In the present study, we investigated the underlying mechanisms for MIP-2 induction. sICAM-1 induced MIP-2 in astrocytes lacking membrane-bound ICAM-1, indicating that its action is due to heterophilic binding to an undescribed receptor rather than homophilic binding to surface ICAM-1. Signal transduction may be mediated by src tyrosine kinases, as the src tyrosine kinase inhibitors herbimycin A and PP2 abolished MIP-2 induction by sICAM-1. Phosphorylation of p42/44 mitogen-activated protein kinase (MAPK), but not of p38 MAPK, occurred further downstream, as evidenced by western blot analysis combined with the use of herbimycin A and specific MAPK inhibitors. By contrast, induction of MIP-2 by tumour necrosis factor-alpha (TNF-alpha) involved both p42/44 MAPK and p38 MAPK. Following stimulation with either sICAM-1 or TNF-alpha, astrocyte supernatants promoted chemotaxis of human neutrophils and incubation of these supernatants with anti-MIP-2 antibodies more efficiently suppressed the migration induced by sICAM-1 than by TNF-alpha. These results show that sICAM-1 induces the production of biologically active MIP-2 in astrocytes by heterophilic binding to an undefined receptor and activation of src tyrosine kinases and p42/44 MAPK.

Animals↗
PubMed2002

Immunological and structural analysis of IgE-mediated cross-reactivity between manganese superoxide dismutases.

BACKGROUND: Allergy results from inappropriate immune responses to normally innocuous proteins. More than 300 IgE-binding proteins have been cloned and shown to cover structurally and functionally heterogeneous protein families including enzymes, backbone and storage proteins as well as proteins with unknown function. Structurally related cross-reactive allergens are involved in the pathogenesis of important clinical syndromes. We found that phylogenetically highly conserved proteins, including human manganese superoxide dismutase (MnSOD), are involved in IgE-mediated autoreactivity. METHODS: MnSOD cloned from different phylogenetically distant species were produced as recombinant proteins in Escherichia coli and used to study IgE-mediated cross-reactivity and proliferative responses in peripheral blood mononuclear cells (PBMC) of individuals sensitised to Aspergillus fumigatus MnSOD. Homology models of the three-dimensional structures of MnSOD from A. fumigatus, Drosophila melanogaster and Saccharomyces cerevisiae using the human MnSOD structure refined at 2.2 A as template were constructed to identify conserved amino acid residues exposed to the solvent. RESULTS: Cross-reactivity between the MnSOD at B and T cell level was demonstrated by inhibition experiments showing shared B cell epitopes and by the capability of the different MnSODs to induce proliferative responses in PBMCs of sensitised individuals. Structural modelling allowed to identify conserved residues exposed to the solvent. The identified residues are scattered over the sequence of the enzyme indicating putative conformational IgE-binding epitopes. CONCLUSIONS: The results obtained corroborate molecular mimicry as a plausible mechanism to explain autoreactivity to human MnSOD. Moreover, homology modelling provides a rational tool to identify conserved residues involved in defining cross-reactive B cell epitopes.

Adult↗
PubMed1995

[Modeling of protein-ligand interaction complexes as a basic of the rational design of drugs]

Most of our today"s drugs have been discovered by the lucky coincidences called serendipity. The drugs itself however, have tremendously changed since the ancient times. From the herbal teas as the remedies of our ancestors, we have switched to modern therapeutics, mostly containing only one, very high active compound. Modern analytical methods and the approaches of recent molecular biology have revealed the molecular mechanisms of a lot of this high active drugs. In most of the cases, interaction of the drug molecule with a special protein in the body, located on the surface of the cell releases the biological response. This may lead to the idea, that knowledge of the structure of the protein-ligand complex enables us to design a drug with certain properties on the computer screen. If successful, serendipity could be considerably reduced as well as animal testing. As a matter of fact, it can be shown, that the combination of biophysics, modeling, and biological in vitro methods brings us very close to that goal. However, basic problems, for instance in the handling of complex systems are opposed to the reductionistic approach in the drug research. They prevent the simple application of the lock and key principle in making a drug by fitting a putative drug molecule into the binding site of a receptor. Nevertheless, whenever a biologically active compound has been discovered, be it by pure chance, serendipity, design or ethnological pharmacy, molecular modeling or drug design will be of great help in optimizing the active compounds and enrichment of the whole new class of therapeutics.

Journal Article↗
PubMedDate not supplied

Methodology and problems of protein-ligand docking: case study of dihydroorotate dehydrogenase, thymidine kinase, and phosphodiesterase 4.

The docking methodology was applied to three different therapeutically interesting enzymes: human dihydroorotate dehydrogenase (DHODH), Herpes simplex virus type I thymidine kinase (HSV1 TK) and human phosphodiesterase 4 (PDE4). Programs FlexX, AutoDock and DOCK where used. The three targets represent three distinct cases. For DHODH and HSV1 TK, the binding modes of substrate and inhibitors within the active site are known, while the binding orientation of cAMP within PDE4 has been solely hypothesized. Active site of DHODH is mainly hydrophobic and the binding mode of the inhibitor brequinar was used as a template for evaluating the docking strategies. The presence of cofactors revealed to be crucial for the definition of the docking site. The HSV1 TK active site is small and polar and contains crystal water molecules and ATP. Docking of thymidine and aciclovir (ACV) within the active site was analyzed by keeping or removing water molecules. It showed the crucial role of water in predicting the binding of pyrimidines and purines. The crystal structure of PDE4 contains magnesium and zinc cations as well as catalytic water molecule but no ligand. Several docking experiments of cAMP and rolipram were performed and the results showed clear-cut dependence between the ligand orientation and the presence of metals in the active site. All three cases show specific problems of the docking methodology, depending on the character of the active site.

3',5'-Cyclic-AMP Phosphodiesterases↗