Biomedical subjects

Hans-Dieter Höltje

Publications and source records attributed to Hans-Dieter Höltje.

At least 19 recordsLinked to original sources

PubMed2006

Allosteric interactions with muscarinic acetylcholine receptors: complex role of the conserved tryptophan M2422Trp in a critical cluster of amino acids for baseline affinity, subtype selectivity, and cooperativity.

In general, the M2 subtype of muscarinic acetylcholine receptors has the highest sensitivity for allosteric modulators and the M5 subtype the lowest. The M2/M5 selectivity of some structurally diverse allosteric agents is known to be completely explained by M2 177Tyr and M2 423Thr in receptors whose orthosteric site is occupied by the conventional ligand N-methylscopolamine (NMS). This study explored the role of the conserved M2 422Trp and the adjacent M2 423Thr in the binding of alkane-bisammonio type modulators, gallamine, and diallylcaracurine V. Experiments were performed with human M2 or M5 receptors or mutants thereof. It was found that M2 422Trp and M2 423Thr independently influenced allosteric agent binding. The presence of M2 423Thr may enhance the affinity of binding, depending on the allosteric agent, either directly or indirectly (by avoiding sterical hindrance through its M5 counterpart 478His). Replacement of M2 422Trp and of the corresponding M5 477Trp by alanine revealed a pronounced contribution of these epitopes to subtype independent baseline affinity in NMS-bound and NMS-free receptors for all agents except diallylcaracurine V. In a few instances, this tryptophan also influenced cooperativity and subtype selectivity. Docking simulations using a three-dimensional M2 receptor model revealed that the aromatic rings of M2 177Tyr and M2 422Trp, in a concerted action, might fix one of the aromatic moieties of alkane-bisammonio compounds between them. Thus, M2 422Trp and the spatially adjacent M2 177Tyr, as well as M2 423Thr, form a cluster of amino acids within the allosteric binding cleft that is pivotal for both M2/M5 subtype selectivity and baseline affinity of allosteric agents.

Allosteric Regulation↗

PubMed2005

Molecular mechanisms of antibiotic resistance: QM/MM modelling of deacylation in a class A beta-lactamase.

Modelling of the first step of the deacylation reaction of benzylpenicillin in the E. coli TEM1 beta-lactamase (with B3LYP/6-31G + (d)//AM1-CHARMM22 quantum mechanics/molecular mechanics methods) shows that a mechanism in which Glu166 acts as the base to deprotonate a conserved water molecule is both energetically and structurally consistent with experimental data; the results may assist the design of new antibiotics and beta-lactamase inhibitors.

Acylation↗

PubMed2005

Molecular dynamics simulations of bovine rhodopsin: influence of protonation states and different membrane-mimicking environments.

G-protein coupled receptors (GPCRs) are a protein family of outstanding pharmaceutical interest. GPCR homology models, based on the crystal structure of bovine rhodopsin, have been shown to be valuable tools in the drug-design process. The initial model is often refined by molecular dynamics (MD) simulations, a procedure that has been recently discussed controversially. We therefore analyzed MD simulations of bovine rhodopsin in order to identify contacts that could serve as constraints in the simulation of homology models. Additionally, the effect of an N-terminal truncation, the nature of the membrane mimic, the influence of varying protonation states of buried residues and the importance of internal water molecules was analyzed. All simulations were carried out using the program-package GROMACS. While N-terminal truncation negatively influenced the overall protein stability, a stable simulation was possible in both solvent environments. As regards the protonation state of titratable sites, the experimental data could be reproduced by the program UHBD (University of Houston Brownian Dynamics), suggesting its application for studying homology models of GPCRs. A high flexibility was observed for internal water molecules at some sites. Finally, interhelical hydrogen-bonding interactions could be derived, which can now serve as constraints in the simulations of GPCR homology models.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2005

Human topoisomerase I poisoning: docking protoberberines into a structure-based binding site model.

Using the X-ray crystal structure of the human topoisomerase I (top1) - DNA cleavable complex and the Sybyl software package, we have developed a general model for the ternary cleavable complex formed with four protoberberine alkaloids differing in the substitution on the terminal phenyl rings and covering a broad range of the top1-poisoning activities. This model has the drug intercalated with its planar chromophore between the -1 and +1 base pairs flanking the cleavage site, with the nonplanar portion pointing into the minor groove. The ternary complexes were geometry-optimized and relative interaction energies, computed by using the Tripos force field, were found to rank in correct order the biological potency of the compounds; in addition, the model is also consistent with the top1-poisoning inactivity of berberine, a major prototype of the protoberberine alkaloids. The model might serve as a rational basis for elaboration of the most active compound as a lead structure, in order to develop more potent top1 poisons as next generation anti-cancer drugs.

Berberine Alkaloids↗

PubMed2005

Mechanisms of antibiotic resistance: QM/MM modeling of the acylation reaction of a class A beta-lactamase with benzylpenicillin.

Understanding the mechanisms by which beta-lactamases destroy beta-lactam antibiotics is potentially vital in developing effective therapies to overcome bacterial antibiotic resistance. Class A beta-lactamases are the most important and common type of these enzymes. A key process in the reaction mechanism of class A beta-lactamases is the acylation of the active site serine by the antibiotic. We have modeled the complete mechanism of acylation with benzylpenicillin, using a combined quantum mechanical and molecular mechanical (QM/MM) method (B3LYP/6-31G+(d)//AM1-CHARMM22). All active site residues directly involved in the reaction, and the substrate, were treated at the QM level, with reaction energies calculated at the hybrid density functional (B3LYP/6-31+Gd) level. Structures and interactions with the protein were modeled by the AM1-CHARMM22 QM/MM approach. Alternative reaction coordinates and mechanisms have been tested by calculating a number of potential energy surfaces for each step of the acylation mechanism. The results support a mechanism in which Glu166 acts as the general base. Glu166 deprotonates an intervening conserved water molecule, which in turn activates Ser70 for nucleophilic attack on the antibiotic. This formation of the tetrahedral intermediate is calculated to have the highest barrier of the chemical steps in acylation. Subsequently, the acylenzyme is formed with Ser130 as the proton donor to the antibiotic thiazolidine ring, and Lys73 as a proton shuttle residue. The presented mechanism is both structurally and energetically consistent with experimental data. The QM/MM energy barrier (B3LYP/ 6-31G+(d)//AM1-CHARMM22) for the enzymatic reaction of 9 kcal mol(-1) is consistent with the experimental activation energy of about 12 kcal mol(-1). The effects of essential catalytic residues have been investigated by decomposition analysis. The results demonstrate the importance of the "oxyanion hole" in stabilizing the transition state and the tetrahedral intermediate. In addition, Asn132 and a number of charged residues in the active site have been identified as being central to the stabilizing effect of the enzyme. These results will be potentially useful in the development of stable beta-lactam antibiotics and for the design of new inhibitors.

Acylation↗

PubMed2005

Different environments for a realistic simulation of GPCRs-application to the M2 muscarinic receptor.

A model of the human M(2) muscarinic receptor was taken as an example for a class A G-protein coupled receptor to explore the influence of different environments in a molecular dynamics simulation (MDS) on the protein structure. The most commonly used environment is the vacuum, although it is very unnatural for a transmembrane protein. As an alternative a membrane-like system, consisting of a lipophilic central layer and two aqueous flanking layers, was tested. The most realistic system that can be applied is a phospholipid bilayer with a surrounding physiological sodium chloride solution. From all systems good protein structures were received, nevertheless clear differences between the systems were detected in the structural comparison of the models. Subsequently it was analyzed whether the observed structural differences influence ligand binding. For this purpose the antagonist (S)-scopolamine was docked into the binding cavity, which is well known by many reported single and multiple point mutations. As expected from the observed structural variations triggered by the type of environment employed in MDS, also differences in the binding mode of (S)-scopolamine were detected, all contacts, however, which are known to be important were found.

Chemistry, Pharmaceutical↗

PubMed2005

Molecular design of two sterol 14alpha-demethylase homology models and their interactions with the azole antifungals ketoconazole and bifonazole.

Sterol 14alpha-demethylase (CYP51) is one of the known major targets for azole antifungals. Therapeutic side effects of these antifungals are based on interactions of the azoles with the human analogue enzyme. This study describes for the first time a comparison of a human CYP51 (HU-CYP51) homology model with a homology model of the fungal CYP51 of Candida albicans (CA-CYP51). Both models are constructed by using the crystal structure of Mycobacterium tuberculosis MT-CYP51 (PDB code: 1EA1). The binding mode of the azole ketoconazole is investigated in molecular dynamics simulations with the GROMACS force field. The usage of special parameters for the iron azole complex binding is necessary to obtain the correct complex geometry in the active site of the enzyme models. Based on the dynamics simulations it is possible to explain the enantioselectivity of the human enzyme and also to predict the binding mode of the isomers of ketoconazole in the active site of the fungal model.

Amino Acid Sequence↗

PubMed2005

Metalloporphyrins inactivate caspase-3 and -8.

Activation of caspases represents one of the earliest biochemical indicators for apoptotic cell death. Therefore, measurement of caspase activity is a widely used and generally accepted method to determine apoptosis in a wide range of in vivo and in vitro settings. Numerous publications characterize the role of the heme-catabolizing enzyme heme oxygenase-1 (HO-1) in regulating apoptotic processes. Different metalloporphyrins representing inducers and inhibitors of this enzyme are often used, followed by assessment of apoptotic cell death. In the present work, we found that caspase-3-like activity, as well as activity of caspase-8 measured in either Fas (CD95) ligand-treated Jurkat T-lymphocytes or by the use of recombinant caspase-3 or -8, was inhibited by different metalloporphyrins (cobalt(III) protoporphyrin IX, tin and zinc(II) protoporphyrin-IX). Moreover, employing the mouse model of Fas-induced liver apoptosis these properties of porphyrins could also be demonstrated in vivo. The metalloporphyrins were shown to inhibit caspase-3-mediated PARP cleavage. Molecular modeling studies demonstrated that porphyrins can occupy the active site of caspase-3 in an energetically favorable manner and in a binding mode similar to that of known inhibitors. The data shown here introduce metalloporphyrins as direct inhibitors of caspase activity. This finding points to the need for careful employment of metalloporphyrins as modulators of HO-1.

Animals↗

PubMed2004

A combined QM/MM approach to protein--ligand interactions: polarization effects of the HIV-1 protease on selected high affinity inhibitors.

HIV-1 protease inhibitors are one of the two widely used therapeutic agents for the treatment of HIV-infected patients. The investigation of HIV-1 protease-inhibitor interactions can provide further insight for developing new compounds that are still required due to the growing problem of drug resistance. To this end, a combined QM/MM approach was used to determine electrostatic and polarization interactions on three high affinity inhibitors, nelfinavir, mozenavir, and tipranavir. The present computational results show that explicit treatment of the polarization effect is particularly important since it can contribute as much as one-third of the total electrostatic interaction energy. Further, an amino acid decomposition analysis was applied to determine contributions of individual residues to the enzyme--inhibitor interactions. It was found that the 4-hydroxy-dihydropyrone substructure of tipranavir is especially suited for extended charge delocalization by interacting with the catalytic aspartates and isoleucines of the HIV-1 protease. The calculated electron density difference maps reaffirm and provide a means of visualizing these results.

HIV Protease↗

PubMed2004

Comparison of a 3D-model of the classical alpha-scorpion toxin V from Leiurus quinquestriatus quinquestriatus with other scorpion toxins.

In the present paper, a study of classical and insect alpha-scorpion toxins is described. A homology model of the classical alpha-toxin LqqV from Leiurus quinquestriatus quinquestriatus was developed. The model was compared to stable and energetically favourable conformations of AaHII from Androctonus australis Hector and LqhalphaIT from Leiurus quinquestriatus hebraeus, which are the most active alpha-toxins in mammals and insects. The conformations were retrieved from molecular dynamics simulations of known structures. The model of LqqV shows a C-terminal conformation similar to LqhalphaIT. This is mainly caused by electrostatic interactions between Lys10 /Lys60 and Glu59, which are comparable to the cation-pi interactions of Tyr10 and Arg64 in LqhalphaIT. During the simulations the structures of AaHII and LqqV were stabilised through electrostatic interactions between Glu32 and Lys50 and especially the loop adjacent to the alpha-helix is affected, which is in contrast to LqhalphaIT. When the molecular electrostatic potentials of the toxins were studied, a possibly important difference between the classical alpha-toxins and the insect alpha-toxin LqhalphaIT was found in the area around Lys30 and Arg56 of AaHII, where a positive potential is missing in LqhalphaIT. A large negative potential caused by Asp3, Glu15 and Asp19 in LqhalphaIT is also unique for this toxin. It is proposed that Arg18, which is important for activity of LqhalphaIT, restricts the negative potential in this area and is not essential for toxins where negatively charged residues in comparable positions are not present.

Amino Acid Sequence↗

PubMed2004

Integrin specificity of the cyclic Arg-Gly-Asp motif and its role in integrin-targeted gene transfer.

Targeted gene transfer, addressing the alphavbeta3 integrin by coupling the appropriate ligand, cRGD (S(2)-bridged cyclic Arg-Gly-Asp containing peptide) motif, on to a DNA condensing sequence was described as early as 1995 by Hart, Harbottle, Cooper, Miller, Williamson and Coutelle [(1995) Gene Ther. 2, 552-554]. Their work was followed by a series of publications, introducing the cRGD motif in polycationic DNA carriers, such as peptides, proteins and liposomes. Polyethylenimine and even adenoviruses were additionally ligated using the cRGD motif. 'Integrin specificity' has been determined from the significantly improved transfection efficiency compared with the DNA carriers with control ligands, mainly the cRGE (S(2)-bridged cyclic-Arg-Gly-Glu-containing peptide) motif. However, by observing the physicochemical appearance of the resulting complexes and their controls such as the poly(L-lysine)-DNA complexes carrying the cRGD and the cRGE motifs, we doubted the integrin-mediated specificity of the increased transfection efficiency. To clarify this contradiction, we investigated the suitability of the cRGD motif for targeted gene transfer. We proved the specificity of the RGD motif and its controls using computational docking procedures and molecular modelling methods. Since we were confident of the motifs used, we improved our transfection method. Since aggregation of the RGD-ligated poly(L-lysine)-DNA complexes under physiological conditions caused an enormous amount of unspecific cell uptake and transfection, a method had to be designed to exclude aggregation processes of the motif-polycation-DNA complexes. Small complex sizes are necessary for receptor-specific uptake. The complexes were therefore recharged using poly(vinyl sulphate). Inhibited aggregation of the targeted DNA carriers under physiological conditions is a necessary prerequisite for successful in vivo gene transfer.

Binding Sites↗

PubMed2003

N-(omega-(4-(2-methoxyphenyl)piperazin-1-yl)alkyl)carboxamides as dopamine D2 and D3 receptor ligands.

The dopamine D(3) receptor is recognized as a potential therapeutic target for the treatment of various neurological and psychiatric disorders. Targetting high affinity and D(3) versus D(2) receptor-preferring ligands, the partial agonist BP 897 was taken as a lead structure. Variations in the spacer and the aryl moiety led to N-alkylated 1-(2-methyoxyphenyl)piperazines with markedly improved affinity and selectivity. Molecular modeling studies supported the structural development. Pharmacophore models for dopamine D(2) and D(3) receptor ligands were developed from their potentially bioactive conformation and were compared in order to get insight into molecular properties of importance for D(2)/D(3) receptor selectivity. For the 72 compounds presented here, an extended and more linear conformation in the aliphatic or aryl spacers turned out to be crucial for dopamine D(3) receptor selectivity. Structural diversity in the aryl moiety (benzamides, heteroarylamides, arylimides) had a major influence on (sub)nanomolar D(3) receptor affinity, which was optimized with more rigid aryl acrylamide derivatives. Compound 38 (ST 280, (E)-4-iodo-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)cinnamoylamide) displayed a most promising pharmacological profile (K(i) (hD(3)) = 0.5 nM; K(i) (hD(2L)) = 76.4 nM; selectivity ratio of 153), and above that, compound 38 offered the prospect of a novel radioligand as a pharmacological tool for various D(3) receptor-related in vitro and in vivo investigation.

Animals↗

PubMed2003

Identification of Glu166 as the general base in the acylation reaction of class A beta-lactamases through QM/MM modeling.

Bacterial class A beta-lactamases are responsible for the most known resistance against beta-lactam antibiotics. With the continuing rise in antibiotic resistance, improved knowledge of the mechanisms of action of these enzymes is needed in the development of effective therapeutic agents and strategies. The mechanism of the deacylation step in class A beta-lactamases is well accepted. In contrast, the mechanism of the acylation step has been uncertain, with several conflicting proposals put forward. We have modeled the acylation step in a class A beta-lactamase, using a combined quantum mechanics/molecular mechanics approach. The results provide an atomic level description of the reaction and show that Glu166 acts as the general base in the reaction, deprotonating Ser70 via an intervening water molecule. Ser70 acts as the nucleophile for attack on the lactam ring in a concerted reaction. The results do not rule out the importance of Lys73 in catalysis, in agreement with experimental data.

Acylation↗

PubMed2003

Glucocorticoid receptor interactions with glucocorticoids: evaluation by molecular modeling and functional analysis of glucocorticoid receptor mutants.

In the treatment of inflammatory skin diseases, there are some glucocorticoid (GC) double esters combining pronounced antiinflammatory activity and minor atrophogenic side effects. The reason, however, is only poorly understood. To investigate interactions of GCs with the ligand-binding domain of the glucocorticoid receptor (GR), we measured receptor-binding potency of a series of GC esters including their metabolites and performed a molecular modeling study using progesterone receptor crystal structure data. Ligand docking to the GR-binding pocket showed good fitting of GC 17-esters corresponding to their high receptor-binding affinity, and unfavorable sterical interactions for GC 21-esters with substituents larger than propionate. Molecular dynamics simulations served to visualize induced fit procedures. Ligand docked GC conformations after dynamics simulations were used for generation of a 3D quantitative structure-activity relationship model. Using a set of 11 steroids, this model showed a correlation coefficient (r(2)) of 0.98, a leave-one-out cross validation (q(2)) of 0.79 and was able to predict binding affinity of further six ligands with a standard error of prediction of 0.33. Moreover, interactions of Asn-564 and Met-639 with the steroids were investigated by studying GR mutants of these amino acids. Met-639 participates in hydrophobic interactions mainly with GC side chains, while Asn-564 forms a hydrogen bond to the C11-OH group of the steroid. Asn-564 is shown to be very important for ligand binding and even more for target gene activation and transcription factor repression.

Amino Acid Substitution↗

PubMed2003

Molecular modelling studies on the ORL1-receptor and ORL1-agonists.

The ORLI (opioid receptor like 1)- receptor is a member of the family of rhodopsin-like G protein-coupled receptors (GPCR) and represents an interesting new therapeutical target since it is involved in a variety of biomedical important processes, such as anxiety, nociception, feeding, and memory. In order to shed light on the molecular basis of the interactions of the GPCR with its ligands, the receptor protein and a dataset of specific agonists were examined using molecular modelling methods. For that purpose, the conformational space of a very potent non-peptide ORL1-receptor agonist (Ro 64-6198) with a small number of rotatable bonds was analysed in order to derive a pharmacophoric arrangement. The conformational analyses yielded a conformation that served as template for the superposition of a set of related analogues. Structural superposition was achieved by employing the program FlexS. Using the experimental binding data and the superposition of the ligands, a 3D-QSAR analysis applying the GRID/GOLPE method was carried out. After the ligand-based modelling approach, a 3D model of the ORL1-receptor has been constructed using homology modelling methods based on the crystal structure of bovine rhodopsin. A representative structure of the model taken from molecular dynamics simulations was used for a manual docking procedure. Asp-130 and Thr-305 within the ORL1-receptor model served as important hydrophilic interaction partners. Furthermore, a hydrophobic cavity was identified stabilizing the agonists within their binding site. The manual docking results were supported using FlexX, which identified the same protein-ligand interaction points.

Amino Acid Sequence↗

PubMed2003

Allosteric site on muscarinic acetylcholine receptors: identification of two amino acids in the muscarinic M2 receptor that account entirely for the M2/M5 subtype selectivities of some structurally diverse allosteric ligands in N-methylscopolamine-occupied receptors.

Two epitopes have been identified recently to be responsible for the high-affinity binding of alkane-bisammonium and caracurine V type allosteric ligands to N-methylscopolamine (NMS)-occupied M2 muscarinic acetylcholine receptors, relative to M5 receptors: the amino acid M2-Thr423 at the top of transmembrane region (TM) 7 and an epitope comprising the second extracellular loop (o2) of the M2 receptor including the flanking regions of TM4 and TM5. We aimed to find out whether a single amino acid could account for the contribution of this epitope to binding affinity. Allosteric interactions were investigated in wild-type and mutant receptors in which the orthosteric binding site was occupied by [3H]NMS (5 mM Na,K,Pi buffer, pH 7.4, 23 degrees C). Using M2/M5 chimeric and point-mutated receptors, the relevant epitope was narrowed down to M2-Tyr177. A double point-mutated M2 receptor in which both M2-Tyr177 and M2-Thr423 were replaced by the corresponding amino acids of M5 revealed that these two amino acids account entirely for the (approximately 100-fold) M2/M5 selectivity of the alkane-bisammonium and the caracurine V type allosteric ligands. At NMS-free M2 receptors, the caracurine V derivative also displayed approximately 100-fold M2/M5 selectivity, but the double point mutation reduced the M2 affinity by only approximately 10-fold; thus, additional epitopes may influence selectivity for the free receptors. A three-dimensional model of the M2 receptor was used to simulate allosteric agent docking to NMS-occupied receptors. M2-Tyr177 and M2-Thr423 seem to be located near the junction of the allosteric and the orthosteric areas of the M2 receptor ligand binding cavity.

Allosteric Regulation↗

PubMed2002

Kappa-opioid receptor model in a phospholipid bilayer: molecular dynamics simulation.

A three-dimensional molecular model of the transmembrane domain of the kappa-opioid receptor in a phospholipid bilayer is presented. The endogenous ligand, dynorphin A (1), and synthetic ligands, benzomorphan-based compounds (2a, 2b) (Figure 1), are docked into the model. We report the results of a 500 ps molecular dynamics simulation of these protein-ligand complexes in a simplified bilayer of 97 molecules of the lipid dipalmitoylphosphatidylcholine and 26 water molecules per lipid. The simulations explore the stability and conformational dynamics of the model in a phospholipid bilayer; we also investigate the interactions of the protein with its ligands. Molecular simulation of the receptor-ligand complexes, endogenous and synthetic, has confirmed the existence of different binding domains for peptide and non-peptide ligands. Similarities are found in the dynamics and binding mode of all conformations of the synthetic ligands studied. The protonated hydrogen of the benzomorphan is always involved in an H-bond with Asp138, and other potentially stabilizing receptor-ligand interactions found involve the hydroxyl substituent on the benzomorphan, which may form an H-bond with Tyr139 or Gly190 according to the different molecules. The ester group of 2a may therefore form an H-bond with Ile316, while the carbonyl group of 2b forms an H-bond with Gln115 and Tyr312. The remaining part of the ligand is located in the extracellular portion of the pocket. It is surrounded by hydrophobic residues in the transmembrane region (TM), and it interacts with different sets of residues. The results obtained are in general agreement with site-directed mutagenesis data that have highlighted the importance of all TM regions for synthetic-ligand affinity with the kappa-opioid receptor.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2002

Molecular modeling and QSAR studies on K(ATP) channel openers of the benzopyran type.

The present paper describes our molecular modeling and quantitative structure-activity relationships (QSARs) studies on K(ATP) channel openers (KCOs) of the benzopyran type. In the first part we performed molecular modeling investigations with seven benzopyrans, varied at the C3- and C4-positions, in order to understand which molecular features at these positions are essentially effecting the biological activity. The impact of C6-substitution on biological activity was studied in the second part via HANSCH analysis. For this purpose physicochemical properties (charge distributions, lowest unoccupied molecular orbital (LUMO) energies, desolvation energies, volumes and dipole moments) were calculated for a set of 50 C6-varied benzopyrans. A QSAR equation was developed showing a relationship between the vasodilator activity and the direction of the dipole vector of the ligands. The conclusion can be drawn that a direct interaction between the C6-substituents and the receptor structure is not of primary importance. However, the substitutents influence the orientation of the whole ligand approaching the binding site. An unfavorably oriented ligand cannot bind to the binding site, thus exhibiting weak activity.

Benzopyrans↗